Characterization of Mice with Altered Dopamine Transporter and Vesicular Monoamine Transporter 2
Levels
by
Shababa Tanzeel Masoud
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Pharmacology and Toxicology University of Toronto
© Copyright by Shababa Tanzeel Masoud 2017
ii
Characterization of Mice with Altered Dopamine Transporter and
Vesicular Monoamine Transporter 2 Levels
Shababa Tanzeel Masoud
Doctor of Philosophy
Department of Pharmacology and Toxicology
University of Toronto
2017
Abstract
Dopamine is a key neurotransmitter that regulates motor coordination and dysfunction of the
dopamine system gives rise to Parkinson’s disease. Nigrostriatal dopamine neurons are
vulnerable to various genetic and environmental insults, suggesting that these cells are inherently
at-risk. A cell-specific risk factor for these neurons is the neurotransmitter, dopamine itself. If
intracellular dopamine is not appropriately sequestered into vesicles, it can accumulate in the
cytosol. Cytosolic dopamine is highly reactive and can trigger oxidative stress, leading to cellular
toxicity. Cytosolic dopamine levels are modulated by the plasma membrane dopamine
transporter (DAT) that takes up dopamine from the extracellular space, and the vesicular
monoamine transporter 2 (VMAT2) that stores dopamine into vesicles. In this thesis, we altered
DAT and VMAT2 levels to investigate the detrimental consequences of potentially amplifying
cytosolic dopamine in transgenic mice. Project 1 focused on selective over-expression of DAT in
dopaminergic cells of transgenic mice (DAT-tg). DAT-tg mice displayed phenotypes of
dopaminergic damage: increased dopamine-specific oxidative stress, L-DOPA-reversible fine
motor deficits and enhanced sensitivity to toxicant insult, suggesting that increasing DAT-
mediated dopamine uptake is detrimental for dopamine cells. As an extension of Project 1,
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Project 2 focused on mice that simultaneously over-express DAT and under-express VMAT2
(DAT-tg/VMAT2-kd mice). These animals were hypothesized to demonstrate exacerbated
dopaminergic toxicity due to buildup of cytosolic dopamine caused by increased uptake and
decreased packaging. While DAT-tg/VMAT2-kd mice displayed detrimental phenotypes (poor
survival, decreased body weight, reduced dopamine tissue content and release) and
compensatory changes (increased dopamine receptors and metabolism), they did not show
dopamine cell loss. This is due to unexpected loss of phenotypes in DAT-tg mice from a new
colony that no longer displayed dopaminergic neurodegeneration. Thus, instead of Parkinsonian
behavior, DAT-tg/VMAT2-kd mice showed novel phenotypes such as hyperactivity and
improved fine-motor and cognitive skills compared to other genotypes. DAT-tg/VMAT2-kd
mice were also highly sensitive to amphetamine-induced locomotion. Hence, in the absence of
neurodegeneration, altered DAT and VMAT2 levels produced unique behavioral changes in
DAT-tg/VMAT2-kd mice, shedding light on the complex function of the dopamine system.
Collectively, these studies demonstrate how perturbations in dopamine compartmentalization can
impact dopamine homeostasis and behavior.
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Acknowledgments
First, I would like to thank my supervisor, Dr. Ali Salahpour, for his immense guidance and
mentorship during my Ph.D. As one of his first students, I have had the privilege of learning
from him directly and seeing the lab grow over the years. His enthusiasm for science, positive
outlook, understanding nature and approachability make him a truly unique supervisor.
To my committee members, Drs. Peter G. Wells, José Nobrega, W. M. Burnham and David S.
Riddick: you have been my guiding light throughout this Ph.D. You have challenged, supported
and encouraged me. I am eternally grateful for having the best Ph.D. supervisory committee I
could ever hope for. A special thank you to Dr. David S. Riddick for playing the dual role of my
co-supervisor and thesis reader. You have always had the time to check up on me, provide
constructive criticism and guide me in the right direction. Also, a special thank you to Dr. W. M
Burnham – I started my scientific journey in your lab as a 4th year project student and since then,
I have shared a great working relationship with you as the TA for PCL475. Thank you for your
kindness and for always having my best interest in mind.
To Dr. Amy Ramsey, thank you for offering your expertise and advice throughout my Ph.D. To
our collaborators: Drs. Gary W. Miller, Jason Richardson, Jonathan Brotchie and Andrei
Starostin – I truly appreciate your invaluable technical help with my projects. I would like to
gratefully acknowledge Dr. Salah El Mestikawy for being my external examiner. A special
mention for Lien Nguyen, my undergraduate project student, for her useful contribution to these
experiments. I am also grateful for my sources of funding from Parkinson Society of Canada,
Canadian Institutes of Health Research and the University of Toronto.
Wendy Horsfall, you are the backbone of our lab – I cannot thank you enough for sharing your
knowledge and being so patient with us. Marija Milenkovic and Dr. Laura Vecchio, thank you
for helping me every day and being my voice of reason. To all members of the Salahpour and
Ramsey labs, I am grateful to have shared this journey with you.
Finally, I would like to extend my deepest gratitude to my family. To my parents, Chowdhury A.
Masud and Shabina M. Masud - you never doubted me even for a moment. You stood by me as
pillars of strength throughout all my struggles and I will forever remain grateful. To Nafees, you
supported me in every way imaginable. Thank you for being my teammate.
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Table of Contents
Acknowledgments.......................................................................................................................... iv
Table of Contents .............................................................................................................................v
List of Figures ..................................................................................................................................x
List of Tables ............................................................................................................................... xiii
List of Appendices ....................................................................................................................... xiv
List of Publications ........................................................................................................................xv
List of Abbreviations ................................................................................................................... xvi
Chapter 1 Introduction .....................................................................................................................1
Introduction .................................................................................................................................1
1.1 Statement of Research Problem ...........................................................................................1
1.2 Literature Review.................................................................................................................2
1.2.1 Dopamine function in the brain ................................................................................2
1.2.1.1 Nigrostriatal pathway and movement ........................................................3
1.2.1.2 Other dopaminergic pathways ...................................................................8
1.2.2 Dopamine homeostasis .............................................................................................9
1.2.2.1 Synthesis ....................................................................................................9
1.2.2.2 Release .....................................................................................................11
1.2.2.3 Degradation .............................................................................................12
1.2.3 Dopamine transport ................................................................................................14
1.2.3.1 Plasma membrane transport ....................................................................14
1.2.3.2 Vesicular membrane transport .................................................................15
1.2.4 Dopamine compartmentalization and its effects ....................................................17
1.2.4.1 Extracellular dopamine ............................................................................17
1.2.4.1.1 Dopamine Receptors............................................................... 17
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1.2.4.2 Intracellular dopamine .............................................................................20
1.2.4.2.1 Cytosolic dopamine ................................................................ 20
Reactivity .................................................................. 21
Toxicity .................................................................... 24
1.2.5 Classical drugs that interact with the dopamine system .........................................28
1.2.5.1 Enzyme ligands .......................................................................................29
1.2.5.2 DAT ligands ............................................................................................30
1.2.5.3 VMAT2 ligands .......................................................................................33
1.2.5.4 Dopamine receptor ligands ......................................................................35
1.2.6 Parkinson’s disease ................................................................................................37
1.2.6.1 Symptoms ................................................................................................38
1.2.6.2 Pathology .................................................................................................39
1.2.6.3 Therapy ....................................................................................................41
1.2.6.4 Etiology ...................................................................................................42
1.2.6.5 Vulnerability of nigrostriatal dopaminergic cells ....................................44
1.2.6.5.1 Role of cytosolic dopamine in Parkinson’s disease................ 47
1.2.6.5.2 Role of dopamine transporters in Parkinson’s disease ........... 49
1.2.7 Animal models with altered transporter levels .......................................................52
1.2.7.1 DAT-knockout mice ................................................................................54
1.2.7.2 DAT-overexpressing transgenic mice .....................................................55
1.2.7.3 VMAT2-knockout homozygote mice ......................................................56
1.2.7.4 VMAT2-knockout heterozygote mice .....................................................57
1.2.7.5 VMAT2-knockdown mice .......................................................................58
1.2.7.6 VMAT2-overexpressing mice .................................................................61
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1.3 Rationale, Hypothesis and Aims ........................................................................................62
Chapter 2 Materials and Methods ..................................................................................................64
Materials and Methods ..............................................................................................................64
2.1 Mice ...................................................................................................................................64
2.1.1 Generation of DAT-tg mice (Project 1) .................................................................64
2.1.2 Generation of DAT-tg/VMAT2-kd mice (Project 2) .............................................64
2.1.3 Body weight ...........................................................................................................65
2.1.4 Survival ......……………………………………………………………………....66
2.2 Biochemistry ......................................................................................................................66
2.2.1 Western blots ..........................................................................................................66
2.2.2 Quantitative reverse transcriptase PCR ..................................................................67
2.2.3 Immunohistochemistry ...........................................................................................68
2.3 Neurochemistry ..................................................................................................................68
2.3.1 High performance liquid chromatography (HPLC) ...............................................68
2.3.2 Fast-scan cyclic voltammetry (FSCV) ...................................................................69
2.4 Stereology ..........................................................................................................................70
2.5 Radioligand binding ...........................................................................................................72
2.6 Behavioral Assessments.....................................................................................................73
2.6.1 Open field locomotor activity ................................................................................73
2.6.2 Wire-hang test ........................................................................................................74
2.6.3 Challenging beam traversal task ............................................................................74
2.6.4 Puzzle box ..............................................................................................................76
2.6.5 Elevated plus maze .................................................................................................77
2.6.6 Abnormal Involuntary Movements Scale ..............................................................78
2.7 Drug treatment ...................................................................................................................79
2.7.1 MPTP .....................................................................................................................79
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2.7.2 Dopaminergic drugs ...............................................................................................79
2.8 Statistics .............................................................................................................................80
Chapter 3 Results ...........................................................................................................................81
Results .......................................................................................................................................81
3.1 Characterization of DAT over-expressing transgenic mice ...............................................81
3.1.1 Presynaptic dopamine homeostasis ........................................................................81
3.1.2 Markers of oxidative stress ....................................................................................84
3.1.3 Motor behavior .......................................................................................................90
3.1.4 Response to MPTP-induced dopaminergic damage ...............................................95
3.2 Characterization of mice that over-express DAT and under-express VMAT2 .................98
3.2.1 Confirmation of transporter levels .........................................................................98
3.2.2 Fitness ...................................................................................................................102
3.2.3 Presynaptic dopamine homeostasis ......................................................................106
3.2.4 Integrity of dopamine neurons .............................................................................116
3.2.5 Dopamine receptor levels .....................................................................................120
3.2.6 Baseline behavior .................................................................................................123
3.2.7 Response to dopaminergic drugs..........................................................................135
Chapter 4 Discussion ...................................................................................................................148
Discussion ...............................................................................................................................148
4.1 Project 1: Characterization of DAT-tg mice ....................................................................148
4.2 Project 2: Characterization of DAT-tg/VMAT2-kd mice ................................................153
4.2.1 Discrepancy between original DAT-tg mice and DAT-tg mice from the DAT-
tg/VMAT2-kd colony ...........................................................................................159
4.2.2 Hypothesis revisited .............................................................................................161
4.3 Conclusion .......................................................................................................................162
4.4 Technical Challenges .......................................................................................................164
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4.5 Future Directions .............................................................................................................165
References ....................................................................................................................................168
Appendix 1 ...................................................................................................................................195
Appendix 2 ...................................................................................................................................201
Copyright Acknowledgements.....................................................................................................208
x
List of Figures
CHAPTER 1
Figure 1-1. Dopaminergic pathways of the brain. .......................................................................... 3
Figure 1-2. Direct and indirect pathways of the basal ganglia. ...................................................... 5
Figure 1-3. Synthetic pathway of dopamine ................................................................................. 11
Figure 1-4. Degradation pathways for dopamine. ........................................................................ 13
Figure 1-5. Dopamine transport in the presynaptic neuron .......................................................... 14
Figure 1-6. Generation of reactive oxygen species in dopamine cells ......................................... 23
Figure 1-7. Substrates for DAT cause selective damage to dopamine neurons. ........................... 32
CHAPTER 2
Figure 2-1. Wire-hang test apparatus. ........................................................................................... 74
Figure 2-2. Challenging beam traversal task. ............................................................................... 75
Figure 2-3. Puzzle box apparatus. ................................................................................................. 76
Figure 2-4. Schematic image of elevated plus maze..................................................................... 78
CHAPTER 3: Project 1 - DAT-tg mice
Figure 3-1. DAT protein expression in the striatum of DAT-tg mice. ......................................... 82
Figure 3-2. Metabolite to dopamine ratios in the striatum of DAT-tg mice. ................................ 83
Figure 3-3. VMAT2 protein expression in the striatum of DAT-tg mice. .................................... 84
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Figure 3-4. Protein carbonylation in the striatum of DAT-tg mice. ............................................. 86
Figure 3-5. Protein nitrosylation and MnSOD levels in DAT-tg mice. ........................................ 87
Figure 3-6. Cysteinyl adducts of dopamine and its metabolites in DAT-tg mice......................... 89
Figure 3-7. Motor behavior of DAT-tg mice. ............................................................................... 91
Figure 3-8. Challenging beam traversal task in DAT-tg mice with L-DOPA treatment. ............. 93
Figure 3-9. Baseline behaviors of DAT-tg mice stratified by sex. ............................................... 94
Figure 3-10. Effect of MPTP treatment on TH protein levels in DAT-tg mice............................ 96
Figure 3-11. Effect of MPTP on striatal dopamine tissue content of DAT-tg mice. .................... 97
CHAPTER 3: Project 2 - DAT-tg/VMAT2-kd mice
Figure 3-12. DAT protein expression in the striatum. .................................................................. 99
Figure 3-13. VMAT2 protein levels in the striatum. .................................................................. 100
Figure 3-14. DAT and VMAT2 mRNA expression in the midbrain. ......................................... 101
Figure 3-15. Survival curve from birth to 12 weeks of age. ....................................................... 104
Figure 3-16. Body weight of adult mice. .................................................................................... 105
Figure 3-17. Striatal tissue content of dopamine and its metabolites. ........................................ 107
Figure 3-18. . Metabolite-to-dopamine ratios in the striatum. .................................................... 108
Figure 3-19. Electrically evoked dopamine release and uptake in the dorsal striatum. .............. 112
Figure 3-20. TH protein expression in the striatum. ................................................................... 113
Figure 3-21. MAO-B protein expression in the striatum. ........................................................... 114
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Figure 3-22. Stereological counts of TH+ cells in the SNc. ....................................................... 117
Figure 3-23. Stereological counts of TH+ and NeuN+ cells in SNpc. ....................................... 118
Figure 3-24. Stereological counts of TH+ and Nissl+ cells in SNpc. ......................................... 119
Figure 3-25. Dopamine receptor levels in the striatum............................................................... 122
Figure 3-26. Open field locomotion and stereotypy. .................................................................. 126
Figure 3-27. Locomotor activity of 12-month old mice. ............................................................ 127
Figure 3-28. Locomotor activity of DAT-tg/VMAT2-het mice. ................................................ 128
Figure 3-29. Fine motor skill evaluated using the challenging beam traversal task. .................. 129
Figure 3-30. Executive function evaluated using the puzzle box. .............................................. 131
Figure 3-31. Anxiety-like behavior assessed using elevated plus maze. .................................... 133
Figure 3-32. Amphetamine-induced locomotion. ....................................................................... 137
Figure 3-33. Amphetamine-induced stereotypy. ........................................................................ 138
Figure 3-34. Abnormal involuntary movements (AIM) induced by 2 mg/kg of amphetamine. 139
Figure 3-35. Locomotor effect of 5 mg/kg amphetamine on WT and DAT-tg mice. ................ 140
Figure 3-36. Locomotion induced by DAT inhibitors, cocaine and methylphenidate. .............. 142
Figure 3-37. Apomorphine-induced stereotypy. ......................................................................... 143
Figure 3-38. Effect of SKF 81297, L-DOPA and saline on locomotor activity of DAT VMAT2
mice. ............................................................................................................................................ 146
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List of Tables
Table 1-1. Summary of mouse models with genetically altered DAT or VMAT2 levels. ........... 53
Table 2-1. Description of tasks on the puzzle box test. ................................................................ 77
Table 2-2. List of dopaminergic drugs administered .................................................................... 80
Table 3-1. Summary of DAT and VMAT2 expression in DAT VMAT2 mice. ........................ 102
Table 3-2. Summary of overall fitness of DAT VMAT2 mice................................................... 106
Table 3-3. Summary of presynaptic dopamine homeostasis in DAT VMAT2 mice. ................. 115
Table 3-4. Summary of dopamine cell counts in SNpc of DAT VMAT2 mice. ........................ 119
Table 3-5. Summary of dopamine receptor levels in the striatum of DAT VMAT2 mice. ........ 122
Table 3-6. Summary of baseline motor and non-motor behaviors in DAT VMAT2 mice. ....... 134
xiv
List of Appendices
Appendix 1: Project 2 - Additional experiments……………………………………………195
Appendix 2: Low copy DAT-tg mice……………………………………………………… 201
xv
List of Publications
Masoud ST, Vecchio LM, Bergeron Y, Hossain MM, Nguyen LT, Bermejo MK, Kile B,
Sotnikova TD, Siesser WB, Gainetdinov RR, Wightman RM, Caron MG, Richardson JR, Miller
GW, Ramsey AJ, Cyr M, Salahpour A. Increased expression of the dopamine transporter leads to
loss of dopamine neurons, oxidative stress and l-DOPA reversible motor deficits. Neurobiol Dis.
2015; 74: 66-75.
Lohr KM*, Masoud ST*, Salahpour A, Miller GW. Membrane transporters as mediators of
synaptic dopamine dynamics: implications for disease. Eur J Neurosci. 2017; 45 (1): 20-33.
Trossbach SV*, Bader V*, Hecher L, Pum ME, Masoud ST, Prikulis I, Schäble S, de Souza
Silva MA, Su P, Boulat B, Chwiesko C, Poschmann G, Stühler K, Lohr KM, Stout KA, Oskamp
A, Godsave SF, Müller-Schiffmann A, Bilzer T, Steiner H, Peters PJ, Bauer A, Sauvage M,
Ramsey AJ, Miller GW, Liu F, Seeman P, Brandon NJ, Huston JP, Korth C. Misassembly of
full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and
behavioral deficits. Mol Psychiatry. 2016; 21 (11): 1561-1572.
Medvedev IO, Ramsey AJ, Masoud ST, Bermejo MK, Urs N, Sotnikova TD, Beaulieu JM,
Gainetdinov RR, Salahpour A. D1 dopamine receptor coupling to PLCβ regulates forward
locomotion in mice. J Neurosci. 2013; 33 (46): 18125-18133.
*co-first author
xvi
List of Abbreviations
AADC aromatic L-amino acid decarboxylase
ADHD attention deficit hyperactivity disorder
AMPT α-methyl-para-tyrosine
ATP adenosine triphosphate
BAC bacterial artificial chromosome
cAMP cyclic adenosine monophosphate
CNS central nervous system
COMT catechol-O-methyltransferase
CREB cAMP response element-binding protein
DAG diacylglycerol
DARPP-32 dopamine- and cAMP-regulated neuronal phosphoprotein
DAT dopamine transporter
DAT-KO dopamine transporter knock-out
DAT-tg dopamine transporter over-expressing transgenic
DAT-tg/VMAT2-kd dopamine transporter overexpressing and vesicular monoamine transporter
2 knockdown
DAT-tg/VMAT2-het dopamine transporter overexpressing and vesicular monoamine transporter
2 heterozygote
DOPAC 3,4-dihydroxyphenylacetic acid
xvii
DOPAL 3,4-dihydroxyphenylacetaldehyde
DOPET 3,4-dihydroxyphenylethanol
FSCV fast scan cyclic voltammetry
GPe globus pallidus external
GPi globus pallidus internal
GPCR G protein coupled receptor
HPLC-EC High performance liquid chromatography with electrochemical detection
5-HT Serotonin
5-HT 2A Serotonin 2A receptor
HVA homovanillic acid
IP3 inositol trisphosphate
LC locus coeruleus
L-DOPA L-3,4-dihydroxyphenylalanine
LRRK2 leucine-rich repeat kinase 2
MAO monoamine oxidase
MDMA 3,4- methylenedioxymethamphetamine
MnSOD Manganese superoxide dismutase
MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
3MT 3-methoxytyramine
NET norepinephrine transporter
xviii
PBS phosphate-buffered saline
PINK1 PTEN-induced kinase 1
PKC protein kinase C
PVDF polyvinylidene difluoride
RGS Regulators of G protein signaling
ROS reactive oxygen species
SN substantia nigra
SNpc substantia nigra pars compacta
SNpr substantia nigra pars reticulata
STN subthalamic nucleus
TH tyrosine hydroxylase
VMAT2 vesicular monoamine transporter 2
VMAT2-het vesicular monoamine transporter 2 knock-out heterozygote
VMAT2-kd vesicular monoamine transporter 2 knock-down
VMAT2-OE vesicular monoamine transporter 2 over-expressor
VTA ventral tegmental area
WT wild-type
1
Chapter 1
Introduction
Introduction
1.1 Statement of Research Problem
Dopamine neurotransmission is important for a variety of physiological functions including
motor coordination and reward-based learning. On the other hand, malfunction of the dopamine
system gives rise to disorders such as Parkinson’s disease. While the pathological loss of
nigrostriatal dopaminergic neurons in Parkinson’s disease is well established, the etiology of this
neurodegeneration typically remains unknown. Both genetic and environmental insults have
been implicated in causing selective damage to dopaminergic neurons even though in most
instances, their mechanism of toxicity could theoretically have more widespread effects. This
suggests that nigrostriatal dopamine neurons possess a unique phenotype with inherent
characteristics that render them susceptible to challenges.
In fact, the endogenous neurotransmitter dopamine itself can act as a cell-specific risk factor for
dopaminergic cells. When intracellular dopamine accumulates in the cytosolic space, it is highly
prone to reactions that give rise to oxidative stress. In particular, cytosolic dopamine has been
shown to produce reactive oxygen species and unstable quinones via metabolic, enzyme-
dependent and autoxidation reactions (Graham, 1978; Graham and Gutknecht, 1978; Stokes et
al., 1999; Ramkissoon and Wells, 2011). Using mostly in vitro and a few in vivo systems,
previous studies have documented the potentially toxic effects of cytosolic dopamine
accumulation (Filloux and Townsend, 1993; Hastings et al., 1996; Chen et al., 2008; Mosharov
et al., 2009). However, typically, these studies injected exogenous dopamine into brain regions
or engineered non-dopaminergic cells to take up the neurotransmitter. Thus, it is unclear whether
dopaminergic cells that routinely handle this neurotransmitter and are capable of degrading it,
may also succumb to cytosolic dopamine-induced toxicity.
Cytosolic dopamine levels are modulated by two key proteins: the dopamine transporter (DAT)
and the vesicular monoamine transporter 2 (VMAT2). DAT is located on the presynaptic
membrane of dopaminergic neurons and functions in the rapid uptake of dopamine from the
2
extracellular space into the nerve terminal. VMAT2 is located on the vesicular membrane of
monoaminergic cells and functions to sequester intracellular neurotransmitters into vesicles for
release. In a simplistic sense, DAT acts to increase cytosolic dopamine levels whereas VMAT2
acts to decrease it. In this work, we propose to use these two transporters as tools to manipulate
the cytosolic pool of dopamine in vivo. Previously in our laboratory, we generated transgenic
mice (DAT-tg) that over-express DAT specifically in dopaminergic neurons (Salahpour et al.,
2008). DAT over-expression led to greater dopamine uptake and loss of midbrain dopamine
neurons, presumably due to the detrimental effects of cytosolic dopamine accumulation (Masoud
et al., 2015). In this work, we investigated phenotypes of DAT-tg mice and assessed markers of
oxidative stress since cytosolic dopamine reactivity typically causes oxidative damage.
Moreover, in a second project, we generated animals with simultaneously increased DAT and
decreased VMAT2 levels to further enhance cytosolic dopamine accumulation in vivo. Instead of
applying external, non-physiological concentrations of dopamine like previous studies, we
modify endogenous dopamine compartmentalization and assess its impact on the function of the
dopamine system. Results from this work will shed light on the role of DAT, VMAT2 and
cytosolic dopamine in the inherent vulnerability of nigrostriatal dopamine neurons to insult.
1.2 Literature Review
1.2.1 Dopamine function in the brain
Dopamine was first discovered in the brain almost 60 years ago (Montagu, 1957; Carlsson et al.,
1958) and was thought to merely act as an intermediate in norepinephrine and epinephrine
synthesis. After years of research challenging this notion, the field finally began to recognize
dopamine itself as a key neurotransmitter (Carlsson et al., 1957). Currently, dopamine is one of
the most studied neurotransmitters due to its role in diverse physiological functions as well as its
contribution to disease states such as Parkinson’s disease, schizophrenia and attention deficit
hyperactivity disorder (ADHD). In the brain, dopamine is involved in a variety of functions
including locomotion, cognition, motivation, neuroendocrine regulation and response to reward.
Outside the central nervous system (CNS), dopamine plays important roles in regulating
vascular, adrenal, renal, cardiac and immune function (Missale et al., 1998; Chang et al., 2006;
Buttarelli et al., 2011).
3
Although dopamine performs several functions, dopamine neurons account for only a minute
percentage (less than 0.001%) of all neurons and are confined to a few discrete regions of the
brain (Surmeier et al., 2010). In the human brain, dopaminergic cell bodies have been detected in
the substantia nigra (SN), ventral tegmental area (VTA), hypothalamus (posterior, arcuate
nucleus, mammillothalamic tract), zona incerta, periventricular nucleus and olfactory bulb (Fuxe,
1965; Björklund and Dunnett, 2007). From these small regions, dopamine neurons project to
various structures in the brain to exert their effects. Classically, dopaminergic projections in the
brain are divided into four major pathways: 1) nigrostriatal, 2) mesolimbic, 3) mesocortical and
4) tuberoinfundibular. The majority of these pathways (nigrostriatal, mesolimbic and
mesocortical) originate in the midbrain which includes the SN and VTA. The midbrain contains
the majority (75%) of dopamine neurons which corresponds to 400,000 to 600,000 cells in adult
humans (German et al., 1983; Pakkenberg et al., 1991).
Figure 1-1. Dopaminergic pathways of the brain.
A schematic of the 4 major dopamine pathways in the brain showing where they originate and
the structures they project to. Image adapted from Genetic Science Learning Center, 2013.
1.2.1.1 Nigrostriatal pathway and movement
The SN is divided into two parts: pars compacta (pc) and pars reticulata (pr). The SNpc consists
of densely packed, neuromelanin-containing dopaminergic cell bodies that appear darker than
4
surrounding tissue, hence justifying the name substantia nigra, which is Latin for “black
substance”. On the other hand, the SNpr primarily contains diffuse GABAergic neurons. The
nigrostriatal pathway refers to dopamine cells that originate in the SNpc and project to the dorsal
striatum, alternatively known as the caudate nucleus and putamen. These dopamine cells are also
classified as A9 neurons according to the nomenclature proposed in 1964 that initially identified
discrete dopamine-containing cell groups in the brain using immunofluorescence (Dahlstroem
and Fuxe, 1964; Fuxe, 1965). Functionally, the nigrostriatal pathway is primarily responsible for
controlling voluntary movement. Indeed, degeneration of these neurons leads to the motor
symptoms that are characteristic of Parkinson’s disease, highlighting the essential role of the
nigrostriatal pathway in motor function.
In order to describe how the nigrostriatal pathway regulates motor activity, it is important to
understand the role of this pathway in the basal ganglia motor loop. The basal ganglia are a
collection of distinct yet interconnected nuclei within the brain that act together to perform
multiple functions, the most notable of which is movement control (Obeso et al., 1997). The
basal ganglia include subcortical structures such as the striatum (caudate/putamen), globus
pallidus internal and external (GPi, GPe), SN and subthalamic nucleus (STN). The basal ganglia
also have strong connections with the thalamus and cortex. There are two central basal ganglia
pathways that modulate movement: the direct and indirect pathways (Calabresi et al., 2014). In
general, the direct pathway facilitates motor activity by removing inhibition on the thalamus and
allowing it to excite the cortex and initiate movement. Conversely, the indirect pathway reduces
unwanted motor activity by enhancing inhibition of the thalamus which prevents subsequent
activation of motor cortices. The balance of these pathways allow for the selection of appropriate
voluntary movements. Nigrostriatal dopamine neurons can influence both the direct and indirect
pathways of movement. In particular, dopaminergic neurons from the SNpc synapse on to
medium spiny GABAergic neurons in the striatum. When dopamine is released, it can activate
D1 dopamine receptors on inhibitory GABAergic neurons. These neurons project to the GPi and
inhibit its activity. Normally, the GPi provides tonic inhibition of the thalamocortical circuit.
However, when dopamine activates the direct pathway, the GPi is strongly inhibited by the
striatum, leading to disinhibition of the thalamus, thus allowing it to excite the motor cortex and
initiate movement. Dopamine can also modulate the indirect pathway of movement by acting on
D2-expressing GABAergic neurons in the striatum. In the indirect pathway, striatal neurons
5
project to the GPe and inhibit its activity. Typically, the GPe is responsible for tonic inhibition of
the STN. However, when striatal neurons transiently inhibit the GPe, this releases the STN and
allows it to excite the GPi. Activating the GPi leads to greater inhibition of thalamocortical
circuits, preventing movement. When dopamine is released, it can inhibit D2-expressing striatal
neurons, weakening the downstream effects of the indirect pathway. Hence, nigrostriatal
dopamine neurons encourage the direct pathway via D1 receptors and suppress the indirect
pathway via D2 receptors. The net effect of these actions by dopamine is to facilitate movement.
In general, nigrostriatal dopamine acts as a crucial modulator of the basal ganglia motor loop.
Figure 1-2. Direct and indirect pathways of the basal ganglia.
Excitatory input is shown as (+) and inhibitory input is shown as (-). Adapted from
Neuroscience, 4th edition (Figure 18.8, Part 2). (Purves et al., 2008).
Besides involvement of the nigrostriatal dopaminergic pathway, other structures also influence
the overall motor loop. For instance, the SN receives input from, and also projects to, the STN
allowing for negative feedback mechanisms that can regulate the amount of dopamine released.
6
The SNpr also participates in basal ganglia connections as one of the major output structures of
the striatum. In addition to the globus pallidus, the striatum also sends GABAergic projections to
the SNpr, forming the striatonigral pathway. The SNpr also receives input from radiating
dendrites of the SNpc (dopaminergic) as well as the GPe (GABAergic) (Deniau et al., 2007;
Beaulieu and Gainetdinov, 2011). Hence, the SNpr is in a unique position to integrate various
basal ganglia signals and send efferent projections to the thalamus, brain stem and superior
colliculus via predominantly, GABAergic output neurons (Deniau et al., 2007). Finally, both the
direct and indirect pathways of movement are under cortical control since the striatum receives
input from the cortex.
Nigrostriatal dopamine neurons possess several unique characteristics that distinguish them from
other types of neurons. Structurally, these neurons are highly branched and support enormous
unmyelinated axonal fields (Matsuda et al., 2009). In humans, it has been estimated that each
SNpc dopamine cell gives rise to approximately 370,000 synapses in the striatum (Arbuthnott
and Wickens, 2007). In rats, each nigrostriatal axon forms 100,000 to 245,000 synapses, which is
orders of magnitude higher than other basal ganglia cells: medium spiny neurons produce 300-
500 synapses and striatal GABAergic interneurons form around 5,000 synapses (Bolam and
Pissadaki, 2012). In fact, even dopamine cells of the VTA produce far fewer synapses (12,000 to
30,000) than their nigral neighbors, highlighting the exceptional morphological phenotype of
SNpc dopamine cells (Moss and Bolam, 2009; Bolam and Pissadaki, 2012). As a result of this
extensive axonal arborization, relatively few nigral dopamine neurons can provide dense
innervation of a large target area, the striatum. In order to maintain this axonal complexity, the
energetic demands of nigrostriatal neurons are exceptionally high. Energy is required for
cytoskeleton maintenance, axonal transport, action potential propagation and synaptic
transmission. Indeed, in comparison to VTA dopamine cells, SNpc dopamine neurons have
higher density of axonal mitochondria, greater rate of mitochondrial oxidative phosphorylation
and elevated ATP production (Pacelli et al., 2015).
Aside from structural complexity, nigrostriatal dopamine neurons also display a distinctive
physiological phenotype. Unlike most neurons, these cells are spontaneously active. Even in the
absence of synaptic input, SNpc dopamine neurons generate regular action potentials at a slow
frequency of 2-4Hz (Guzman et al., 2009). This self-generated pacemaking activity is thought to
be responsible for maintaining baseline dopamine levels in the striatum. Most pacemakers,
7
including VTA dopamine neurons, rely on monovalent cations such as sodium for their
pacemaking activity (Khaliq and Bean, 2010). However, adult SNpc dopamine neurons also
engage voltage dependent L-type Ca channels containing the rare Cav1.3 subunit. This allows
the channel to open at relatively hyperpolarized membrane potentials. Hence, calcium enters
these cells at subthreshold membrane potentials, allowing for rhythmic oscillations to drive
pacemaking in between spikes. Typically, intracellular calcium concentration is under tight
homeostatic control due to its involvement in a variety of cellular processes. In most cells,
calcium levels are manageable because the ion enters the cell only during evoked action
potentials. However, nigrostriatal dopamine neurons experience a constant influx of calcium due
to autonomous pacemaking. Therefore, these cells have increased pressure to regulate calcium
levels and are more likely to accumulate intraneuronal calcium that can have detrimental effects
(Surmeier et al., 2010; Bolam and Pissadaki, 2012). Inside the cell, calcium is buffered by
membrane, mitochondrial and endoplasmic reticulum pumps that are metabolically expensive,
making nigrostriatal neurons particularly reliant on ATP generation. Thus, similar to the
extensive axonal arborization, this unusual calcium-dependent, tonic firing also imposes high
energetic demands on nigrostriatal neurons.
Lastly, the most obvious factor that differentiates dopamine neurons from other cells is the
neurotransmitter dopamine itself. While extracellular dopamine serves important functions in
signaling and neurotransmission, intracellular dopamine also has significant consequences. As a
highly reactive molecule, cytosolic dopamine can be auto-oxidized or undergo enzymatic
reactions to produce volatile intermediates such as dopamine-quinones and 3,4-
dihydroxyphenylacetaldehyde (DOPAL). These reactive derivatives of dopamine can modify
cellular proteins, lipids and nucleic acids producing oxidative stress and damage (Graham, 1978;
Burke et al., 2003). While all dopaminergic neurons contain dopamine, nigrostriatal neurons are
suggested to intrinsically handle higher amounts of the neurotransmitter. In fact, when treated
with the precursor of dopamine, L-3,4-dihydroxyphenylalanine (L-DOPA), studies reveal that
SN neurons display 2 to 3 times higher accumulation of cytosolic dopamine in comparison to
their counterparts in the VTA (Mosharov et al., 2009). Increased content of cytosolic dopamine
in nigrostriatal neurons can affect cellular health and lead to toxicity, as discussed in detail in
subsequent chapters.
8
In summary, nigrostriatal neurons play an essential role in voluntary movement by participating
in the basal ganglia motor loop. These cells also have a distinctive phenotype that sets them apart
from other cells as well as other dopaminergic pathways. While complex axonal branching,
calcium-dependent pacemaking and high cytosolic dopamine content are unique and necessary
features of nigrostriatal dopamine neurons, they can also act as risk factors for these cells
(Mosharov et al., 2009; Surmeier et al., 2010; Bolam and Pissadaki, 2012). In fact, healthy
humans demonstrate approximately a 40% loss of midbrain dopamine neurons between 40 and
60 years of age, suggesting that these cells are inherently vulnerable (Bogerts et al., 1983; Chinta
and Andersen, 2005). Nigrostriatal dopamine neurons are prone to oxidative stress due to the
handling of a reactive neurotransmitter and heavy dependence on mitochondrial oxidative
phosphorylation to meet their energetic demands. These cell-specific factors may contribute to
the vulnerability of nigrostriatal neurons not only in normal aging, but also in disorders such as
Parkinson’s disease.
1.2.1.2 Other dopaminergic pathways
In addition to the nigrostriatal pathway, the mesolimbic and mesocortical pathways also originate
in the midbrain (Björklund and Dunnett, 2007). However, instead of the SN, the cell bodies of
these dopaminergic projections are contained in the VTA. In particular, the mesolimbic tract
mainly sends projections to the nucleus accumbens, as well as the amygdala and hippocampus.
The nucleus accumbens is a major component of the ventral striatum and plays an important role
in reward and motivation. Thus, mesolimbic dopamine is involved in modulating response to
rewarding stimuli and is strongly implicated in the behavioral effects of reinforcing drugs. It has
been shown that psychostimulants such as cocaine and amphetamine stimulate release of
mesolimbic dopamine, whereas withdrawal of these drugs dampens dopamine transmission
(Adinoff, 2004; Sulzer, 2011). Mesocortical dopamine neurons primarily innervate the prefrontal
cortex in addition to the cingulate and perirhinal cortices. This pathway is involved in cognitive
processes such as attention, executive function, learning and memory. Since mesolimbic and
mesocortical pathways are closely related and can share overlapping functions, they are
collectively referred to as the mesocorticolimbic system. Finally, the fourth classical
dopaminergic pathway in the brain is the tuberoinfundibular pathway. These dopamine cells
arise from the arcuate and periventricular nuclei of the hypothalamus and send axons to the
infundibular region, also known as the median eminence of the hypothalamus. Dopamine is
9
released in the capillary circulation that connects the hypothalamus to the pituitary gland, where
it influences hormonal release. In particular, dopamine negatively regulates the release of
prolactin, a hormone involved in lactation and reproductive functions. In summary, dopamine is
a vital neurotransmitter that performs a variety of functions through different neuronal pathways.
1.2.2 Dopamine homeostasis
Dopamine belongs to a family of catecholamines which is part of a larger class of
neurotransmitters known as monoamines. Monoamines are synthesized from particular amino
acids and structurally contain an amino group that is connected to an aromatic ring through an
ethyl chain. Monoamines include histamine, serotonin, dopamine, epinephrine and
norepinephrine. The latter 3 compounds are further classified as catecholamines because they
possess a catechol group (which is a benzene ring with 2 hydroxyl groups), that is conjugated
with the side chain amine. Catecholamines are derived from the aromatic amino acid, l-tyrosine.
Tyrosine can directly be obtained from protein-rich dietary sources or synthesized from the
essential amino acid, phenylalanine. For dopaminergic cells, production of dopamine is the
ultimate objective, however for other catecholaminergic systems, it serves as an intermediate
step. Indeed, dopamine is a precursor in the sequential synthesis of norepinephrine and
epinephrine. Specifically, dopamine is converted to norepinephrine by dopamine β hydroxylase
while norepinephrine is transformed to epinephrine by the enzyme, phenylethanolamine N-
methyltransferase.
1.2.2.1 Synthesis
The life-cycle of dopamine spans multiple stages including synthesis, vesicular storage, release,
uptake and degradation. Synthesis of dopamine is a two-step process that occurs in the cytosol of
catecholaminergic cells. The first step involves addition of a hydroxyl group on the phenol ring
of the amino acid, L-tyrosine, to convert it to L-DOPA. This reaction is catalyzed by the rate-
limiting enzyme, tyrosine hydroxylase (TH) and uses molecular oxygen (O2), iron (Fe) and
tetrahydrobiopterin (BH4) as cofactors (Daubner et al., 2011). L-DOPA is then rapidly converted
to dopamine by DOPA decarboxylase, generally known as aromatic L-amino acid
decarboxylase. This reaction requires pyridoxal phosphate, the active form of vitamin B6, as a
cofactor and generates CO2 as a by-product of decarboxylation. Synthesis of dopamine is tightly
10
regulated because it is a major contributor to overall dopamine homeostasis within a cell. As the
rate-limiting enzyme in dopamine production, TH expression and activity are under complex
regulatory control. For instance, levels of fully synthesized neurotransmitter can influence TH
activity, allowing for feedback mechanisms to control intracellular dopamine accumulation
(Daubner et al., 2011). Specifically, dopamine competes with the TH cofactor BH4, to bind iron
at the catalytic site of TH. Thus, in the presence of dopamine, the essential cofactor BH4 cannot
associate with TH, leading to reversible inhibition of the synthetic enzyme. This provides
negative feedback and inhibits further production of dopamine. Activity of TH can also be
regulated by the protein’s state of phosphorylation. Phosphorylation of TH at particular sites
(Ser19, 31, 40) can enhance its activity and lead to greater production of dopamine, whereas
dephosphorylation of TH is correlated with reduced dopamine synthesis. Various signals can
influence the phosphorylation of TH. For example, increased extracellular dopamine levels
activate the D2 autoreceptor which inhibits phosphorylation of TH at Ser40 and thereby,
dampens dopamine production (Lindgren et al., 2001). Conversely, membrane depolarization
leads to influx of calcium that activates calcium-dependent kinases which phosphorylate TH and
increase dopamine synthesis (Salvatore et al., 2016). Hence, the process of dopamine production
is highly responsive to diverse stimuli. Once dopamine is synthesized within the cytosolic space,
it is readily sequestered into dense core vesicles by the vesicular monoamine transporter 2
(VMAT2). Vesicular storage of dopamine serves the dual function of protecting the
neurotransmitter from degradation and maintaining a high concentration of dopamine for
eventual release. The process of vesicular storage is discussed in detail in a subsequent section.
11
Figure 1-3. Synthetic pathway of dopamine
Adapted from Carlson, Physiology of Behavior 11th ed. (Carlson, 2012)
1.2.2.2 Release
Vesicular dopamine is released from the presynaptic neuron into the synaptic cleft through the
process of exocytosis. This release is triggered by the arrival of an action potential that stimulates
the dopaminergic nerve terminal. Depolarization causes voltage-gated calcium channels to open,
increasing the presynaptic calcium concentration. Influx of calcium produces a cascade of
intracellular events, including the mobilization of dopamine-containing vesicles. These vesicles
migrate towards the presynaptic membrane where they are docked and primed. Subsequently, the
vesicular membrane fuses with the plasma membrane, releasing dopamine contents into the
synaptic cleft. This exocytotic dopamine release is dependent on generation of action potentials
and calcium influx as demonstrated by in vitro experiments. In particular, studies show that
evoked dopamine release can be blocked by: 1) tetrodotoxin, an inhibitor of voltage dependent
sodium channels and 2) removal of extracellular calcium (Chen and Rice, 2001). Exocytosis is
the predominant mechanism of dopamine release and is common to other neurotransmitters as
12
well. While typical neurotransmitter release occurs at the axon terminal (i.e. striatum in
nigrostriatal pathway), dopamine can also be released from soma and dendrites in the SN and
VTA. Similar to striatal dopamine release, midbrain somatodendritic dopamine release is also
reported to regulate voluntary movement through basal ganglia circuits.
Presynaptic dopamine release plays an instrumental role in overall dopamine neurotransmission.
Intensity of the dopamine signal relies on multiple factors including the amount of dopamine
released, the time course of release events and the neuronal firing rate. Effects of extracellular
dopamine on pre- and post-synaptic dopamine receptors are discussed in a subsequent section.
1.2.2.3 Degradation
To terminate the actions of released extracellular dopamine, the neurotransmitter must be
removed from the synaptic cleft. This is achieved through 2 processes: 1) recycling dopamine
back into the presynaptic neuron through the dopamine transporter (DAT), after which it can be
re-packaged into vesicles or degraded and 2) metabolism of dopamine by glial cells. In either
case, degradation serves as the final step in the life-cycle of dopamine. Degradation not only
concludes the effects of dopamine but also limits buildup of the neurotransmitter to maintain
homeostasis.
Within the presynaptic neuron, if dopamine is not sequestered into vesicles, it is available for
degradation by metabolic enzymes in the cytosolic space (Eisenhofer et al., 2004a). Dopamine
accumulates in the cytosol during synthesis, following extracellular reuptake or as a result of
vesicular leakage. One of the key enzymes involved in monoamine catabolism is monoamine
oxidase (MAO). MAO exists in two forms: MAO-A and MAO-B. In humans, dopamine is
mostly metabolized by MAO-B, which is located on the outer mitochondrial membrane (Glover
et al., 1977). MAO-B catalyzes the oxidative deamination of dopamine to produce the aldehyde,
DOPAL as well as hydrogen peroxide. Both products are highly reactive and can contribute to
oxidative stress in the cell (Goldstein et al., 2013). DOPAL can be deactivated to its
corresponding alcohol, DOPET by aldehyde reductase. However, the more prevalent reaction is
the rapid oxidation of DOPAL to a carboxylic acid, DOPAC by aldehyde dehydrogenase.
DOPAC is one of the major intracellular metabolites of dopamine. Another important enzyme
involved in dopamine degradation is catechol-O-methyltransferase (COMT) which is mainly
expressed in glial cells. COMT transfers a methyl group donated from S-adenosylmethionine to a
13
hydroxyl group on DOPAC, generating another major metabolite, homovanillic acid (HVA). In
the striatum, metabolism of dopamine primarily begins in the presynaptic neuron. However, a
small proportion of circulating extracellular dopamine can also be taken up by glial cells. Since
glia express both MAO and COMT, dopamine can be sequentially degraded to DOPAC and
HVA as discussed. In an alternative, less significant metabolic pathway, COMT acts on
dopamine before MAO. In this case, dopamine is methylated to 3-methoxytyramine (3MT) and
then deaminated and oxidized to HVA. Additionally, some reports suggest that COMT is also
expressed on post-synaptic neurons where it could participate in metabolism of released
dopamine (Elsworth and Roth, 1997). Hence, prevalence of specific dopamine metabolites and
preference of particular catabolic pathways depend on the abundance, activity and localization of
key metabolic enzymes in different brain regions.
Figure 1-4. Degradation pathways for dopamine.
Image adapted from Pérez-Mañá et al., 2015. COMT, catechol-O-methyltransferase; MAO,
monoamine oxidase; ALDH, aldehyde dehydrogenase; ADH, alcohol dehydrogenase.
14
1.2.3 Dopamine transport
Transport of dopamine across different cellular compartments is an integral process that
contributes to dopamine homeostasis, compartmentalization and neurotransmission. Cellular
transport of dopamine occurs at 2 levels: 1) the plasma membrane and 2) the vesicular
membrane.
Figure 1-5. Dopamine transport in the presynaptic neuron
Dopamine levels are modulated by 2 transporters: the dopamine transporter (DAT, shown in
blue) on the plasma membrane and the vesicular monoamine transporter 2 (VMAT2, shown in
green) on the vesicular membrane. Adapted from Rilstone et al., 2013, NEJM (Rilstone et al.,
2013).
1.2.3.1 Plasma membrane transport
Transport of dopamine across the plasma membrane is mediated by the dopamine transporter
(DAT, SLC6A3), a membrane protein located on dopaminergic cells. Similar to other
monoamine transporters, DAT has 12 transmembrane domains with intracellular amino- and
carboxyl- termini and belongs to the SLC6A family of Na+/Cl--dependent symporters
(Gainetdinov and Caron, 2003). In particular, DAT couples the active transport of dopamine with
the movement of one Cl- and two Na+ ions along the concentration gradient. This concentration
gradient is created by the plasma membrane Na+/K+ ATPase and serves as the driving force for
15
DAT-mediated dopamine uptake (Kanner and Schuldiner, 1987; Gether et al., 2006). Dopamine
translocation across the plasma membrane occurs as a result of conformational changes in DAT.
The uptake cycle begins when DAT is open to the extracellular space in an outward facing state
(Reith et al., 2015). In this conformation, Na+ and Cl- ions bind to DAT and prepare the
transporter for dopamine binding. Upon binding of dopamine, the extracellular gate closes,
generating an occluded DAT state. Importantly, dopamine binding induces a conformational
change allowing the transporter to open on the cytosolic side. In this inward facing state,
dopamine and the ions dissociate from DAT. Finally, the cycle is reset once DAT returns to the
outward facing conformation (Reith et al., 2015).
The function of DAT is to rapidly transport dopamine from the extracellular space into the
cytosol of the presynaptic neuron. At the plasma membrane, DAT is located peri-synaptically,
where it removes extracellular dopamine and provides spatial and temporal control of the
dopamine signal (Hersch et al., 1997; Jones et al., 1998a; Cragg and Rice, 2004). In
dopaminergic brain regions such as the striatum, DAT provides the principal mechanism of
clearing extracellular dopamine and terminating neurotransmission (Giros et al., 1996). Aside
from modulating the dynamics of released dopamine, DAT is also responsible for recycling the
neurotransmitter back into the dopaminergic cell, allowing it to be reused (Sotnikova et al.,
2006). By loading the presynaptic neuron with dopamine, DAT directly contributes to the
buildup of cytosolic dopamine and indirectly influences vesicular dopamine as well.
Accumulation of cytosolic dopamine can produce neurotoxicity as discussed in the next section.
Hence, DAT is a key player in dopamine compartmentalization that can have significant
consequences for the presynaptic neuron. Collectively, DAT regulates the concentrations of both
1) extracellular dopamine at the synapse and 2) intracellular dopamine within the presynaptic
neuron.
1.2.3.2 Vesicular membrane transport
Another transporter that plays an essential role in maintaining dopamine homeostasis and
neurotransmission is the vesicular monoamine transporter 2 (VMAT2, SLC18A2) (Erickson and
Eiden, 1993; Wimalasena, 2011). VMAT2 is a membrane protein that is expressed on synaptic
vesicles of monoaminergic neurons. Structurally, it contains 12 transmembrane helices with
cytosolic amino- and carboxyl- termini. VMAT2 is responsible for transporting intracellular
16
monoamines such as dopamine, norepinephrine, epinephrine, serotonin and histamine from the
cytosolic space into synaptic vesicles. Synaptic vesicles are small spherical lipid bilayers that are
approximately 40nm in diameter. These vesicles are filled with neurotransmitters at the nerve
terminal where they are released through exocytosis upon stimulation of the cell. VMAT2
belongs to the SLC18 family of transporter proteins that also include VMAT1 and the vesicular
acetylcholine transporter. VMAT1 (SLC18A1) is predominantly located in neuroendocrine cells
of the peripheral nervous system including chromaffin cells of the adrenal gland and melatonin-
synthesizing cells of the pineal gland (Lawal and Krantz, 2013). Conversely, VMAT2 is
primarily expressed in monoaminergic neurons of the CNS as well as platelets, β pancreatic cells
and histaminergic cells of the gastric mucosa (Peter et al., 1995).
The process of vesicular filling serves dual functions as it accumulates dopamine for eventual
release and also controls buildup of cytosolic levels. It is estimated that vesicular concentrations
of monoamines are 10,000 fold higher than cytoplasmic levels due to VMAT2 loading (Parsons,
2000). VMAT2 packages high concentrations of dopamine within small vesicles through active
transport which relies heavily on the electrochemical gradient generated by the vesicular H+-
ATPase. Using the energy from ATP hydrolysis, the vesicular H+-ATPase preferentially moves
H+ ions into vesicles. This establishes an acidic environment (pH 5.5) within the vesicular lumen
and creates proton and electrochemical gradients across the vesicular membrane that serve as an
energy source for VMAT2 activity. Specifically, as a H+-antiporter, VMAT2 couples the uptake
of each dopamine molecule with the expulsion of 2 protons from the vesicular lumen. Transport
is initiated by the efflux of the first H+ ion from the vesicular lumen which alters the
conformation of the transporter and enables binding of dopamine on the cytosolic side
(Wimalasena, 2011). Following translocation of the second proton, the transporter undergoes a
conformational switch to move dopamine from the cytosolic side to the luminal side
(Wimalasena, 2011). This type of VMAT2 uptake cycle applies to other monoamines as well.
Several factors can influence vesicular uptake including: 1) magnitude of the transmembrane
proton and electrochemical gradients, 2) cytoplasmic concentrations of neurotransmitter and 3)
VMAT2 expression and activity (Wimalasena, 2011). Vesicular storage is a dynamic process
because although VMAT2 actively loads dopamine into vesicles, the neurotransmitter also
passively leaks through vesicular membrane back into the cytoplasm at a constant rate. It is
estimated that 90% of leaked molecules are re-captured into vesicles by VMAT2 and the
17
remaining 10% persist in the cytosol where they can be degraded (Eisenhofer et al., 2004b).
Hence, overall vesicular content of dopamine is determined by a balance of VMAT2-uptake and
passive leakage. Appropriate vesicular storage is fundamental to extracellular as well as
cytosolic dopamine dynamics. Quantal release of dopamine has been shown to be closely tied to
the expression of VMAT2, where increased VMAT2 levels lead to larger vesicular stores and
greater dopamine release and knock-down of VMAT2 translates to lower dopamine release
(Caudle et al., 2007; Lohr et al., 2014). Aside from influencing extracellular dopamine levels,
vesicular storage is also a crucial mechanism of maintaining low levels of cytosolic dopamine
and protecting cells from dopamine-induced toxicity as discussed in subsequent sections.
1.2.4 Dopamine compartmentalization and its effects
Appropriate compartmentalization of dopamine is essential to neuronal homeostasis. At a
cellular level, dopamine can exist in distinct compartments: 1) extracellular and 2) intracellular
that is further divided into vesicular and cytosolic fractions. Movement of dopamine between
these compartments is a dynamic process that is mediated by DAT and VMAT2. Notably, based
on the compartment, dopamine produces different effects that have important consequences for
the cell. Generally, extracellular dopamine is given most importance as it plays a pivotal role in
dopamine signaling. However, intracellular dopamine, specifically the cytosolic portion, has
been shown to influence neuronal health and potentially contribute to the vulnerability of
dopaminergic cells.
1.2.4.1 Extracellular dopamine
Once dopamine is released into the extracellular space, it participates in neurotransmission by
acting on specific receptors.
1.2.4.1.1 Dopamine Receptors
Dopamine receptors belong to the superfamily of G protein coupled receptors (GPCRs). GPCRs
are membrane proteins containing 7 transmembrane domains, an extracellular amino terminal
and intracellular carboxyl tail (Kobilka, 2007). GPCRs can exist and function as monomers or
oligomeric complexes (Angers et al., 2002). These metabotropic receptors receive signals from
18
the extracellular environment and respond by activating intracellular signal transduction
pathways. Notably, GPCRs are associated with a heterotrimeric G protein complex consisting of
α, β and γ subunits. Whether this coupling occurs before or after ligand binding to the GPCR is a
matter of controversy (Kobilka, 2007; Qin et al., 2011). Nonetheless, once the GPCR is activated
by an agonist, the receptor undergoes a conformational change that catalyzes the exchange of
GDP for GTP on the Gα subunit. This activates the G protein, and according to the classical
theory, causes Gα to dissociate from the receptor and the Gβγ dimer (Gilman, 1987; Digby et al.,
2006). However, some studies also indicate that physical dissociation of subunits may not be
necessary for signaling (Levitzki and Klein, 2002; Bunemann et al., 2003). Importantly,
activated Gα and Gβγ subunits then bind different intracellular proteins and propagate the signal
via second messengers. Specific signaling pathways are dependent on the type of G protein that
the receptor is coupled to. Signal transmission can be terminated by the GTPase activity of Gα
that hydrolyzes GTP to GDP and converts the receptor to an inactive conformation. In this state,
G protein subunits may re-associate and bind to the GPCR once again. Regulators of G protein
signaling (RGS) are proteins that can accelerate the GTPase activity of Gα, thus, encouraging G-
protein inactivation and termination of downstream signaling pathways (Beaulieu and
Gainetdinov, 2011).
There are at least 5 different types of dopamine receptors including D1, D2 (which exists in 2
isoforms; long and short), D3, D4 and D5. Classically, dopamine receptors are divided into two
families, D1 and D2, based on their structure, sequence homology, pharmacology and most
importantly, signaling properties (Kebabian and Calne, 1979). Typically, dopamine receptors
signal through G-proteins that are associated with adenylyl cyclase, an enzyme that converts
ATP to cyclic adenosine monophosphate (cAMP). cAMP is a second messenger that regulates
proteins such as protein kinase A (PKA). When activated, PKA phosphorylates downstream
targets including ion channels, CREB and DARPP-32 that can amplify the signal. The D1-like
family, consisting of D1 and D5, signal through Gαs/olf to stimulate adenylyl cyclase and PKA
activity. Conversely, the D2-like family, including D2 long, D2 short, D3 and D4, are coupled to
Gαi/o which inhibits adenylyl cyclase and reduces PKA activity. Generally, dopamine activation
of D1 receptors produces a stimulatory effect whereas D2 receptors produce an inhibitory effect.
Aside from cAMP-mediated signaling, D1-like receptors can also engage Gαq which regulates
phospholipase C (PLC) (Sahu et al., 2009; Medvedev et al., 2013). Upon activation, PLC leads
19
to synthesis of inositol trisphosphate (IP3) and diacylglycerol (DAG). These second messengers
activate protein kinase C (PKC) and mobilize intracellular calcium stores, triggering a cascade of
downstream effects. While traditionally, dopamine receptors function as GPCRs, accumulating
evidence suggests that they also engage G protein-independent pathways, such as β-arrestin
signaling. Studies demonstrate that β-arrestin 2 contributes to D2 receptor signaling by
regulating the Akt/glycogen synthase kinase 3 (GSK-3) pathway (Beaulieu et al., 2005).
Dopamine receptors are expressed on both pre- and post-synaptic neurons. Pre-synaptic receptors
on dopaminergic neurons allow these cells to regulate their own function through negative
feedback mechanisms. In response to changes in extracellular dopamine, autoreceptors can
adjust neuronal firing rate, dopamine synthesis and release accordingly (Missale et al., 1998).
Autoreceptors are present along the dopaminergic neuron and therefore can respond to both
terminal and somatodendritic dopamine release. Presynaptic dopamine receptors belong to the
class of D2 receptors while D1 receptors are exclusively post-synaptic. Generally, D2
autoreceptors are activated by a lower concentration of dopamine than post-synaptic receptors,
allowing for high sensitivity to extracellular dopamine levels (Elsworth and Roth, 1997).
Stimulation of autoreceptors leads to reduction of neuronal firing, inhibition of dopamine
synthesis and diminished release of dopamine. Taken together, these actions dampen
extracellular dopamine signaling. With regards to the nigrostriatal pathway, D1 receptors are
expressed on medium spiny neurons that project to the GPi and constitute the direct pathway of
movement, while D2 receptors are expressed on striatal projections to the GPe which is the
indirect pathway. Hence, extracellular dopamine promotes movement by stimulating the direct
pathway via D1 and suppressing the indirect pathway via D2 receptors. Generally, activation of
post-synaptic D1 receptors has a stimulatory effect on locomotion. However, effects of D2
receptors are more complex since they are expressed both pre-and post-synaptically. While
activation of post-synaptic D2 receptors promotes locomotor activity, stimulation of D2
autoreceptors produces the opposite effect.
Although D1 and D2 receptors are typically divided into two distinct families, recent evidence
suggests that their actions may be interconnected. When D1 and D2 receptors were co-expressed
in the same cell, dual stimulation elevated intracellular calcium via a pathway that could not be
activated by either receptor individually (Lee et al., 2004). These findings led to the discovery of
D1-D2 heteromeric receptor complexes in the brain, specifically the striatum, that were found to
20
be coupled to Gαq/11 (So et al., 2005; Rashid et al., 2007). Through this signaling pathway,
concurrent agonist binding to both receptors activates PLC and causes release of intracellular
calcium, which then stimulates Ca2+/calmodulin-dependent protein kinase II, an important
mediator of synaptic plasticity and learning. Blockade of D1 or D2 receptors with antagonists
prevented this cascade, illustrating the necessity of both receptor types for rapid activation of the
Gαq/11 pathway (Rashid et al., 2007). Despite ongoing controversy regarding dopamine receptor
heterodimerization, some studies suggests that these heteromers may play important roles in
pathological conditions such as schizophrenia, depression and drug addiction (Grymek et al.,
2009; Pei et al., 2010; Perreault et al., 2010; Hasbi et al., 2011).
1.2.4.2 Intracellular dopamine
Although extracellular dopamine serves important functions in dopamine signaling, the majority
of synaptic dopamine is stored intracellularly within dopamine neurons. Intracellular dopamine is
divided into two compartments: vesicular and cytosolic. Vesicular dopamine is a reflection of
overall dopamine tissue content because at any given moment, most neurotransmitters are stored
within vesicles. Dopamine is accumulated in vesicles for eventual release. In fact, vesicular
dopamine has been shown to directly determine the amount of neurotransmitter released from a
cell (Caudle et al., 2007; Lohr et al., 2014). Hence, vesicular dopamine not only represents the
largest cellular repository of dopamine, it also impacts neurotransmitter signaling. The process of
vesicular storage is dynamic and involves active uptake as well as passive leakage as discussed
in the next section. Importantly, when dopamine is sequestered into vesicles, it is protected from
metabolic reactions that can occur in the cytosol.
1.2.4.2.1 Cytosolic dopamine
Cytosolic dopamine represents a small fraction of presynaptic dopamine since the
neurotransmitter is usually readily packaged into vesicles. However, there are multiple
circumstances when dopamine can accumulate in the cytosolic space: 1) during synthesis, 2)
following reuptake from the extracellular space, and 3) after vesicular leakage. In the cytoplasm,
dopamine is exposed to various reactions that can propagate oxidative stress and potentially have
damaging consequences for the dopaminergic cell.
21
Reactivity
Dopamine is a highly reactive molecule that can undergo enzymatic reactions or direct auto-
oxidation. The predominant metabolic pathway of cytosolic dopamine involves deamination by
the enzyme, MAO. This reaction gives rise to 2 products: 1) DOPAL, a volatile aldehyde and 2)
hydrogen peroxide, a reactive oxygen species (ROS) (Stokes et al., 1999). If hydrogen peroxide
is not rapidly eliminated by anti-oxidant pathways such as glutathione peroxidase, it can react
with transition metals, such as iron to generate more reactive oxidants (Halliwell, 1992). In
addition, the other product of dopamine metabolism, DOPAL has been shown to produce
quinones and radical species, cause protein cross-linking and damage mitochondria (Kristal et
al., 2001; Rees et al., 2009; Anderson et al., 2011). Moreover, in the presence of hydrogen
peroxide, DOPAL generates highly reactive hydroxyl radicals that can cause further
macromolecular damage (Burke et al., 2004). This demonstrates the synergistic and potentially
harmful effects of these dopamine metabolites. In addition, emerging in vitro and in vivo
evidence suggest that DOPAL is toxic to cells (Dauer et al., 2002). Injection of low doses of
DOPAL in rat SN resulted in loss of TH staining, which is indicative of dopaminergic toxicity
(Burke et al., 2003). One hypothesis also postulates that DOPAL contributes to the loss of
dopamine-containing terminals in Parkinson’s disease (Goldstein et al., 2011, 2013). Indeed,
post-mortem analysis of patients reveal increased DOPAL:DOPAC ratios, suggesting impaired
detoxification of this reactive metabolite (Goldstein et al., 2011). Taken together, the normal
degradation of cytosolic dopamine directly produces chemicals that can propagate oxidative
stress in dopaminergic cells.
Aside from metabolic deamination, cytosolic dopamine is also prone to oxidation reactions.
These reactions yield ROS (such as superoxide, hydroxyl radicals and hydrogen peroxide) and
quinone products (Tse et al., 1976; Barzilai et al., 2001). In particular, the catechol ring of
dopamine can undergo oxidation to produce electron deficient dopamine-quinones that are
highly unstable. These chemical species readily react with nucleophilic sulfhydryl groups on free
cysteine, glutathione or cysteinyl residues of proteins (Graham, 1978; Hastings and Zigmond,
1994; Stokes et al., 1999). Addition reactions between dopamine quinones and cysteine
predominantly occur at the number 5 position of the catechol ring, leading to the formation of 5-
S-cysteinyl-dopamine, an indicator of dopaminergic oxidative stress (Fornstedt et al., 1986).
Conjugation between dopamine-derived quinones and cysteine residues on glutathione can
22
reduce the levels and effectiveness of this important antioxidant. Furthermore, given that
cysteinyl residues often reside at the active site of proteins, covalent modification of these
residues by dopamine-quinones can alter protein structure and inhibit normal function. Aside
from dopamine, its catechol-containing precursor, L-DOPA, and metabolite, DOPAC, are also
capable of forming quinones that bind cysteinyl residues (Fornstedt et al., 1986).
Oxidation of cytosolic dopamine can occur spontaneously or via enzymatic activation. Enzymes
that catalyze the conversion of dopamine to dopamine-quinones include: 1) tyrosinase, which is
involved in melanin formation, 2) prostaglandin H synthase, also known as cyclooxygenase,
which possesses peroxidase activity and catalyzes prostaglandin production, 3) xanthine oxidase,
which is involved in purine catabolism and generates ROS and 4) lipoxygenase, which mediates
metabolism of eicosanoids (Korytowski et al., 1987; Rosei et al., 1994; Hastings, 1995; Foppoli
et al., 1997; Gonçalves et al., 2009; Ramkissoon and Wells, 2011). Thus, diverse enzymatic
pathways are capable of dopamine oxidation, highlighting the reactivity of this neurotransmitter.
In fact, even in the absence of catalysts, dopamine can be directly autoxidized to produce
superoxide anions and reactive quinones (Graham and Gutknecht, 1978; Barzilai et al., 2001).
Superoxide can be converted to H2O2 by superoxide dismutase or it can react with nitric oxide, to
generate peroxynitrite, a highly reactive nitrogen species. In comparison to other catecholamines,
the dopamine molecule displays the highest rate of autoxidation, suggesting that it is most likely
to spontaneously form reactive quinones (Graham and Gutknecht, 1978). Conversely, dopamine
exhibits the slowest rate of internal cyclization, a process by which the quinone reacts with its
own side chain amine group producing leukochromes or aminochromes. This slow detoxification
pathway prolongs the longevity of the dopamine quinone, allowing it access to react with
sulfhydryl groups on cellular macromolecules and generate cysteinyl adducts (Graham, 1978).
Indeed, rates of addition reactions between dopamine-quinones and external sulfhydryl groups
on glutathione or cysteine residues is at least 3 fold higher than internal cyclization reactions
(Tse et al., 1976). Moreover, cellular conditions such as the presence of transition metals (e.g.
iron, copper or manganese) and a pro-oxidant background (e.g. hydroxyl radical) can accelerate
dopamine autoxidation. In particular, dopaminergic neurons routinely produce hydrogen
peroxide as a result of MAO-mediated metabolism. Through the Fenton reaction, H2O2 reacts
with transition metals such as iron to produce reactive hydroxyl radicals that can greatly enhance
dopamine oxidation rates (Nappi et al., 1995). Given that dopaminergic neurons are also an
23
abundant source of iron, the contribution of this transition metal in promoting dopamine
oxidation can be significant (Halliwell and Gutteridget, 1984; Velez-Pardo et al., 1997).
Furthermore, increased iron content was found in the substantia nigra of Parkinson’s disease
patients versus control subjects, suggesting that iron-mediated production of reactive dopamine
intermediates may impact disease pathogenesis (Sofic et al., 1988; Dexter et al., 1989b).
Figure 1-6. Generation of reactive oxygen species in dopamine cells
Dopamine neurons are inherently prone to oxidative stress due to a variety of reactions that
convert dopamine to ROS. (Top) Enzyme dependent and independent reactions of cytosolic
dopamine (DA) lead to the production of cytotoxic molecules including superoxide anions (O2•),
dopamine–quinone species (SQ•), and hydroxyl radicals (OH•). Hydrogen peroxide (H2O2), a
by-product of dopamine metabolism by monoamine oxidase (MAO), can lead to formation of
hydroxyl radicals via the Fenton reaction. Antioxidant systems include glutathione (GSH)
peroxidase, glutathione reductase, superoxide dismutase and catalase. (Bottom) An imbalance
between the production and elimination of ROS may propagate oxidative stress and render
dopamine cells vulnerable to cell death. These pathways may play a role in the pathogenesis of
neurodegenerative disorders such as Parkinson’s disease. Image adapted from Lotharius and
Brundin, 2002.
24
In summary, when dopamine accumulates in the cytosolic space, it is available for a variety of
reactions including metabolic deamination, enzymatic oxidation and autoxidation. Due to an
unstable catechol ring, the structure of dopamine easily lends itself to the formation of radical
species, quinones and reactive intermediates. These products can propagate oxidative stress and
compromise normal cell function by binding and inactivating important macromolecules. As
such, cytosolic dopamine inherently possesses the potential to cause toxicity in cells. In fact, one
of the most toxic compounds known to damage dopaminergic neurons is 6-hydroxydopamine, a
structural analog of dopamine that differs from the neurotransmitter only by a single hydroxyl
group.
Toxicity
The theory that accumulation of cytosolic dopamine can be deleterious to neuronal function and
survival is one that has been discussed for some time. In 1978, a study by Graham et al showed
that dopamine was much more cytotoxic to neuroblastoma cells than the other catecholamines,
epinephrine and norepinephrine (Graham and Gutknecht, 1978). This toxicity correlates with
dopamine’s higher rate of autoxidation and lower rate of internal cyclization, strongly
implicating oxidative damage and quinone formation as a mechanism for dopamine-induced cell
death (Graham, 1978). Indeed, in a catecholaminergic cell line, application of dopamine
produced signs of oxidative stress such as lipid peroxidation, DNA base damage and increased
intracellular peroxides (Masserano et al., 1996, 2000). These changes were accompanied by
dopamine-induced apoptosis that could be inhibited by: 1) catalase, an anti-oxidant enzyme that
catalyzes the decomposition of H2O2, and 2) N-acetylcysteine, a precursor in the formation of the
anti-oxidant, glutathione. Furthermore, a study by Lai et al confirmed that dopamine-induced
cytotoxicity in catecholaminergic neuroblastoma cells could be reversed by application of
various anti-oxidants such as glutathione, L-ascorbic acid and N-acetylcysteine or anti-oxidant
enzymes such as catalase and superoxide dismutase (Lai and Yu, 1997). The connection between
dopamine-induced toxicity and oxidative stress is further supported by a study showing that
pretreatment with a glutathione-depleting compound, L-buthionine sulfoximine, exacerbates the
detrimental effects of dopamine in a neuroblastoma cell line (Stokes et al., 2000). This indicates
a circular relationship where dopamine reactivity can lead to oxidative stress, but a pro-oxidant
background can also further promote dopaminergic toxicity.
25
A number of other studies lend additional support to the toxic potential of dopamine in vitro. Ziv
et al proposed that physiologically-relevant concentrations of dopamine were capable of causing
apoptosis-like cell death in cultured embryonic sympathetic neurons as shown by axonal
degeneration, nuclear fragmentation and severe shrinkage of cell bodies (Ziv et al., 1994;
Barzilai et al., 2001). In this system, dopamine was the most toxic in comparison to
norepinephrine, epinephrine and serotonin, highlighting the inherent ability of this
neurotransmitter to cause cellular damage (Zilkha-Falb et al., 1997). Similar results of
dopamine-induced cell death were also observed in mesencephalic, dorsal root ganglion, cortical
and striatal primary neuronal cultures (Michel and Hefti, 1990; Tanaka et al., 1991; Alagarsamy
et al., 1997; McLaughlin et al., 2002). In a study on human neuroblastoma cells that selectively
take up dopamine, treatment with the neurotransmitter produced changes in cell morphology,
shrinkage, atrophy, accumulation of apoptotic particles and cell death (Simantov et al., 1996).
However, when dopamine uptake was inhibited by the application of antisense DAT-specific
oligonucleotides, it dose-dependently decreased the toxic effects of dopamine. This clearly
suggests that dopamine uptake into the cytosol is necessary for dopaminergic toxicity (Simantov
et al., 1996; Porat et al., 2001).
The most convincing evidence linking cellular damage and cytosolic dopamine was provided by
Mosharov and colleagues in 2009. This research had some unique advantages: 1) it is the only
study that directly measured cytoplasmic dopamine using a novel technique, intracellular patch
electrochemistry and 2) it used cells that naturally handle dopamine: cultured cells from the
ventral midbrain which mostly contain dopaminergic neurons (Mosharov et al., 2009). Initially,
they treated these cells with increasing concentrations of L-DOPA, the precursor of dopamine,
and reported corresponding increases in cytosolic dopamine levels, as expected. Importantly,
they also showed that L-DOPA-induced surges in cytosolic dopamine were correlated with
progressive loss of TH-positive dopaminergic neurons. Interestingly, dopaminergic neurons from
the SN were more susceptible to toxicity than VTA neurons, as observed in Parkinson’s disease.
This vulnerability was related to greater accumulation of cytosolic dopamine in SN versus VTA
cells when treated with L-DOPA. Pharmacologic and genetic manipulations revealed a close
correlation between cytoplasmic dopamine content and cell death. For instance, treatment with
pargyline, an inhibitor of MAO, effectively terminated the metabolism of dopamine to DOPAC,
thus increasing cytosolic dopamine levels and enhancing neuronal loss when treated with L-
26
DOPA. Conversely, blockade of dopamine synthesis using NSD-1015 or benserazide, inhibitors
of DOPA decarboxylase, reduced cytosolic dopamine levels and prevented L-DOPA induced cell
death. Genetic over-expression of VMAT2, enhanced vesicular uptake of dopamine and
significantly decreased cytosolic dopamine levels. Consequently, this protected cells from L-
DOPA mediated neurotoxicity. Taken together, these results convincingly demonstrate that
cytosolic levels of dopamine can directly determine toxic outcomes in dopaminergic cells.
Despite possessing the unique technical advantage of being able to measure cytosolic dopamine,
it should be noted that in this study, cells needed to be preloaded with L-DOPA for cytosolic
dopamine levels to reach the threshold for detection. This highlights the difficulty of measuring
such a small and transient pool of dopamine even in neuronal cultures.
Expanding on the findings of in vitro studies, in vivo research has also validated the ability of
dopamine to cause detrimental effects in intact biological systems. The simplest of these studies
involve direct application of dopamine to the brain. In an extreme example,
intracerebroventricular injection of dopamine in rats pretreated with pargyline, an MAO
inhibitor, caused dose-dependent mortality of animals (Ben-Shachar et al., 1995). Conversely, in
the absence of pargyline, dopamine-induced mortality was reduced, suggesting that blocking
dopamine degradation allows the neurotransmitter to accumulate and propagate toxicity. In these
rats, dopamine also inhibited activity of NADH dehydrogenase, a crucial enzyme involved in
mitochondrial respiration. This reveals the ability of dopamine to modulate the function of the
electron transport chain, which may play a role in its toxicity. Several studies have assessed the
effects of dopamine injections in the striatum as it is rich in dopaminergic nerve terminals. In
rats, intrastriatal application of dopamine produces apoptotic cell death and DNA damage while
activating transcription factors that are responsive to oxidative stress (Hattori et al., 1998; Luo et
al., 1999). These changes are proportional to the concentration of dopamine applied and the
length of exposure, indicating dose-dependent effects of dopamine in triggering cellular toxicity
(Hattori et al., 1998). In other studies, following intrastriatal injection of dopamine in rats, both
free and protein-bound cysteinyl-dopamine and cysteinyl-DOPAC were markedly increased (22
to 37-fold), suggesting enhanced oxidation of the neurotransmitter and subsequent conjugation
with cellular macromolecules (Hastings et al., 1996; Rabinovic et al., 2000). This was correlated
with dose-dependent loss of TH-immunoreactivity and gliosis surrounding the injection site
(Filloux and Townsend, 1993; Hastings et al., 1996). Furthermore, these authors also
27
demonstrated selective degeneration of dopaminergic terminals as evidenced by 1) reduction in
the dopamine-specific marker, DAT which correlates with loss of TH and 2) amino-cupric silver
staining showing neuronal degeneration specifically in the area of reduced TH signal (Rabinovic
et al., 2000). This illustrates that even in dopaminergic neurons that are equipped with protective
mechanisms to handle dopamine such as vesicular storage and degradation, an overload of the
molecule can instigate cellular damage. Interestingly, extracellular dopamine reached peak levels
within 30 minutes post-injection after which it rapidly declined, pointing to DAT-mediated
uptake of dopamine as a mechanism of clearing extracellular levels and accumulating the
neurotransmitter exclusively in dopaminergic cells (Rabinovic et al., 2000). Also, dopamine-
induced oxidative modifications seemed to precede the earliest signs of degeneration suggesting
that oxidative stress plays a causal role in dopaminergic toxicity (Rabinovic et al., 2000). This
idea is further cemented by findings showing that the extent of dopamine-induced protein
modification and neurodegeneration can be rescued by co-injection of the antioxidants
glutathione and ascorbate, strongly implicating oxidative stress as the mechanism of cell death
(Hastings et al., 1996).
Direct measurement of cytosolic dopamine levels has not yet been possible in vivo due to
technical constraints. Despite this limitation, other indicators can be used to indirectly gauge
cytosolic dopamine levels in vivo, such a metabolite to dopamine ratios, presence of cysteinyl-
dopamine adducts and activity of dopamine transporters. To study the effects of cytosolic
dopamine in vivo, Chen et al generated transgenic mice with ectopic and inducible DAT
expression in the forebrain (Chen et al., 2008). In these mice, GABAergic striatal neurons were
engineered to take up extracellular dopamine released from nigrostriatal dopaminergic terminals.
Since striatal neurons lack the regulatory mechanisms to effectively sequester and metabolize
dopamine, once taken up, the neurotransmitter accumulates in the cytoplasm. These mice
showed significant signs of oxidative stress in the striatum as demonstrated by dramatic
increases in cysteinyl dopamine and cysteinyl DOPAC and reduction of glutathione content.
Oxidative modifications were accompanied by progressive neurodegeneration in the striatum and
impaired locomotor activity. In order to assess whether the amount of cytosolic dopamine could
impact cellular and behavioral outcomes in these mice, dopamine supply was enhanced through
L-DOPA treatment. L-DOPA accelerated neurodegeneration, exacerbated loss of body weight
and further deteriorated motor function. Conversely, when unilateral 6-hydroxydopamine lesions
28
to the medial forebrain bundle were used to reduce dopaminergic input to the striatum, motor
dysfunction was attenuated. These results show that exposure to dopamine is the single
determining factor in producing neurodegeneration and motor disability in these animals. The
damaging effects of cytosolic dopamine are evident in these engineered striatal cells since they
lack the protective mechanisms to appropriately sequester and manage the neurotransmitter.
Various other animal models have been developed with altered levels of dopamine transporters
such as VMAT2-knockdown and DAT-overexpressing transgenic mice (Caudle et al., 2007;
Salahpour et al., 2008). By modifying the transport and compartmentalization of dopamine, the
neurotransmitter can be forced to accumulate within the cytosolic compartment. While published
results from these studies are discussed in subsequent sections, novel findings are also
characterized in this thesis.
In summary, a substantial body of evidence suggests that cytosolic dopamine is highly reactive
and can cause toxicity in vitro and in vivo. However, most of these studies possess important
caveats: 1) they exogenously apply non-physiological concentrations of dopamine or its
precursor, and 2) they use systems that are not intrinsically equipped to handle dopamine. Thus,
it is unclear whether the findings from these studies are relevant to physiological conditions of
dopaminergic neurons. The primary aim of this thesis is to address these limitations and
investigate the effects of altered dopamine transport and potential cytosolic dopamine
accumulation in vivo.
1.2.5 Classical drugs that interact with the dopamine system
Since dopamine neurotransmission plays important roles in brain function and is also implicated
in various disease conditions, the dopamine system serves as an important pharmacological
target. Various drugs can modify dopamine synthesis, degradation, transport or receptor
function, to ultimately impact dopaminergic signaling and related behaviors. Dopaminergic
drugs are used therapeutically, recreationally or as research tools to manipulate and investigate
dopaminergic function in vitro and in vivo. The following section describes selected classical
drugs that affect the dopamine system by interacting with enzymes, transporters or receptors.
29
1.2.5.1 Enzyme ligands
Dopamine synthesis and degradation are fully dependent upon the activity of key enzymes.
Different drugs can bind to these enzymes and alter their function, thus directly affecting
dopamine homeostasis. The initial step in dopamine production in the conversion of tyrosine to
L-DOPA by the rate-limiting enzyme, TH. A well-known inhibitor of TH activity is the
compound, α-methyl-para-tyrosine (AMPT) (Brogden et al., 1981). As a structural relative of
tyrosine, AMPT competes for the substrate binding site and blocks enzymatic activity, thus
preventing dopamine synthesis (Bloemen et al., 2008). Since TH is also involved in
norepinephrine production, application of AMPT results in depletion of dopamine,
norepinephrine and their metabolites. Therapeutically, AMPT is approved for clinical use in
pheochromocytoma, a rare catecholamine-secreting tumor in the adrenal gland that leads to
hypertensive crisis (Brogden et al., 1981). In healthy humans, an acute dose of AMPT has been
reported to produce slightly negative mood, anxiety, sleepiness and decreased attention and
alertness (Bloemen et al., 2008). For research purposes, AMPT is often used as a
pharmacological challenge to evaluate the consequence of catecholamine depletion on outcomes
of interest.
The second enzyme involved in dopamine anabolism is DOPA decarboxylase. Two commonly
used inhibitors of DOPA decarboxylase are benserazide and carbidopa. Both these drugs act
peripherally as they are unable to cross the blood brain barrier. Hence, these drugs block the
conversion of L-DOPA to dopamine in the body without affecting the brain. Clinically, they are
used as adjunctive therapy in combination with L-DOPA for the management of Parkinson’s
disease (Birkmayer and Hornykiewicz, 1961). Parkinson’s disease is characterized by reduced
dopaminergic transmission. Therefore, to replenish dopamine levels, its precursor, L-DOPA, is
applied since dopamine itself cannot cross the blood brain barrier and produces peripheral side
effects. Normally, exogenously administered L-DOPA would be converted to dopamine in the
periphery before it reaches the brain. However, by co-administering benserazide or carbidopa,
peripheral decarboxylation of L-DOPA can be blocked, allowing the dopamine precursor to enter
the CNS where it can be converted to dopamine and exert its therapeutic effect. Further details
on Parkinson’s disease and its therapy are included in a later section. It should be noted that
DOPA decarboxylase is generally known as aromatic L-amino acid decarboxylase (AADC)
because it catalyzes several decarboxylation reactions and is involved in serotonin synthesis as
30
well. Thus, in addition to inhibiting dopamine production, benserazide and carbidopa can also
prevent the decarboxylation of 5-hydroxytryptophan to serotonin in the periphery.
The major pathways responsible for dopamine degradation involve the enzyme, MAO. MAO not
only catalyzes the inactivation of monoamines, but is also involved in metabolizing exogenous
compounds, such as MPTP. In humans, MAO is found within the brain and in the periphery and
exists in two forms: MAO-A and MAO-B. MAO-B mainly degrades dopamine and
phenethylamine, a trace amine, while MAO-A also metabolizes serotonin and norepinephrine
(Glover et al., 1977; Di Monte et al., 1996). Drugs that target MAO can be non-specific or
selective for a particular isoform. Non-selective and irreversible MAO inhibitors such as
isocarboxacid, phenelzine and tranylcypromine are clinically used as antidepressants and
anxiolytics. Since dopamine, norepinephrine and serotonin signaling contribute to normal mood,
by blocking their metabolism, MAO inhibitors elevate levels of these neurotransmitters.
Selective MAO-B inhibitors include selegiline (also known as deprenyl), rasagiline and
pargyline. Selegiline and rasagiline are used in the treatment of Parkinson’s disease because they
specifically increase dopamine levels by blocking MAO-B-mediated metabolism. At higher
doses, some of these drugs lose their selectivity and also bind to MAO-A. Selegiline can also be
used to combat acute MPTP exposure (Przedborski et al., 2001). MPTP is converted to its toxic
metabolite, MPP+ by MAO-B. Therefore, administration of selegiline prevents formation of the
active metabolite and reduces toxicity. A possible side effect of MAO inhibitor use is
hypertension due to increased catecholamine levels.
1.2.5.2 DAT ligands
DAT is the primary target for many compounds including psychostimulants, medications and
neurotoxicants (Miller et al., 1999b; Torres et al., 2003). Since uptake of dopamine is dependent
on DAT, pharmacological manipulation of DAT can produce profound effects on dopamine
neurotransmission. Two classical psychostimulants that operate by altering DAT function are
cocaine and amphetamine. Cocaine binds to DAT and blocks the transport of dopamine from the
extracellular space to the presynaptic neuron (Ritz et al., 1987). Cocaine is a competitive
inhibitor of dopamine transport because its binding site overlaps with dopamine’s site of action,
precluding the endogenous substrate from binding (Beuming et al., 2008). As a result, dopamine
accumulates in the extracellular space where it can reinforce downstream signaling. Conversely,
31
amphetamines (amphetamine, methamphetamine, MDMA) compete with dopamine to enter
dopaminergic cells, acting as a substrate for DAT (Sulzer et al., 2005). Once inside the cell,
amphetamine disrupts the proton gradient required for vesicular storage thus causing dopamine
to leak from the vesicles into the cytoplasm (Sulzer et al., 1995). Ultimately, accumulation of
cytosolic dopamine in combination with the actions of amphetamine on DAT cause a reversal of
the transporter, resulting in efflux of intracellular dopamine into the extracellular space. This
DAT-mediated release of dopamine produces a surge in dopamine signaling in response to
amphetamine. While cocaine and amphetamine can also produce other effects in the CNS, it is
the manipulation of DAT function that directly enhances dopamine neurotransmission and is
thought to underlie the reinforcing properties of these psychostimulants (Donovan et al., 1999;
Howell and Kimmel, 2008). In addition to enhancing extracellular dopamine levels, these
psychostimulants have also been shown to activate phasic dopamine signaling events causing
release of dopamine that contributes to drug reinforcement (Aragona et al., 2008; Wanat et al.,
2009; Daberkow et al., 2013).
Along with these examples, several compounds can inhibit DAT function with varying levels of
selectivity and potency. Initially, it was postulated that all DAT inhibitors would have cocaine-
like stimulant and reinforcing properties (Ritz et al., 1987). However, over the past 10-15 years,
accumulating evidence has challenged this notion, showing heterogeneity among DAT inhibitors
(Schmitt et al., 2013). In fact, different compounds preferentially bind and stabilize distinct
structural states of DAT. Typical DAT inhibitors such as cocaine and methylphenidate have been
shown to stabilize the outward facing conformation and produce locomotor stimulation and
behavioral reinforcement (Loland et al., 2007). However, atypical DAT inhibitors such as
modafinil, bupropion and vanoxerine (GBR12909) tend to promote occluded/inward facing
conformations (Schmitt et al., 2013). Interestingly, these compounds also lack cocaine-like
behavioral effects and possess limited rewarding properties (Schmitt and Reith, 2011). Recently,
ligands that bind to allosteric sites on DAT have been identified and shown to block dopamine
uptake as well (Janowsky et al., 2016). Hence, it seems that the specific pharmacological profile
of a drug and its behavioral effects are heavily dependent on how the drug interacts with DAT
and which structural conformation is favored. In general, DAT antagonists lock the transporter in
a particular structural state, preventing the conformational transitions that are required to shuttle
dopamine across the plasma membrane (Reith et al., 2015). Taken together, these data exemplify
32
1) the diversity of DAT ligands and 2) the responsiveness of DAT to different types of
pharmacological manipulation.
As a plasma membrane transporter, DAT also provides a gate of entry into dopaminergic cells.
The most potent dopaminergic toxicants, 6-hydroxydopamine and MPP+, are substrates of DAT
(Gainetdinov et al., 1997; Miller et al., 1999b; Schober, 2004). These compounds are used to
mimic symptoms of Parkinson’s disease in animal models because they cause robust
degeneration of dopaminergic cells. Since 6-hydroxydopamine is a structural analog of
dopamine, it can hijack the DAT-mediated uptake mechanism to access dopamine cells. It should
be noted that 6-hydroxydopamine is also a substrate for the norepinephrine transporter (NET)
and thus, must be administered specifically to dopaminergic regions to exert selective toxicity.
With regards to MPTP, after crossing the blood brain barrier, this compound is converted to its
toxic metabolite MPP+ by glial MAO-B. MPP+ is specifically translocated into dopaminergic
neurons by DAT (Javitch et al., 1985). Once these toxins accumulate in dopaminergic cells, they
cause oxidative stress and mitochondrial dysfunction, culminating in neurotoxicity (Miller et al.,
1999a; Simola et al., 2007; Abdulwahid Arif and Ahmad Khan, 2010). Hence, DAT provides a
molecular gateway for toxicants to selectively access and damage dopaminergic cells.
Figure 1-7. Substrates for DAT cause selective damage to dopamine neurons.
Mechanisms of MPTP and 6OHDA induced toxicity in dopamine cells. Image adapted from
Rangel-Barajas et al., 2015.
33
In summary, various compounds produce significant effects in the brain as a result of their
actions on DAT. Given this rich pharmacology, manipulation of DAT function occurs in
different ways: 1) by inhibiting DAT and causing buildup of extracellular dopamine, 2) by
reversing DAT and causing release of dopamine and 3) by acting as substrate of DAT and using
the transporter to specifically access dopamine cells. Interestingly, compounds that increase
DAT activity are currently lacking.
1.2.5.3 VMAT2 ligands
Vesicular storage of dopamine can be modified by drugs that act on VMAT2. Similar to DAT
pharmacology, VMAT ligands include inhibitors, psychostimulants and toxins. However, since
VMAT transports serotonin and norepinephrine in addition to dopamine, drugs that manipulate
this transporter can produce more widespread effects.
Reserpine and tetrabenazine are two well-established inhibitors of VMAT function. Reserpine is
a potent VMAT inhibitor that binds at, or very close to, the cytoplasmic monoamine binding site
of VMAT. Reserpine acts on both VMAT1 and VMAT2 although it has a higher affinity for the
latter. Reserpine-induced inhibition of VMAT is long-lasting as it is thought to bind irreversibly.
By disrupting monoamine uptake and storage in vesicles, reserpine and other VMAT inhibitors
can substantially diminish monoamine signaling. Previously, reserpine was used to treat
hypertension since it reduces catecholamine signaling in the peripheral sympathetic nervous
system (Freis, 1954). However, a reported side effect of reserpine treatment was depression due
to depletion of monoamines in the CNS. Indeed, the monoamine hypothesis of depression was
derived, at least in part, from the negative effect of reserpine on mood. In rats, reserpine
administration is used to model Parkinson’s disease since it produces profound hypokinesia and
rigidity (Colpaert, 1987). This highlights the crucial role of VMAT2 in maintaining appropriate
dopaminergic tone for locomotion. In contrast to reserpine, tetrabenazine is relatively selective
for VMAT2, has a shorter half-life and reversibly binds to a site that is distinct from the substrate
binding site. Clinically, tetrabenazine is used for symptomatic control of hyperkinetic disorders
such as Huntington’s disease (Paleacu, 2007). Its therapeutic effect in controlling involuntary
movements is at least partially mediated by VMAT2 inhibition and the consequent dampening of
monoaminergic, and particularly dopaminergic, transmission. Other drugs such as lobeline and
structurally related compounds, have also been shown to inhibit VMAT2 by binding the same
34
site as tetrabenazine. Lobeline derivatives are suggested to reduce the addictive effects of
methamphetamine by decreasing drug-induced dopamine release (Wilhelm et al., 2008; Nickell
et al., 2010).
Aside from acting on plasma membrane transporters, psychostimulants like amphetamine can
also influence the movement of substrates across the vesicular membrane. Although the precise
mechanisms of vesicular involvement are controversial, evidence suggests multiple ways that
amphetamine and its derivatives, methamphetamine and MDMA, can interact with VMAT2: 1)
by acting as a substrate to gain access to the vesicular lumen, 2) by inhibiting dopamine uptake
from the cytosol, 3) by dissipating the proton gradient that drives vesicular monoamine uptake
and 4) by promoting efflux of transmitters from vesicles (Sulzer et al., 2005; Lawal and Krantz,
2013; Nickell et al., 2014). In addition, amphetamines have been shown to displace reserpine or
tetrabenazine binding to VMAT2, indicating that they interact at overlapping sites on the
transporter. However, since amphetamines are highly lipophilic, they can also potentially
permeate membranes without the engagement of carriers. According to one hypothesis, since
amphetamine is a weak base, once it enters the vesicle, it becomes protonated in the acidic
environment of the vesicular lumen. Thus, amphetamine binds free protons and alkanizes the
vesicular interior, disrupting the activity of the proton pump that is necessary for monoamine
uptake. Another hypothesis suggests that since amphetamines are themselves transported by
VMAT2, they cause dopamine release via a carrier-mediated exchange mechanism (Partilla et
al., 2006). Regardless of the particular mechanism, administration of amphetamine increases
cytosolic dopamine levels by 5-fold as measured by intracellular patch electrochemistry
(Mosharov et al., 2003). These results demonstrate the ability of amphetamine to displace
dopamine from vesicles into the cytosol. Following this increase in cytoplasmic dopamine, DAT
activity is reversed leading to amphetamine-evoked dopamine release. Hence, amphetamine acts
by not only engaging DAT, but also manipulating vesicular transport to deplete dopamine stores
and redistribute the neurotransmitter to the cytosol. It should be noted that amphetamines also
produce other pharmacological effects such as NET reversal and MAO inhibition, which can
contribute to their mechanism of action.
Toxins can also act as ligands for VMAT2. Interestingly, the VMAT sequence shares close
homology with bacterial toxin-extruding antiporters, suggesting that VMAT has evolved from
proteins that function to protect the cell from exogenous compounds. Hence, it is not surprising
35
that VMAT2 also possesses a neuroprotective role in monoaminergic cells. Indeed, VMAT was
identified on the basis of conferring resistance to MPP+ toxicity (Liu et al., 1992; Stern-Bach et
al., 1992). MPP+, the active metabolite of MPTP, causes cell death by inhibiting complex I of
the mitochondrial electron transport chain, disrupting energy production and generating ROS.
MPP+ is not only a substrate of DAT, but it is also sequestered into vesicles by VMAT2. This
prevents MPP+ from interacting with mitochondria and causing cellular damage. VMAT2
expression is closely correlated with the extent of MPTP-induced toxicity: mice with low
VMAT2 levels are particularly vulnerable while those with high VMAT2 levels are protected
(Gainetdinov et al., 1998; Mooslehner et al., 2001; Lohr et al., 2014, 2016). Besides MPP+,
VMAT2 is also a target for environmental toxins such as organochlorine pesticides (e.g.
heptachlor), structurally-related polychlorinated biphenyls and brominated flame retardants.
These compounds have been reported to bind and inhibit VMAT2 function thus, reducing
vesicular dopamine uptake and storage.
In summary, while the physiological role of VMAT2 is to package monoamines, this transporter
also responds to drugs that have important consequences for dopamine compartmentalization and
transmission. There exists a bidirectional relationship between VMAT2 and its ligands: certain
drugs manipulate VMAT2 function and vesicular dopamine while other compounds are
sequestered by VMAT2 for cellular protection.
1.2.5.4 Dopamine receptor ligands
The drugs described so far can impact synaptic dopamine levels by altering dopamine uptake,
storage, synthesis or degradation. However, another large class of drugs acts on specialized
dopamine receptors without directly modifying neurotransmitter levels. These ligands are
classified according to: 1) the type of dopamine receptors they target and 2) their actions at the
receptor. Dopamine receptors are typically divided into 2 families: D1 (includes D1 and D5) and
D2 (includes D2, D3 and D4). While ligands generally show preference for one of the two
families of dopamine receptors, they are rarely selective for a single receptor type within the
same family.
Apomorphine is a non-selective dopamine receptor agonist that binds both receptor types but has
higher affinity for D2. In humans, apomorphine has been used in the treatment of Parkinson’s
disease, as it stimulates dopamine receptor signaling despite low extracellular dopamine levels in
36
patients (Deleu et al., 2004). However, apomorphine also has powerful emetic effects probably
due to its actions on dopamine receptors in the chemoreceptor trigger zone. In rodents,
administration of apomorphine produces stereotypy and climbing behavior, which is regarded as
a readout of striatal dopamine receptor activation (Protais et al., 1976). D2 receptor agonists
include quinpirole, bromocriptine, carbergoline, pramipexole, lisuride, ropinirole and others.
Although these agents may also act at other receptors at higher doses, they show strong affinity
for D2-like receptors. Clinically, many of these drugs are used as adjunctive therapy or even
monotherapy in Parkinson’s disease since they can directly activate D2 receptors in the striatum
to restore dopaminergic signaling even though presynaptic dopamine-releasing cells have
degenerated (Hisahara and Shimohama, 2011). Specifically, D2 agonists suppress the activity of
the indirect pathway, thus promoting locomotion. In comparison to L-DOPA, the traditional
treatment for Parkinson’s disease, certain D2 agonists have much longer half-lives making them
an attractive alternative. However, D2 receptors function as both: 1) presynaptic autoreceptors on
dopamine cells and 2) post-synaptic receptors on striatal GABAergic neurons (De Mei et al.,
2009). Depending on the dose, some D2 agonists such as apomorphine, have been reported to
preferentially bind autoreceptors versus post-synaptic receptors (Skirboll et al., 1979).
Autoreceptor activation dampens dopamine synthesis, release and neuronal firing, ultimately
diminishing the dopamine signal, whereas post-synaptic receptor stimulation promotes dopamine
signaling pathways and encourages motor behavior. Hence, some D2 agonists can produce
paradoxical behavioral effects due to engagement of both types of D2 receptors (Skirboll et al.,
1979).
Another important class of clinically relevant drugs that interact with the dopamine system are
D2 antagonists used in the treatment of schizophrenia. According to the dopamine hypothesis of
schizophrenia, positive symptoms such as hallucinations and delusions, are caused by over-
activity of the mesolimbic dopaminergic pathway (Howes and Kapur, 2009). In fact, this
hypothesis emerged from the discovery that 1) major antipsychotic drugs are D2 blockers and 2)
clinical effectiveness of these drugs was directly correlated with their affinity for D2 receptors
(Creese et al., 1976; Seeman et al., 1976; Howes and Kapur, 2009). The first generation of
typical antipsychotic drugs such as haloperidol, chlorpromazine and fluphenazine, were effective
against psychosis by blocking post-synaptic D2-mediated transmission in the nucleus
accumbens. However, these drugs also antagonize D2 signaling in other pathways that terminate
37
in the striatum and hypothalamus, producing adverse effects such as extrapyramidal symptoms
and hyperprolactinemia, respectively. As such, second generation atypical antipsychotic drugs
(clozapine, risperidone, olanzapine, quetiapine) were generated with a presumably ameliorated
side effect profile. These drugs are also primarily D2 antagonists although they act at other
receptors such as 5HT2A (serotonin 2A) as well. For research purposes, raclopride and spiperone
are commonly used to bind and inhibit D2 receptors for radioligand binding and other
experiments.
There are also selective ligands for D1-like receptors. D1 agonists include dihydrexidines and
benzazepines such as SKF81297. Dihydrexidine shows anti-parkinsonian effects in MPTP-
treated monkeys, suggesting that stimulation of D1 signaling in the direct pathway can
compensate for presynaptic dopaminergic damage (Taylor et al., 1991). SKF 81297 treatment in
WT animals has been reported to produce stimulant-like effects such as hyperactivity and self-
administration, showcasing the role of dopamine neurotransmission in motor behavior and
reward (Weed and Woolverton, 1995). The synthetic compound, SCH23390 was the first
selective D1 antagonist and has been a useful research tool (Bourne, 2006). Several D1 ligands
also show affinity for D2-like receptors. For example, various typical and atypical antipsychotic
drugs block D1 receptors in addition to D2 receptors.
1.2.6 Parkinson’s disease
Parkinson’s disease is the most common neurodegenerative movement disorder in humans that
affects approximately 1% of the population over the age of 60 (de Lau and Breteler, 2006). It is a
progressive disease and its incidence significantly increases with age (Dauer and Przedborski,
2003). The clinical features of Parkinson’s disease were described by James Parkinson in his
“Essay on the Shaking Palsy” in 1817 (Parkinson, 1817). However, it took over a century to link
this disease to the neurotransmitter, dopamine. Indeed, dopamine was first synthesized in 1910
and was identified in the mammalian brain in the late 1950s (Carlsson et al., 1958;
Hornykiewicz, 1986). Even after its discovery, dopamine remained in the shadows of the other
two popular catecholamines, norepinephrine and epinephrine, as an intermediary. A major
breakthrough occurred in the 1960s when Ehringer and Hornykiewicz demonstrated that post-
mortem brains of Parkinson’s disease patients showed almost a complete loss of dopamine in the
caudate putamen (Ehringer and Hornykievicz, 1960). This link between Parkinson’s disease and
38
striatal dopamine depletion established the essential physiological role of this neurotransmitter in
controlling motor function.
1.2.6.1 Symptoms
Overall, Parkinson’s disease is a progressive neurological disorder that affects multiple
neurotransmitter systems and is often coupled with psychiatric, cognitive, sensory and autonomic
symptoms. However, the cardinal symptom of the disease is the impairment of voluntary
movement that is attributed to reduced dopaminergic tone in the basal ganglia (Dauer and
Przedborski, 2003). This arises from the specific loss of dopaminergic neurons projecting from
the SNpc to the striatum. Clinically, Parkinson’s disease is characterized by motor deficits
including muscle rigidity, postural instability, impaired gait, resting tremor, bradykinesia
(slowness of movement) and ultimately, akinesia (loss of movement) (Jankovic, 2008). Deficits
in initiating and executing voluntary movements significantly impact the patient’s quality of life
as everyday tasks become difficult to perform. Motor symptoms only become apparent when
dopaminergic tone in the striatum is depleted by ~80% and ~60% of nigrostriatal dopamine
neurons have degenerated. Since motor disability serves as the most robust clinical feature of the
disease, typically, when patients are diagnosed, the majority of SNpc dopamine cells have
already been lost. This reduces the therapeutic window for intervention and emphasizes the need
to recognize other symptoms of Parkinson’s disease that may arise earlier.
Although clinical diagnosis of Parkinson’s disease relies on the presence of motor deficits, many
patients also experience non-motor symptoms that substantially contribute to their disability.
Even James Parkinson’s early description of the disease identifies non-motor symptoms
(Parkinson, 1817). These include sleep abnormalities such as disrupted nocturnal sleep,
excessive daytime somnolence and REM sleep behavioral disorder, which occurs in a third of
patients (Schenck et al., 1996; Olson et al., 2000; Chaudhuri et al., 2006). Patients also report
neuropsychiatric problems such as anhedonia, apathy, depression, anxiety and cognitive
impairment. While depressive symptoms can partially be attributed to reaction upon diagnosis of
the disease, it is believed that monoaminergic deficiency plays a prominent role (Poewe, 2008).
The rate of dementia increases with older patients and is 6-times higher in those with Parkinson’s
disease compared to healthy individuals (Emre, 2003; Chaudhuri et al., 2006). Also, a variety of
symptoms are related to autonomic dysfunction such as orthostatic hypotension, sexual
39
impairment and constipation, one of the most common non-motor symptoms (Poewe, 2008). A
prospective study on 7000 men showed that those with initial constipation were 3-times more
likely to develop Parkinson’s disease after 10 years (Abbott et al., 2001). A particularly notable
olfactory symptom is hyposmia, the reduced ability to detect and discriminate odors. Hyposmia
affects up to 90% of patients and may be used as a preclinical marker for Parkinson’s disease
(Chaudhuri et al., 2006). Although various non-motor symptoms are evident prior to motor
impairment and diagnosis, others reveal themselves with disease progression. While dopamine
depletion contributes to the development of motor disability, widespread pathology in other
neurotransmitter systems are likely responsible for non-motor symptoms of Parkinson’s disease.
1.2.6.2 Pathology
The pathological hallmark of Parkinson’s disease is a loss of nigrostriatal dopaminergic neurons.
Interestingly, dopamine neurons in the adjacent VTA are relatively spared, suggesting a specific
vulnerability of SNpc dopamine cells. Degeneration of SNpc dopamine neurons leads to
diminished dopaminergic innervation of the striatum, which disrupts the basal ganglia motor
loop. Specifically, D1 receptors on GABAergic medium spiny neurons are not adequately
stimulated while D2-expressing striatal neurons are not sufficiently inhibited by dopamine. The
net effect of this nigrostriatal imbalance is to enhance the inhibitory output of the basal ganglia to
the thalamus, thus subsequently reducing cortical activity which impedes voluntary movement.
These changes eventually produce the motor deficits that characterize Parkinson’s disease.
Studies report that the reduction of dopamine terminals in the striatum is greater than the loss of
dopamine cell bodies in the SNpc (Bernheimer et al., 1973). This suggests that terminals are
more sensitive to damage in Parkinson’s disease and cell body degeneration may occur in a
“dying back” process that is initiated in the axon terminals (Dauer and Przedborski, 2003; Cheng
et al., 2010). It is interesting to note that DAT is mostly present in dopaminergic terminals and
serves as a gateway for dopamine and toxicants to enter the cytosol.
Another prominent pathological feature and diagnostic marker of Parkinson’s disease is the
presence of neuronal cytoplasmic protein inclusions known as Lewy bodies. These inclusions
were first described by Frederic Lewy in the early 1900s and are also found in other diseases
such as Lewy body dementia (Holdorff, 2002). Histologically, typical Lewy bodies appear as
eosinophilic spherical masses surrounded by a halo of radiating fibrils. They are composed of
40
proteins such as ubiquitin and most notably, α-synuclein. α-Synuclein is abundant in the brain
and is predominantly found in neuron terminals where it acts as a synaptic modulator with
chaperoning abilities (Souza et al., 2000). α-Synuclein knockout mice show deficits in vesicle
mobilization and synaptic transmission, highlighting the physiological role of this protein in
synaptic function (Cabin et al., 2002). Also, α-synuclein interacts with various cellular
components such as SNARE complexes, chaperone proteins, tubulin and DAT (Norris et al.,
2004; Burré et al., 2010). Natively, α-synuclein is proposed to exist in a soluble form as an
unstructured monomer or tetramer (Bartels et al., 2011; Fauvet et al., 2012). However, in
pathological conditions, the protein undergoes conformational changes and misfolding to
aggregate into high molecular weight oligomers and insoluble fibrils that give rise to Lewy
bodies (Spillantini et al., 1997; Norris et al., 2004). Lewy bodies have been proposed to
contribute to disease pathogenesis in multiple ways: 1) abnormal aggregates of α-synuclein
reduce availability of the normal protein and thus, disrupt its physiological effects and 2)
intracellular proteinaceous inclusions directly obstruct cellular functioning leading to
degeneration (Norris et al., 2004; Luk et al., 2012). In Parkinson’s disease, Lewy bodies are not
only found in the remaining dopaminergic cells of the SNpc, but have also been detected in other
neurotransmitter systems (noradrenergic, serotonergic and cholinergic) and other brain regions
including the olfactory bulb, locus coeruleus (LC), raphe nucleus, dorsal nucleus of the vagus,
pedunculopontine nucleus, hypothalamus, nucleus basalis, cerebral cortex and autonomic ganglia
(Forno, 1996; Norris et al., 2004). Interestingly, a recent study has shown that α-synuclein fibrils
travel from cell-to-cell, providing a mechanism for the spread of Lewy bodies throughout
interconnected brain structures (Luk et al., 2012).
Similar to Lewy body pathology, neurodegeneration in Parkinson’s disease is also not limited to
the SN. While the extent of nigrostriatal degeneration is dramatic and undeniably responsible for
motor deficiency in Parkinson’s disease, neuronal loss also extends to other brain regions.
Degeneration of noradrenergic cells of the LC is comparable to the SN and tends to precede
dopaminergic degeneration (Ehringer and Hornykievicz, 1960; Zarow et al., 2003).
Noradrenergic loss is postulated to contribute to motor deficits as well since nigral dopamine
neurons can receive noradrenergic input through α2-adrenergic receptors (Delaville et al., 2011).
Furthermore, activation of the LC has been shown to alter firing of SN neurons (Grenhoff et al.,
1993). In mice, loss of norepinephrine produced greater motor deficits than MPTP treatment
41
which causes specific damage to dopaminergic cells. This highlights a possible contribution of
norepinephrine in controlling motor activity (Rommelfanger et al., 2007). The LC also plays
important roles in cognition, circadian rhythm and mood. Thus, loss of these cells in Parkinson’s
disease could give rise to non-motor symptoms such as cognitive impairment, sleep disorders,
anxiety and depression (Gesi et al., 2000; Delaville et al., 2011; Del Tredici and Braak, 2013).
Aside from catecholamine systems, degeneration has also been reported in other types of cells
such as serotonergic cells of the raphe nucleus and cholinergic cells of the nucleus basalis and
dorsal nucleus of the vagus (Jellinger, 1991; Bohnen and Albin, 2011). It is interesting to note,
that in addition to cell loss, Lewy body pathology is evident in most, if not all, of these pathways
as well. Importantly, these pathways connect to diverse brain regions and can account for the
variety of functional deficits seen in Parkinson’s disease. For instance, loss of hippocampal
structures and cholinergic cortical inputs have been reported to contribute to increased rates of
dementia in older patients with Parkinson’s disease (Braak et al., 1996; Dauer and Przedborski,
2003). Also, serotonergic lesions are speculated to contribute to depressive symptoms in
Parkinson’s disease.
In summary, Parkinson’s disease is a multi-system disorder with widespread pathology and a
spectrum of symptoms. However, the cardinal diagnostic symptom of this disease is impairment
of voluntary movement that is caused by degeneration of nigrostriatal dopamine neurons. Loss of
SNpc dopamine cells is often coupled with degeneration and Lewy body pathology in extranigral
regions as well.
1.2.6.3 Therapy
The symptoms of Parkinson’s disease are predominantly treated using dopamine replacement
therapy. L-DOPA, the precursor of dopamine, is the gold standard pharmacological treatment. It
is typically administered with AADC inhibitors (e.g. carbidopa or benserazide) to block
peripheral metabolism and allow maximal amounts of the precursor to reach the brain, where it is
converted to dopamine. The therapeutic effects of L-DOPA were revealed almost concurrently
with the discovery that striatal dopamine levels were severely depleted in Parkinson’s disease
patients (Hornykiewicz, 1986). In fact, the efficacy of a dopamine precursor in rescuing the
motor deficits of Parkinson’s disease provided the final evidence for the involvement of striatal
dopaminergic transmission in motor control (Hornykiewicz, 1986). Despite its popularity as the
42
treatment of choice since the 1960s when it was first introduced in Parkinson’s disease, L-DOPA
administration has noteworthy limitations: 1) it only provides symptomatic control without
addressing underlying pathology or disease progression 2) with chronic use, efficacy is often
diminished, producing motor fluctuations and 3) long-term use can also lead to the development
of adverse effects such as dyskinesias (Hornykiewicz, 1986; Marsden, 1994). Dopamine receptor
agonists are also used in Parkinson’s disease predominantly as adjunctive therapy. When patients
no longer respond adequately to pharmacotherapy, surgical approaches, namely deep brain
stimulation, is used to manage late stages of the disease. In particular, high frequency deep brain
simulation of target regions such as the subthalamic nucleus or globus pallidus internal,
ameliorates the function of the basal ganglia motor loop in Parkinson’s disease (The Deep-Brain
Stimulation for Parkinson’s Disease Study Group, 2001). However, none of these treatment
approaches tackle the neurodegeneration that gives rise to motor symptoms in Parkinson’s
disease. Furthermore, since it is a progressive disorder, the pathology is ongoing even when
patients are on medication. Thus, it is important to attack the root cause of the disease to prevent
or at least, decelerate the loss of nigrostriatal dopamine neurons. In order to accomplish this task,
the cause of dopaminergic neurodegeneration in Parkinson’s disease needs to be better
understood.
1.2.6.4 Etiology
While the pathological loss of nigrostriatal dopaminergic neurons is well-established in
Parkinson’s disease, the etiology of this degeneration remains elusive in the majority of cases.
(Surmeier et al., 2010). Approximately 90% of Parkinson’s disease is termed sporadic or
idiopathic, without a known cause. Over the last decade, the role of genetics in Parkinson’s
disease pathogenesis has been progressively explored. Using linkage and genome wide
association analyses, different genetic variants have been identified that either cause familial
forms of the disease or are associated with increased risk of developing “sporadic” Parkinson’s
disease (Hardy et al., 2006; Shulman et al., 2011; Klein and Westenberger, 2012). The first locus
shown to cause Parkinson’s disease was SNCA, the gene responsible for generating α-synuclein.
Given the significant role of α-synuclein in Lewy bodies, a pathological marker of Parkinson’s
disease, it is not surprising to find that some familial forms of the disorder are caused by
dominantly-inherited mutations or multiple copies of the SNCA gene. Missense mutations in
leucine-rich repeat kinase 2 (LRRK2) are also commonly associated with autosomal dominant
43
parkinsonism, whereas mutations in genes such as parkin, PTEN-induced kinase 1 (PINK1) and
DJ-1 give rise to early-onset autosomal recessive forms of the disease (Nuytemans et al., 2010).
Since exclusively monogenic cases of Parkinson’s disease are rare, the contribution of genetic
polymorphisms as risk factors has also been considered using genome wide association analyses.
Variants in SNCA, LRRK2 and β-glucocerebrosidase (GBA) genes have been associated with an
increased susceptibility of developing Parkinson’s disease, although these polymorphisms have
also been detected in asymptomatic individuals (Shulman et al., 2011). Given the presence of
genetic heterogeneity and variants with incomplete penetrance, genetic mutations only account
for 5-10% of Parkinson’s disease cases (Dauer and Przedborski, 2003; Hardy et al., 2006;
Shulman et al., 2011).
In addition to genetic influence, there is also substantial evidence for an environmental
component in Parkinson’s disease. In particular, the landmark discovery of MPTP (1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine) in the 1980s brought to the forefront a role of exogenous
toxicants in Parkinson’s disease pathogenesis (Langston et al., 1983; Schober, 2004). MPTP was
accidentally generated as a by-product in an illegal attempt to synthesize MPPP, an opioid drug.
After using MPTP-contaminated drugs intravenously, young adults developed rapid-onset,
irreversible and chronic parkinsonism. Due to their dramatic symptoms and complete inability to
move, they were referred to as the “frozen addicts”. Post-mortem analyses revealed specific
damage to dopaminergic neurons of the substantia nigra, identical to advanced stage Parkinson’s
disease (Langston et al., 1983). It was later discovered that the metabolite of MPTP, MPP+, is a
substrate for DAT that selectively gains access to dopamine cells through the plasma membrane
transporter. Once inside the cell, MPP+ disrupts complex I of the electron transport chain,
inhibits energy production and exacerbates oxidative stress eventually leading to cell death.
Since its discovery, MPTP has been frequently used to model Parkinson’s disease in animal
research due to its ability to cause potent and selective toxicity in nigrostriatal dopamine
neurons. Aside from MPTP, epidemiological and case-control studies have suggested that
environmental conditions such as exposure to pesticides, residing in rural areas and drinking well
water could be significant risk factors for developing Parkinson’s disease (Rajput et al., 1987;
Semchuk et al., 1992; Priyadarshi et al., 2000; Tanner et al., 2011). Indeed, a meta-analysis of 19
distinct studies found that exposure to pesticides approximately doubled the risk of disease
(Priyadarshi et al., 2000). Furthermore, a dose dependent relationship has been reported between
44
lifetime cumulative exposure to paraquat, a widely used herbicide, and susceptibility to
Parkinson’s disease (Liou et al., 1997). Residential exposure to maneb (a fungicide) and
paraquat, was reported to increase the risk of Parkinson’s disease by 75% in a Californian study
(Costello et al., 2009). Chronic treatment of animal models with pesticides such as rotenone was
shown to recapitulate many of the fundamental features of Parkinson’s disease, including
selective nigrostriatal neuronal degeneration, hypokinesia, and cytoplasmic Lewy body-like
inclusions (Greenamyre et al., 2000; Alam and Schmidt, 2002; Cicchetti et al., 2009). Together,
these studies highlight the significance of environmental risk factors in the etiology of
Parkinson’s disease.
In summary, although the symptomatology and pathology of Parkinson’s disease are well-
elucidated, the precise cause of this disorder remains unknown in the majority of cases. It is a
complex and multifactorial disease that is influenced by age, genetics and the environment.
1.2.6.5 Vulnerability of nigrostriatal dopaminergic cells
Despite recent advances in identifying factors that increase the risk of developing Parkinson’s
disease, the question remains as to why the specific population of nigrostriatal dopaminergic
neurons are the most susceptible to insult. Both genetic and environmental factors could
theoretically have widespread implications on different neuronal populations of the CNS,
however they tend to produce particularly selective effects. For instance, parkin encodes a
protein involved in the ubiquitin-proteasome system that mediates protein degradation, however
how mutations in this gene accounts for specific dopaminergic cell loss is unknown (Paris et al.,
2009). Genetic studies also reveal that mutations in PINK1 are associated with early onset
Parkinson’s disease suggesting that the loss of this mitochondrial protein kinase has very
particular implications on the nigrostriatal tract of neurons in comparison to other cell types
(Nuytemans et al., 2010). In the case of environmental toxicants, apart from MPTP, which is
metabolized to form a substrate for DAT, other compounds associated with increasing
Parkinson’s disease susceptibility do not display any precise characteristics that would target
them to dopaminergic neurons only. For instance, rotenone is a highly lipophilic compound that
can cross the blood brain barrier and inhibit complex I of the electron transport chain (Alam and
Schmidt, 2002; McCormack et al., 2002; Richardson et al., 2005; Ramachandiran et al., 2007).
Although rotenone can theoretically accumulate in the entire brain, it causes selective damage to
45
dopaminergic neurons of the SNpc. Indeed, it is interesting to note that a variety of genetic and
environmental factors with diverse mechanisms of action, all seem to converge in damaging a
small group of discrete neurons in the SNpc which gives rise to the symptoms of Parkinson’s
disease. This indicates that apart from general risk factors, there must exist “cell-specific” factors
that render nigrostriatal dopamine neurons highly sensitive to toxicity (Surmeier et al., 2010). In
order to better understand the etiology of nigrostriatal neurodegeneration in Parkinson’s disease,
it is important to uncover the inherent characteristics of these dopamine neurons that permit them
to be easily targeted by genetic and exogenous insults.
As previously discussed, dopamine neurons of the SNpc display several unique features such as:
1) L-type calcium channel dependent pacemaking activity, 2) morphological complexity of axon
terminals, 3) high bioenergetic demands, 4) increased basal oxidative stress and 5) an extremely
reactive cytosolic substrate, dopamine. These characteristics may shape the intrinsic vulnerability
of SNpc dopamine neurons to insult. Indeed, even healthy humans experience around a 40%
reduction in nigrostriatal dopamine neurons between the ages of 40 and 60, illustrating the
susceptibility of these cells to age-dependent degeneration (Bogerts et al., 1983; Stark and
Pakkenberg, 2004; Chinta and Andersen, 2005). In Parkinson’s disease, the differential
vulnerability of SNpc dopamine neurons could be mediated by the distinctive physiological
nature of these cells. Unlike most neurons of the brain, adult SNpc dopamine cells rely on L-type
calcium channels with a Cav1.3 subunit, to generate rhythmic action potentials (Guzman et al.,
2009). A case-control study demonstrates that subjects prescribed L-type calcium channel
blockers for the treatment of hypertension, were 27% less likely to develop Parkinson’s disease,
indicating a neuroprotective effect (Ritz et al., 2010). Furthermore, in vitro and in vivo inhibition
of L-type calcium channels protects SNpc dopamine cells from damage induced by rotenone and
MPTP, two toxicants used to model Parkinson’s disease (Chan et al., 2007). These data suggest
that the unique dependence of SNpc neurons on L-type calcium channels may contribute to their
vulnerability.
In addition, nigrostriatal dopamine neurons display an extensive, unmyelinated axonal arbor that
is orders of magnitude larger than other neurons (Bolam and Pissadaki, 2012). Together,
autonomous pacemaking and massive axonal complexity impose high metabolic costs on these
cells, rendering them particularly sensitive to any perturbation in mitochondrial energy
production (Pissadaki and Bolam, 2013). Indeed, mitochondrial dysfunction is suggested to
46
participate in the mechanisms of toxicity underlying Parkinson’s disease (Keeney, 2006;
Winklhofer and Haass, 2010). Post mortem analyses of Parkinson’s disease patients indicate a
significant reduction of mitochondrial complex I in the substantia nigra (Mizuno et al., 1989;
Schapira et al., 1990; Janetzky et al., 1994). Moreover, mitochondria derived from patients show
increased oxidative damage of complex I catalytic subunits that correlate with reduced
functionality (Keeney, 2006). Interestingly, several genetic and environmental risk factors
associated with Parkinson’s disease, have also been shown to directly or indirectly affect
mitochondrial integrity. The most obvious examples are rotenone and MPP+, two toxicants that
have been implicated to cause Parkinson’s disease in humans and are commonly used to model
the disorder in animals. Both compounds exert their toxicity by inhibiting complex I of the
electron transport chain, reducing ATP production and enhancing generation of reactive oxygen
and nitrogen species (Greenamyre et al., 2003; Winklhofer and Haass, 2010). The genes, PINK1
and parkin, that give rise to familial forms of Parkinson’s disease, have also been shown to
regulate mitochondrial function (Clark et al., 2006; Deng et al., 2008; Poole et al., 2008). In
summary, nigrostriatal dopamine neurons are heavily dependent on mitochondrial ATP
production to meet their high energy demands and therefore, any interruption of mitochondrial
activity seems to differentially affect these cells. Furthermore, accumulating evidence of
impaired mitochondrial function in Parkinson’s disease suggests that it may participate in disease
pathogenesis.
Although the precise mechanisms underlying nigrostriatal degeneration in Parkinson’s disease
remain to be fully elucidated, oxidative stress has emerged as a crucial player (Dias et al., 2013).
Oxidative stress is defined as an imbalance between the production of ROS and the ability of
anti-oxidant mechanisms to detoxify these volatile chemicals. The resulting disequilibrium
produces detrimental consequences as reactive oxygen and nitrogen species modify key cellular
macromolecules, disrupt their function and eventually lead to cell death. Postmortem tissue from
Parkinson’s disease patients demonstrate extensive oxidative and nitrosative injury to
dopaminergic regions like the SN as indicated by: 1) increased protein carbonyls, 2) reduced
levels of antioxidants: glutathione, glutathione peroxidase and catalase 3) elevated protein
adducts, 4) enhanced lipid peroxidation and 5) increased oxidative modifications of DNA and
RNA molecules (Dexter et al., 1989a; Sian et al., 1994; Yoritaka et al., 1996; Alam et al., 1997a,
1997b; Zhang et al., 1999; Asanuma et al., 2003). Moreover, almost all toxicants that are
47
associated with nigrostriatal damage and are used to model Parkinson’s disease – such as MPTP,
6-hydroxydopamine, rotenone and paraquat – induce oxidative stress as their mechanism of
toxicity. These findings strongly suggest that nigrostriatal dopamine neurons are particularly
sensitive to oxidative injury and may be exposed to increased basal levels of oxidative stress. As
the primary cellular consumer of oxygen and armed with several redox enzymes, mitochondria
represent a major source of ROS. While the electron transport chain transfers electrons onto
molecular oxygen to generate superoxide anions, mitochondria also contain antioxidant defense
systems to detoxify the ROS generated. However, in cases of mitochondrial dysfunction, as
observed in Parkinson’s disease, antioxidant defense mechanisms become compromised while
ROS production is exacerbated to give rise to oxidative stress (Lin and Beal, 2006; Yan et al.,
2013). Perhaps the most notable source of ROS in nigrostriatal dopamine neurons is the
endogenous neurotransmitter, dopamine itself. As previously mentioned, MAO-B-mediated
degradation of dopamine routinely gives rise to H2O2. Also, TH-dependent synthesis of
dopamine has been shown to catalyze production of ROS through hydroxylation reactions in
vitro (Haavik et al., 1997). Furthermore, as a highly unstable molecule, dopamine is exposed to
oxidation reactions in the cytosolic space, which contribute to generation of reactive quinones
and radical species, as previously discussed. Thus, it is possible that the constant handling of
cytosolic dopamine may render SNpc neurons particularly susceptible to oxidative stress and
predispose them to degeneration in Parkinson’s disease.
1.2.6.5.1 Role of cytosolic dopamine in Parkinson’s disease
Accumulation of cytosolic dopamine has been shown to produce cellular toxicity in vitro and in
vivo, as described in previous sections. Given the toxic potential of cytosolic dopamine and the
differential loss of nigrostriatal dopaminergic neurons in Parkinson’s disease, it is possible that
cytosolic dopamine may play a role in disease pathogenesis. Several findings indicate a link
between cytosolic dopamine reactivity and Parkinson’s disease pathology. For instance cysteinyl
adducts of dopamine, L-DOPA and DOPAC are significantly increased in the SN of Parkinson’s
disease patients, demonstrating increased oxidation of cytosolic dopamine, its precursor and
metabolite (Spencer et al., 1998). These cysteinyl conjugates have been shown to elevate ROS
generation and enhance DNA base modification, causing neuronal damage (Spencer et al., 2002).
Hence, in Parkinson’s disease, nigrostriatal neurons are exposed to dopamine-induced oxidative
modifications that can potentially contribute to their degeneration. Dopamine turnover is also
48
increased in the brains of Parkinson’s disease patients (Goldstein et al., 2011, 2013). Since
intracellular metabolism specifically occurs on the cytosolic fraction of dopamine, enhanced
turnover may represent efforts to detoxify higher basal levels of cytosolic dopamine in
Parkinson’s disease patients. While dopamine neurons of the SN undergo substantial degradation
in Parkinson’s disease, those in the VTA are relatively spared. Although both subsets of neurons
manage and transmit dopamine, nigral cells are more susceptible suggesting a unique
physiological nature. Indeed, when treated with L-DOPA, studies reveal that SN neurons
accumulate 2 to 3 times higher levels of cytosolic dopamine in comparison to their counterparts
in the VTA (Mosharov et al., 2009). This clearly illustrates that not all dopamine neurons are
created equal. Furthermore, it sheds light on the possibility that since SNpc neurons intrinsically
handle higher quantities of cytosolic dopamine, they probably also experience greater oxidative
stress and therefore, become more sensitive to insult in Parkinson’s disease versus VTA neurons.
The continuous oxidative trauma present in SNpc dopamine cells may account for the exquisite
vulnerability of these neurons to complex I inhibitors (e.g. rotenone, MPTP) or general inducers
of oxidative stress (e.g. paraquat) in Parkinson’s disease (Tanner et al., 2011).
Although nigrostriatal damage is the most striking feature of Parkinson’s disease, other notable
signs include the presence of α-synuclein filled Lewy bodies and considerable degeneration of
noradrenergic neurons in the LC (Dauer and Przedborski, 2003). Interestingly, studies suggest
that cytosolic dopamine levels can potentially impact these factors as well (Sulzer, 2001). Xu et
al report that α-synuclein exhibits neurotoxic effects in dopaminergic neurons while providing
neuroprotection in non-dopaminergic cells (Xu et al., 2002). This consolidates the role of cell-
specific risk factors that are unique to dopaminergic neurons. Moreover, α-synuclein-induced
apoptosis of dopaminergic neurons was shown to be dependent on the presence of dopamine,
since blocking dopamine synthesis protected these cells from toxicity (Xu et al., 2002). In
addition, α-synuclein-transfected dopamine neurons showed marked increases in ROS and
application of antioxidants inhibited α-synuclein-induced cell death (Xu et al., 2002). This
suggests that the toxic actions of dopamine and α-synuclein both converge at a common
mechanism of generating oxidative stress which can lead to cell death. Another study
demonstrates that dopamine can form oxidative adducts with α-synuclein (Conway et al., 2001).
These reactive adducts stabilize the toxic protofibril form of α-synuclein, while inhibiting the
formation of benign fibrils. As a result, in the presence of cytosolic dopamine and an oxidative
49
environment, α-synuclein assumes a pathogenic role (Conway et al., 2001). α-Synuclein is not
only the main component of Lewy bodies, but has also been shown to cause familial forms of
Parkinson’s disease. These results indicate that cytoplasmic dopamine concentration plays an
important role in triggering the pathological accumulation of α-synuclein in Parkinson’s disease.
A significant loss of LC noradrenergic cells has also been detected in Parkinson’s disease. This
deterioration is held responsible for some non-motor phenotypes such as REM sleep
disturbances, a common early symptom of Parkinson’s disease. Interestingly, a shared attribute
between noradrenergic and dopaminergic neurons is the molecule dopamine, which is the direct
precursor to norepinephrine. In noradrenergic neurons, dopamine is converted to norepinephrine
by dopamine β hydroxylase in the vesicular lumen. Hence, noradrenergic neurons also possess
an intracellular pool of dopamine, like dopaminergic neurons. Thus, theoretically, the loss of
noradrenergic neurons in Parkinson’s disease could be related to the fact that these cells also
handle the highly unstable and reactive molecule, dopamine, that can instigate oxidative stress.
Overall, the damaging effects of cytosolic dopamine can potentially contribute to various aspects
of Parkinson’s disease pathology. Indeed, cytosolic dopamine-induced toxicity not only offers an
explanation for the differential susceptibility of nigrostriatal dopamine neurons, but may also
mediate α-synuclein pathology and noradrenergic cell loss in Parkinson’s disease.
1.2.6.5.2 Role of dopamine transporters in Parkinson’s disease
Accumulation of dopamine within the cytoplasm is controlled by two transporters: DAT and
VMAT2. DAT increases the cytosolic pool of dopamine by taking it up from the extracellular
space while VMAT2 reduces cytosolic accumulation by sequestering intracellular dopamine into
vesicles. Since cytosolic dopamine is postulated to contribute to the vulnerability of nigrostriatal
dopamine neurons in Parkinson’s disease, the balance of DAT and VMAT2 activity may also
impact disease pathogenesis. Furthermore, these transporters are also targeted by various drugs
including toxicants that have been implicated in Parkinson’s disease.
In general, the DAT protein sequence appears to be highly conserved, possibly as an
evolutionary mechanism to preserve appropriate function of the dopamine system (Vandenbergh
et al., 2000). The first genetic condition directly caused by loss-of-function mutations in the
DAT gene is DAT deficiency syndrome, a complex motor disorder of progressive parkinsonism-
dystonia that typically manifests in infancy and severely reduces life expectancy (Kurian et al.,
50
2009, 2011; Ng et al., 2014). The striking phenotypes in these patients convincingly demonstrate
the significance of DAT genetics in controlling motor behavior. Unlike DAT deficiency
syndrome, concrete evidence of a causal link between genetic DAT mutations and Parkinson’s
disease is lacking. This is probably because the etiology of Parkinson’s disease is multifactorial
and genetic mutations account for only a small proportion of cases (5-10%) (Dauer and
Przedborski, 2003; Sulzer, 2007). However, several lines of evidence suggest that DAT may act
as a risk factor in Parkinson’s disease. For instance, neuroanatomical analyses indicate that
regions of the human brain containing the highest levels of DAT protein – the caudate and
putamen – are most sensitive to damage in Parkinson’s disease (Bernheimer et al., 1973; Miller
et al., 1997). The pattern of dopaminergic cell loss in the midbrain also appears to parallel the
expression of DAT; nigral neurons display higher DAT mRNA than VTA neurons, which are
relatively spared in Parkinson’s disease (Uhl et al., 1994). These findings indicate a correlation
between DAT expression and vulnerability to insult in Parkinson’s disease. The potential role of
DAT in enhancing susceptibility of dopamine neurons is two-fold: first, it functions to increase
the pool of cytosolic dopamine, which is highly reactive and second, it allows toxicants such as
MPTP selective access to dopaminergic cells. Hence, DAT activity could sensitize dopamine
neurons to both intrinsic oxidative stress as well as extrinsic environmental insult. In fact, a study
by Ritz et al demonstrates that DAT genetic variants in combination with pesticide exposure can
increase risk of Parkinson’s disease by several fold (Ritz et al., 2009). These DAT variants
include single nucleotide polymorphisms in the 5’ region as well as variable number tandem
repeats at the 3’ region of the gene. Although the functional consequences of these DAT variants
are unclear, these results highlight the synergistic influence of DAT in Parkinson’s disease
especially in conjunction with environmental insults (Kelada et al., 2006; Sulzer, 2007). In a rare
example, DAT coding variants were also identified in an individual with comorbid early-onset
parkinsonism and ADHD (Hansen et al., 2014). In vitro, these variants resulted in reduced
dopamine uptake capacity, indicating a role of DAT function in disease pathogenesis (Hansen et
al., 2014). In summary, while DAT mutations typically produce drastic childhood-onset motor
syndromes like DAT deficiency syndrome, in a progressive age-related disorder like Parkinson’s
disease, DAT is more likely to play a modulatory role in combination with other risk factors.
Similar to DAT, mutations within the VMAT2 coding region are rare, consolidating the
fundamental role of these transporters in neurotransmission. In a unique case, members of a
51
consanguineous family were discovered to possess a particular VMAT2 mutation that
compromised vesicular transport of monoamines (Rilstone et al., 2013). These members suffered
from infantile-onset parkinsonism with severe cognitive, autonomic and psychiatric disturbances,
reflecting defects in monoamine transmission (Rilstone et al., 2013). Once again, similar to
DAT, these rare VMAT2 mutations give rise to dramatic pediatric-onset movement disorders
that confirm the necessity of VMAT2 function for appropriate motor behavior. Various findings
suggest that vesicular function is involved in mechanisms leading to nigrostriatal degeneration in
Parkinson’s disease. Vesicular uptake of dopamine and tetrabenazine (VMAT2 ligand) binding
were both severely reduced in isolated synaptic vesicles from Parkinson’s disease patients, even
after correcting for dopamine terminal loss (Pifl et al., 2014). This defect in VMAT2 function
can impair vesicular dopamine storage, causing it to accumulate in the cytosolic space where it is
exposed to oxidative reactions. Hence, reduced VMAT2 activity in Parkinsonian patients can
potentially influence disease progression. Conversely, gain-of-function haplotypes in the
VMAT2 promotor region were found to decrease the risk of Parkinson’s disease in females
(Glatt et al., 2006). In another study, two specific polymorphisms in the VMAT2 promotor
sequence were also found to confer a reduced risk of developing sporadic Parkinson’s disease
(Brighina et al., 2013). These studies indicate that increased VMAT2 function is protective for
Parkinson’s disease probably by 1) sequestering intracellular dopamine into vesicles and thereby,
reducing cytosolic dopamine content and 2) isolating toxicants such as MPP+ away from cellular
machinery. In addition, toxicants such as organochlorine pesticides and polybrominated biphenyl
compounds that have been associated with Parkinson’s disease and detected in post-mortem
brains of patients, have also been shown to inhibit VMAT2 activity and produce nigrostriatal
damage (Bemis and Seegal, 2004; Richardson and Miller, 2004; Hatcher et al., 2008; Guillot and
Miller, 2009; Cannon and Greenamyre, 2011; Hatcher-Martin et al., 2012; Bradner et al., 2013;
Wilson et al., 2014). Hence, environmental toxicants may exert part of their damaging effects in
dopaminergic cells by altering VMAT2 activity. In general, VMAT2 protects dopaminergic
neurons from endogenous and exogenous insults and dysregulation of its function may contribute
to the vulnerability of dopaminergic neurons in Parkinson’s disease.
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1.2.7 Animal models with altered transporter levels
The function of DAT and VMAT2 in regulating dopamine dynamics is best elucidated by animal
models with varying levels of these transporters. Over the past few years, several such mouse
models have been generated allowing for controlled titration of transporter expression and
elucidation of its effects on the dopaminergic system. Findings from these animal models and
from this thesis, have been summarized in Table 1-1 (Lohr et al., 2017) .
53
Table 1-1. Summary of mouse models with genetically altered DAT or VMAT2 levels.
54
1.2.7.1 DAT-knockout mice
The critical role of DAT in maintaining appropriate dopaminergic function is clearly
demonstrated by DAT knockout mice (DAT-KO). Genetic ablation of this plasma membrane
transporter produces dramatic changes in extracellular and intracellular dopamine dynamics
(Giros et al., 1996; Jaber et al., 1997; Jones et al., 1998a). DAT-KO mice display 5-fold elevated
extracellular dopamine levels due to lack of uptake. Additionally, dopamine remains in the
extracellular space 300 times longer since diffusion is the only mechanism to clear the
neurotransmitter in DAT-KO mice. Conversely, intracellular dopamine content is reduced by
95% demonstrating that DAT-mediated recycling of dopamine is chiefly responsible for
maintaining presynaptic dopamine levels (Sotnikova et al., 2005). Due to depleted intracellular
stores, evoked dopamine release is also diminished by 75% in DAT-KO mice (Jones et al.,
1998a). These neurochemical changes illustrate the vital role of DAT in balancing dopamine
levels across different cellular compartments.
Furthermore, lack of DAT activity also triggers compensatory alterations in other pre- and post-
synaptic markers of the dopamine system. Striatal post-synaptic D1 and D2 receptors are
downregulated by 60% and 40% respectively, to adapt to high extracellular dopamine (Ghisi et
al., 2009). Presynaptic D2 autoreceptors are also desensitized, disrupting regulatory negative
feedback mechanisms (Giros et al., 1996; Jones et al., 1999). Levels of dopamine metabolites,
HVA and 3-MT, are increased suggesting that dopamine degradation may be altered in these
mice (Jones et al., 1998a). Without DAT-mediated dopamine recycling, presynaptic dopamine
levels in DAT-KO mice are solely dependent on synthesis by TH. Paradoxically, while TH
expression is reduced, dopamine synthesis rates are doubled, highlighting major adaptive
changes in attempts to stabilize dopamine levels in DAT-KO mice (Jones et al., 1998a; Jaber et
al., 1999). Behaviorally, these animals show spontaneous hyperlocomotion and impaired
habituation as a result of increased extracellular dopamine (Giros et al., 1996). DAT-KO mice
also display disturbances in cognition and sensorimotor gating (Ralph et al., 2001; Barr et al.,
2003; Yamashita et al., 2006; Weiss et al., 2007). Pharmacologically, DAT-KO mice are
insensitive to the classical stimulant actions of cocaine and amphetamine but show paradoxical
calming effects instead (Giros et al., 1996; Jones et al., 1998b; Gainetdinov et al., 1999). In
particular, when treated with cocaine or amphetamine, dopamine release and locomotor activity
55
are not enhanced in DAT-KO mice, validating that transporter function is compulsory for
psychostimulant effects. Also, DAT-KO mice are completely resistant to nigrostriatal damage
induced by MPTP demonstrating that DAT-mediated uptake of MPP+ is required for neurotoxic
effects (Gainetdinov et al., 1997; Bezard et al., 1999). Collectively, mice lacking DAT show
dramatic neurochemical, adaptive and behavioral changes in the dopamine system. Investigation
of DAT-KO mice has contributed essential knowledge on the physiological role of DAT as well
as the importance of this transporter as a pharmacological target.
1.2.7.2 DAT-overexpressing transgenic mice
On the other end of the spectrum, our laboratory has created transgenic mice that over-express
DAT allowing for in vivo analysis of increased dopamine uptake. Specifically, DAT over-
expressing transgenic (DAT-tg) mice were generated by pronuclear injection of a bacterial
artificial chromosome (BAC) containing the 40-kb mouse DAT locus along with 80kb of
flanking DNA sequences (Salahpour et al., 2008). Since the promotor region of DAT is not well-
characterized, this approach allows for DAT transgene expression to be driven by the
endogenous promotor. Hence, DAT is selectively over-expressed in dopaminergic neurons as
confirmed by immunohistochemical data showing similar tissue localization of DAT between
wild type (WT) and DAT-tg mice, although the extent of expression is higher in the latter as
expected (Salahpour et al., 2008). Southern blots estimate that DAT-tg mice display 3-fold
higher genomic DAT expression in comparison to WT mice. Since WT animals contain 2
endogenous copies of DAT, this suggests that DAT-tg mice possess a total of 6 DAT copies
consisting of 2 endogenous and 4 transgenic copies. Similar to genomic levels, DAT-tg mice also
display approximately 3-fold greater total DAT protein in the striatum. However, when DAT
levels were assessed specifically in the synaptic plasma membrane fraction, the increase was
much more modest (30%) suggesting that not all transgenic DAT is expressed at the plasma
membrane (Salahpour et al., 2008). Consistent with this, the amount of functional DAT in DAT-
tg mice was increased by 38% as measured by radioligand binding. This translates to
approximately a 50% increase in the rate of dopamine uptake attesting to enhanced DAT activity
in DAT-tg mice. Due to greater dopamine clearance, these mice display about a 40% reduction
in extracellular dopamine levels (Salahpour et al., 2008). To compensate for this reduction in
dopaminergic tone, DAT-tg mice also demonstrate a 30% increase in D1 and 60% increase in D2
receptors in the striatum (Ghisi et al., 2009). In response to dopamine receptor agonists such as
56
apomorphine (non-selective) or a combination of SKF 81297 (D1 agonist) and quinpirole (D2
agonist), DAT-tg mice demonstrate increased climbing behavior further supporting enhanced
dopamine receptor function in these animals. Furthermore, when treated with amphetamine,
DAT-tg mice show marked enhancement of dopamine release and concomitantly increased
locomotor activity. These results indicate that DAT over-expression enhances the sensitivity of
these animals to the psychostimulant effects of amphetamine (Salahpour et al., 2008).
While the effects of DAT over-expression on extracellular dopamine, post-synaptic receptors and
psychostimulant response have been summarized in two manuscripts (Salahpour et al., 2008;
Ghisi et al., 2009), its consequences on presynaptic dopamine dynamics were unclear when I
began my doctoral thesis. Unpublished findings suggested that although increased DAT-
mediated uptake is expected to enhance dopamine accumulation in the presynaptic neuron, both
striatal dopamine tissue content and evoked dopamine release were reduced in DAT-tg mice.
Furthermore, stereological counts of dopaminergic neurons in the SN and VTA revealed a 30-
40% loss in DAT-tg animals compared to WT mice. In light of a large body of literature
suggesting that accumulation of cytosolic dopamine can produce neurotoxicity, we investigated
whether increased DAT expression may lead to greater intracellular loading of dopamine and
provide a mechanism for the neuronal loss in DAT-tg mice. The fine motor behavior of these
mice was also assessed as a readout of nigrostriatal dopaminergic function. In addition, the
sensitivity of DAT-tg mice to exogenous toxicant insult was evaluated by treating them with
MPTP, a compound known to cause Parkinson’s disease. Further details on these experiments
and their results are outlined in subsequent chapters and summarized in a manuscript (Masoud et
al., 2015). In summary, DAT-tg mice represent a useful in vivo model to understand the
consequences of increased dopamine uptake and probably, cytosolic dopamine accumulation, in
neurons that routinely handle this neurotransmitter.
1.2.7.3 VMAT2-knockout homozygote mice
In addition to DAT, a series of studies have investigated the role of VMAT2 in vivo by varying
levels of the transporter in genetically modified mice. The first and most extreme example of this
is genetic ablation of VMAT2 expression in VMAT2-knockout (VMAT2-KO) mice (Fon et al.,
1997; Takahashi et al., 1997; Wang et al., 1997). Strikingly, lack of VMAT2 results in postnatal
death with most mice dying within 1-3 days after birth. VMAT2-KO mice also appear small,
57
feed poorly, are hypoactive and show severely stunted growth. These results emphasize the
physiological necessity of normal VMAT2 function for survival and development. While
immunohistochemical measures of dopaminergic cell bodies and projections appear normal,
whole brain tissue content of dopamine, norepinephrine and serotonin are drastically reduced by
over 95% in VMAT2-KO mice (Fon et al., 1997; Wang et al., 1997). Although rates of synthesis
are almost doubled in these animals, it cannot compensate for the loss of vesicular storage, which
is the main determinant of monoamine content in the brain. Despite the severe decline in
monoamine levels, metabolite concentrations are unchanged or increased, suggesting enhanced
monoamine degradation in VMAT2-KO mice. Since neurotransmitters are no longer protected in
vesicles, they are extremely vulnerable to metabolic reactions in the cytosol. Furthermore,
electrically-evoked dopamine efflux was completely abolished in striatal slices from VMAT2-
KO animals, highlighting the critical role of vesicular loading in exocytotic neurotransmitter
release. Hence, as a regulator of vesicular uptake, storage and release, VMAT2 activity directly
impacts both intracellular and extracellular dopamine levels. Interestingly, treatment with
amphetamine, enhances locomotion, feeding and survival of VMAT2-KO mice (Fon et al.,
1997). Since amphetamine produces non-vesicular dopamine release, treatment with this drug
circumvents the lack of VMAT2-mediated exocytotic neurotransmitter release, alleviating the
severe symptoms in VMAT2-KO mice (Fon et al., 1997). Given that VMAT2 is involved in
regulating all monoamines, effects on serotonergic and noradrenergic systems may also influence
the phenotypes in these mice.
1.2.7.4 VMAT2-knockout heterozygote mice
Since homozygote VMAT2-KO mice survive for only a few days, detailed experimental
analyses could not be conducted on these animals. Instead, heterozygote VMAT2-knockout
(VMAT2-het) mice containing one functional VMAT2 allele were studied as they survive to
adulthood. VMAT2-het mice show normal development and are indistinguishable from WT mice
in appearance and locomotion (Takahashi et al., 1997). Western blots confirm that in comparison
to WT animals, VMAT2-het mice show 50% VMAT2 protein expression, as expected. Although
the reduction in VMAT2 produces alterations in dopamine homeostasis, the changes are less
severe than homozygote VMAT2-KO mice. For instance, VMAT2-het mice display 50% lower
dopamine uptake in striatal vesicular preparations, indicating a reduction in VMAT2-uptake
activity that parallels the decrease in expression. Extracellular dopamine in the striatum is also
58
reduced by 40% in heterozygotes suggesting impaired VMAT2-mediated dopamine release.
While there are conflicting reports regarding tissue levels, in general, striatal dopamine content
seems to be reduced by approximately 25%, demonstrating diminished VMAT2 storage capacity
in these mice (Wang et al., 1997). However, DOPAC levels were increased by 36%, suggesting
an enhancement of dopamine turnover similar to VMAT2-KO mice. In addition to moderate
changes in dopamine homeostasis, these mice display striking phenotypes in response to drugs
that target the dopamine system. For example, VMAT2-het mice show pronounced hyperactivity
when treated with psychostimulants such as cocaine or amphetamine in comparison to WT mice
(Takahashi et al., 1997; Wang et al., 1997). However, despite the behavioral effect,
amphetamine-induced dopamine release is diminished in VMAT2-het mice (Wang et al., 1997).
A possible explanation of the locomotor sensitivity in these mice is post-synaptic receptor up-
regulation, which may have developed to compensate for chronically lower extracellular
dopamine levels. Indeed, pretreatment with either SCH23390 (D1 antagonist) or raclopride (D2
antagonist), prevented cocaine-induced hyperlocomotion in both WT and VMAT2-het mice,
indicating that this response is driven by dopamine receptor function (Wang et al., 1997). In
addition, administration of MPTP, a Parkinson’s disease-inducing neurotoxin, produced greater
dopaminergic damage in VMAT2-het mice than WT animals as indicated by: 1) loss of nigral
dopaminergic neurons, 2) reduction of striatal dopamine tissue content and 3) decreased DAT
protein expression, a marker of dopaminergic nerve terminals (Takahashi et al., 1997;
Gainetdinov et al., 1998). Enhanced susceptibility to toxicant insult demonstrates the protective
role of vesicular transport in packaging exogenous compounds like MPP+ into vesicles to
prevent their interaction with cellular machinery.
1.2.7.5 VMAT2-knockdown mice
Interestingly, in an attempt to knockout the VMAT2 gene, serendipitous recombination events
gave rise to transgenic mice that express only 5% of normal VMAT2 levels (Mooslehner et al.,
2001; Caudle et al., 2007). These VMAT2-knockdown (VMAT2-kd) mice possess a
hypomorphic VMAT2 allele with insertion of the neomycin cassette in the third intron of the
VMAT2 gene. Unlike VMAT2-KO mice, these animals are viable into adulthood, allowing for
long-term assessment of vesicular deficiency. When these mice were first created by Mooslehner
and colleagues, they unintentionally used an inbred strain of C57BL/6 mice that was later found
to be lacking the α-synuclein gene locus (Mooslehner et al., 2001; Specht and Schoepfer, 2001).
59
Since α-synuclein is a ubiquitous and important protein that can interact with cytosolic dopamine
and contribute to Parkinson’s disease, deletion of this gene limited the utility of these mice.
Subsequently, the Miller laboratory at Emory University strategically bred mice that were
heterozygous for the α-synuclein and VMAT2 genes to eliminate all traces of the α-synuclein
mutation and generate VMAT2-kd mice on a normal α-synuclein background (Caudle et al.,
2007). While most of these studies were performed on VMAT2-kd mice maintained on a
C57BL/6 and 129SV mixed genetic background (Caudle et al., 2007; Guillot et al., 2008; Taylor
et al., 2009, 2014), one recent manuscript and the work outlined in this thesis pertain to VMAT2-
kd mice that were back-crossed to C57BL/6 for several generations (Lohr et al., 2016).
Even though 5% VMAT2 protein expression allows VMAT2-kd mice to survive into adulthood,
they display prominent age-related neurochemical, compensatory and behavioral changes as well
as altered response to toxicants. Concurrent with reduced VMAT2 expression, functional
vesicular uptake of dopamine is also decreased by 80% in VMAT2-kd mice (Caudle et al.,
2007). Tissue levels of dopamine, serotonin and norepinephrine are dramatically diminished
(over 80%) throughout the brain as a result of depleted vesicular stores (Mooslehner et al., 2001;
Caudle et al., 2007; Taylor et al., 2014). Furthermore, the reduction in striatal dopamine content
is age-dependent with 6 and 12 month old animals showing progressively lower dopamine levels
than 2 month old VMAT2-kd mice (Caudle et al., 2007). Reduced monoamine tissue content was
accompanied by increased monoamine turnover as indicated by higher DOPAC/dopamine,
HVA/dopamine, 5HIAA/5HT (serotonin) and DHPG/norepinephrine ratios in various brain
regions (striatum, cortex, hippocampus), similar to VMAT2-KO and VMAT2-het mice
(Mooslehner et al., 2001; Caudle et al., 2007; Taylor et al., 2009, 2014). In comparison to WT
animals, stimulated dopamine release was also significantly lower in VMAT2-kd mice implying
that deficient vesicular filling results in smaller quantal release of neurotransmitters that can
dampen extracellular levels (Lohr et al., 2016). As a mechanism to compensate for reduced
dopaminergic tone, VMAT2-kd mice show enhanced TH activity to upregulate dopamine
synthesis (Caudle et al., 2007).
Presynaptically, a severe reduction in vesicular storage is expected to result in accumulation of
dopamine in the cytoplasm, which can produce negative consequences for the cell. When the
integrity of dopaminergic neurons was assessed in VMAT2-kd mice lacking the α-synuclein
gene, no evidence of dopamine cell loss was found (Mooslehner et al., 2001). However,
60
VMAT2-deficient mice with normal α-synuclein expression showed progressive nigrostriatal
degeneration (Caudle et al., 2007). Specifically, in comparison to WT mice, TH-positive cells of
the SN were reduced by 12% and 26% in 18 and 24-month old VMAT2-kd mice, respectively
(Caudle et al., 2007). However, neighboring dopamine neurons of the VTA were spared, as seen
in Parkinson’s disease (Taylor et al., 2014). This suggests that cytosolic dopamine in
combination with the presence of α-synuclein can exacerbate dopaminergic toxicity in the SN.
Previous studies have shown that cytosolic dopamine can undergo oxidative modifications to
interact with α-synuclein and stabilize the protofibril form of the protein which is neurotoxic
(Conway et al., 2001). Indeed, aged VMAT2-kd mice show pathological accumulation of α-
synuclein in the SN, which is a hallmark of Lewy body pathology in Parkinson’s disease (Caudle
et al., 2007). Notably, markers of dopaminergic oxidative stress, cysteinyl-DOPA and cysteinyl-
DOPAC are also increased in the striatum of VMAT2-kd mice prior to the onset of dopaminergic
neurodegeneration. Taken together, these results demonstrate that vesicular deficiency can cause
dopamine to buildup in the cytosolic space where it produces oxidative damage and eventually
leads to loss of nigrostriatal dopamine cells. Behaviorally, VMAT2-kd mice show deficits in
novelty-induced locomotion that are reversed by L-DOPA, the precursor of dopamine and
principal treatment for Parkinson’s disease. Hence, this establishes that the motor deficiency in
VMAT2-kd mice is due to reduced dopaminergic tone. Aside from basal dopaminergic toxicity,
these mice are also particularly sensitive to the effects of the neurotoxin, MPTP. In particular,
MPTP treatment produces 1) decreases in DAT levels, a marker of dopaminergic nerve terminals
in the striatum and 2) loss of TH-immunopositive cells of the SNpc (Mooslehner et al., 2001;
Lohr et al., 2016). MPTP-induced damage is exacerbated in VMAT2-kd mice compared to their
WT littermates, showing that reduced VMAT2 expression enhances the vulnerability of
dopaminergic cells to toxicant insult, as demonstrated by both VMAT2-kd and VMAT2-het
mice. Thus, VMAT2 confers cellular protection by sequestering reactive cytosolic dopamine and
exogenous toxins into vesicular compartments.
Since dopamine is not the only monoamine transported by VMAT2, VMAT2-kd mice also
display phenotypes that relate to noradrenergic and serotonergic transmission. Preceding
nigrostriatal cell loss, these mice show progressive noradrenergic neurodegeneration in the LC
(Taylor et al., 2014). Interestingly, dopamine is generated in the cytosol of these neurons as well
since it is the direct precursor of norepinephrine. In Parkinson’s disease, motor symptoms are
61
attributed to dopaminergic cell loss, while non-motor symptoms are often due to noradrenergic
cell loss. Similarly, VMAT2-kd mice also show non-motor deficits such as progressively
diminished olfactory discrimination, altered sleep latency and delayed gastrointestinal emptying
(Taylor et al., 2009). In addition, these mice also display anxiety-like and depressive behavior as
assessed on the elevated plus maze and forced swim test, respectively (Taylor et al., 2009).
These phenotypes are a reflection of disrupted serotonergic, dopaminergic and noradrenergic
transmission. In summary, given the crucial role of VMAT2 in regulating intracellular and
extracellular monoamine concentrations, reduced vesicular storage leads to a variety of
detrimental consequences in VMAT2-kd mice.
1.2.7.6 VMAT2-overexpressing mice
Taken together, the evidence from VMAT2-KO, VMAT2-het and VMAT2-kd mice highlight the
adverse effects of decreased VMAT2 levels, suggesting that enhancing VMAT2 function can
potentially be beneficial for dopaminergic cells. A BAC transgenic approach was used to
generate mice that over-express VMAT2 in monoaminergic cells (VMAT2-OE) (Lohr et al.,
2014). Physiologically, VMAT2-OE mice seem healthy and have normal body weight. As
expected, these animals display increased VMAT2 expression in the nigrostriatal dopaminergic
pathway, as well as serotonergic and noradrenergic cell bodies. Genomic quantitative PCR
results suggest that VMAT2-OE mice have incorporated 3 copies of the BAC to possess a total
of 5 copies of the VMAT2 gene. This translates to increases in VMAT2 mRNA (3.5 fold) and
VMAT2 protein in striatal homogenates (3-fold) and vesicular fractions (3-fold). Functionally,
VMAT2-OE mice display 2-fold higher vesicular dopamine uptake, 56% larger maximal
vesicular capacity for dopamine and 33% greater dopamine vesicle volume in comparison to WT
mice. These results demonstrate that increased VMAT2 expression is capable of inducing
functional changes in dopamine vesicular transport and storage. Since the majority of dopamine
in the brain is stored within vesicles, greater vesicular volume in VMAT2-OE mice is reflected
in a 21% higher dopamine tissue content in the striatum, which is an indication of presynaptic
dopamine stores. Moreover, enhanced vesicular efficiency also promotes dopamine
neurotransmission in the striatum as evidenced by increased stimulated dopamine release (84%)
in slices and greater extracellular dopamine levels (44%). Interestingly, other dopaminergic
markers such as striatal DAT and TH protein expression, number of TH-positive cells in the
midbrain and dopamine metabolite levels, remain unchanged in these mice, suggesting absence
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of major compensatory modifications to the dopamine system. Behaviorally, these mice show
41% increased locomotor activity selectively in the active, dark cycle, indicating that elevated
vesicular filling of dopamine can enhance motor behavior. Regarding tests of anxiety-like
behaviors, VMAT2-OE mice portray no changes on the elevated plus maze, however they depict
reduced basal anxiety on the marble burying task. In addition, these animals show reduced
depressive-like behavior on the forced swim task in comparison to WT mice. In combination,
VMAT2-OE mice portray improved outcomes on motor, anxiety and depression measures,
unlike VMAT2-kd mice. Lastly, VMAT2-OE mice are also relatively resistant to MPTP-induced
neurotoxicity since they showed smaller decreases in 1) TH and DAT protein expression in the
striatum and 2) TH+ positive cell bodies in the SNpc, when compared to WT mice (Lohr et al.,
2014, 2016). Thus, upregulation of VMAT2 protects dopaminergic cells from toxicant insult
while reduced transporter expression is damaging as shown by VMAT2-het and VMAT2-kd
mice. Together, the spectrum of mouse models with altered VMAT2 expression summarize the
significance of vesicular storage in preserving the health of vulnerable nigrostriatal dopamine
neurons and maintaining appropriate neurotransmission for monoaminergic behaviors.
1.3 Rationale, Hypothesis and Aims
As discussed, nigrostriatal dopamine neurons are inherently susceptible to a wide variety of
insults. A cell-specific risk factor that may contribute to their intrinsic vulnerability is the highly
reactive cytosolic neurotransmitter, dopamine. Studies show that buildup of dopamine in the
cytosol can produce oxidative stress and deleterious consequences for the cell. However, these
studies typically apply exogenous concentrations of dopamine or use non-dopaminergic systems
that are not equipped to handle the neurotransmitter. To address these limitations, we propose to
alter endogenous dopamine compartmentalization in genetically modified mice and investigate
its effects on dopaminergic function. In particular, by enhancing dopamine uptake through DAT
over-expression (Project 1) and reducing dopamine storage through VMAT2 knockdown
(Project 2), we aim to increase cytosolic dopamine levels and investigate its outcomes on
dopamine homeostasis, dopamine-related behaviors and response to dopaminergic drugs and
toxicants. Based on previous findings indicating the toxic potential of cytosolic dopamine
reactivity, our hypothesis is as follows:
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Hypothesis: Genetic manipulations of transporter expression that potentially increase
cytosolic dopamine levels, will lead to dopaminergic toxicity (e.g. loss of dopamine cells,
altered dopamine homeostasis, oxidative stress, poor motor behavior).
This thesis has been separated into two projects:
Project 1. Aim: To evaluate the consequences of dopamine transporter (DAT) over-
expression on the dopamine system of transgenic mice (DAT-tg) and assess their response
to MPTP
Previous characterization of DAT-tg mice reveals enhanced uptake of dopamine and 36%
reduction in the number of midbrain dopamine neurons. In this Project, we extended previous
findings by investigating: markers of oxidative stress (as a potential mechanism of dopaminergic
neurodegeneration), level of dopamine metabolism and motor behavior of DAT-tg mice. In
addition, their sensitivity to exogenous insult was also evaluated by administering a Parkinson’s
disease-inducing toxicant, MPTP.
Project 2. Aim: To investigate the dual effect of DAT over-expression and VMAT2 knock-
down on the dopamine system of genetically modified mice
Adult DAT-tg mice display a 36% loss of dopamine neurons while aged (24 month old)
VMAT2-kd mice also show comparable (26%) loss of nigrostriatal dopamine neurons. In this
project, DAT-tg and VMAT2-kd mice were intercrossed to generate animals that would
potentially accumulate more cytosolic dopamine and display greater toxicity than either
genotype alone. Characterization of these mice included assessments of overall fitness,
presynaptic and postsynaptic markers of the dopamine system, baseline behaviors and response
to dopaminergic drugs.
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Chapter 2
Materials and Methods
Materials and Methods
The following sections include all methods used for Projects 1 and 2. Some sections may apply
exclusively to one project.
2.1 Mice
2.1.1 Generation of DAT-tg mice (Project 1)
Generation of DAT-tg mice using BAC transgenesis has been described in Salahpour et al.,
2008. Briefly, transgenic animals were created by pronuclear injection of a BAC containing the
DAT locus and 80kb of upstream and downstream genomic sequences. This approach was used
since the promotor region of the DAT gene was not well characterized. The DNA was isolated
from the BAC (obtained from Genome Sciences) and injected in pronuclei of C57BL/6J embryos
at the Duke Transgenic Mouse Facility. Once a positive transgene founder was identified using
PCR-based genotyping, it was further bred to generate the mouse colony. For most experiments,
adult (3-5 months old) DAT-tg mice and their wild-type (WT) littermates were used, unless
otherwise specified. Animals were age and sex-matched across groups. Animals were provided
food and water ad libitum and maintained on a 12-hour light-dark cycle. Experiments were
conducted in accordance with the Canadian Council on Animal Care and approved by the
Faculty of Medicine Animal Care Committee at the University of Toronto.
2.1.2 Generation of DAT-tg/VMAT2-kd mice (Project 2)
DAT-tg/VMAT2-kd double transgenic mice were generated by interbreeding DAT-tg and
VMAT2-kd mice. Generation of DAT-tg mice has been discussed above. VMAT2-kd mice were
generously donated from our collaborator, Dr. Miller at Emory University. VMAT2-kd animals
were generated using gene targeting as previously described (Caudle et al., 2007). Briefly,
insertion of the neomycin cassette in the third intron of the VMAT2 gene results in hypomorphic
mice that only show 5% of normal VMAT2 protein expression (Caudle et al., 2007). Initial
generation of these mice occurred on an inbred strain of C57BL/6 mice that was later found to be
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lacking the α-synuclein gene locus (Mooslehner et al., 2001; Specht and Schoepfer, 2001).
Subsequently, the Miller laboratory at Emory University strategically bred mice that were
heterozygous for the α-synuclein and VMAT2 genes to eliminate all traces of the α-synuclein
mutation and generate VMAT2-kd mice on a normal α-synuclein background (Caudle et al.,
2007). These VMAT2-kd mice were maintained on a C57BL/6 and 129SV mixed genetic
background. In particular, DAT tg/VMAT2-kd mice were produced from two rounds of cross
breeding. First DAT-tg mice (normal VMAT2) were crossed with VMAT2-kd (VMAT2 -/-)
animals to produce DAT-tg/VMAT2-heterozygous mice (DAT-tg/VMAT2 +/-). These DAT-
tg/VMAT2 +/- mice were then crossed with VMAT2 +/- (normal DAT, DAT-ntg) animals
producing 6 possible genotypes:
DAT-ntg/ VMAT2 +/+ (WT, 12.5%), DAT-ntg/ VMAT2 -/- (VMAT2-kd, 12.5%), DAT-tg/
VMAT+/+ (DAT-tg, 12.5%), DAT-tg/VMAT2 -/- (DAT-tg/VMAT2-kd, 12.5%), DAT-
tg/VMAT2 +/- (25%) and DAT-ntg/VMAT2+/- (25%) mice.
This breeding strategy yields the 4 necessary genotypes that are used for experiments: WT,
DAT-tg, VMAT2-kd and DAT-tg/VMAT2-kd, indicating that littermates can serve as
experimental controls. However, the probability of obtaining an animal of each genotype is only
1/8 or 12.5%. Collectively these mice are referred to as the DAT VMAT2 colony. Since the
original VMAT2-kd mice were on a mixed background, mice were back-crossed to C57BL/6J
for several generations to produce a mouse colony exclusively on the C57BL/6 background.
For most experiments, adult (2-4 month old) DAT VMAT2 mice were used, unless otherwise
specified. Animals were age and sex-matched across groups. Animals were provided food and
water ad libitum and maintained on a 12-hour light-dark cycle. Experiments were conducted in
accordance with the Canadian Council on Animal Care and approved by the Faculty of Medicine
Animal Care Committee at the University of Toronto.
2.1.3 Body weight
Adult DAT VMAT2 mice (2 to 4 months old) were weighed from all 4 genotypes: WT, DAT-tg,
VMAT2-kd and DAT-tg/VMAT2-kd. The average age of animals from each genotype was
calculated and matched across groups. Results were stratified according to sex since male mice
tend to be larger than female mice.
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2.1.4 Survival
Kaplan-Meier survival curves were generated using retrospective data from DAT VMAT2
animals born between 2012 and 2015. Survival data were assimilated from mice that were
naturally found dead and mice that were intentionally sacrificed to conduct experiments. The age
at which the animal was found dead or was sacrificed was noted. The analysis focused on the
time frame between birth and 12 weeks of age, after which adult mice are usually sacrificed for
experiments. Results were shown for both sexes combined and for each sex separately. Results
were tabulated and analysed using GraphPad Prism 6.
2.2 Biochemistry
2.2.1 Western blots
Western blots were used to quantify expression of various proteins in striatal tissue. Western
blots were performed as previously described (Masoud et al., 2015). The striatum was dissected
and tissue was mechanically homogenized in RIPA buffer with protease inhibitors. For most
proteins, samples were centrifuged at 15,000 rpm for 15 minutes and the supernatant was used to
analyze protein concentration (BCA protein assay, Pierce). However, for VMAT2 and its
corresponding loading control, GAPDH, striatal tissue was mechanically homogenized in
320mM sucrose, 5mM HEPES buffer with protease inhibitors. Homogenized samples were
centrifuged at 3500rpm for 5 minutes and the supernatant was again centrifuged at 14,000rpm
for 1 hour. The pellet was resuspended in homogenization buffer and used to analyze protein
concentration (BCA protein assay, Pierce).
Protein extracts (20-30ug) were separated by 8.5 - 10% SDS/PAGE and transferred onto
polyvinylidene difluoride (PVDF) membranes. Nonspecific binding was blocked using either 5-
7.5% milk, 5% BSA (specifically Na/K ATPase) or Rockland blocking buffer (specifically for
DAT). Immunoblots were incubated overnight at 4°C with the following primary antibodies: rat
anti-DAT (1:750, Millipore), rabbit anti-TH (1:3000, Millipore), rabbit anti-VMAT2 (1:20,000,
obtained from Miller lab, Lohr et al., 2014), mouse anti-manganese superoxide dismutase
(MnSOD, 1:1000, BD Transduction), goat anti-MAOb (1:1000, Santa Cruz), mouse anti-
GAPDH (1:4000, Sigma), mouse anti-α-tubulin (1:2000, Hybridoma Bank) and rabbit anti-
sodium/potassium ATPase (Na/K ATPase, 1:2000, Cell Signaling). Species appropriate
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secondary antibodies (1:5000, Alexa Fluor 680 or IRDye 800CW, Rockland) were used and
blots were developed using the LI-COR Odyssey Imaging System (LI-COR). Densitometric
analysis of protein bands were performed using Image-J software (National Institutes of Health).
Immunoblots of loading controls (GAPDH, α-tubulin, Na/K ATPase) were used to normalize
protein loading across samples.
Protein carbonyl and 3-nitrotyrosine levels were also evaluated in DAT-tg mice (Project 1) using
western blots. The striatum was dissected and synaptic plasma membrane (SPM) fractions were
prepared at 4oC using protease inhibitors according to Salahpour et al., 2008. Briefly, striata from
3-4 mice were combined, homogenized in 4mM HEPES /0.32M sucrose buffer (pH7.4) and
centrifuged at 900 x g. The resulting supernatant was centrifuged at 10,000 × g. The pellet was
resuspended in 0.32 M sucrose/HEPES and lysed with water. Membranes were layered on a
discontinuous sucrose gradient, ultracentrifuged at 200,000 x g (2hrs), and the 1.2M sucrose
interphase was collected. The SPM fraction was added to 0.32M sucrose, centrifuged at 200,000
x g (30min), and the pellet was resuspended in 50mM HEPES/2mM EDTA solution. Protein
concentration was determined using the BCA Protein Assay (Thermo Scientific). For protein
carbonyl detection, the SPM samples (20ug) were further derivatized to 2,4-
dinitrophenylhydrazone (DNP) by reaction with 2,4-dinitrophenylhydrazine (DNPH) according
to the Oxyblot Protein Oxidation Detection Kit (Millipore). Western blots were used to quantify
both DNP levels (from SPM derivatized samples) and 3-nitrotyrosine levels (from SPM
samples). Proteins were separated by 10% SDS/PAGE and transferred onto PVDF membranes.
Membranes were incubated with primary antibodies (rabbit anti-DNP, 1:300, Millipore or mouse
anti-3-nitrotyrosine, 1:350, Abcam) and corresponding secondary antibodies (1:5000, Rockland).
Immunostaining was developed using the LI-COR and quantified using Image-J. DNP levels
were used as a measure of protein carbonylation.
2.2.2 Quantitative reverse transcriptase PCR
Quantitative reverse transcriptase PCR was used to determine mRNA expression of DAT and
VMAT2 in DAT VMAT2 mice. Since these transporters are dopaminergic markers, mRNA was
isolated from the midbrain which contains dopamine cell bodies. Brain regions were
microdissected and homogenized in Tri-Reagent (BioShop) to isolate RNA. RNA isolation steps
were performed as previously described (Rio et al., 2010). Briefly, homogenates are centrifuged and
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chloroform is added to isolate RNA in the aqueous phase. The collected RNA phase is concentrated
in a pellet and dissolved in DEPC water. RNA concentration was measured using optical density
readings (260/280 nm). cDNA was constructed from RNA samples using SuperScript III Reverse
Transcriptase according to manufacturer’s protocol (Invitrogen). Primer sets were generated for
each gene of interest (including a housekeeping gene, phosphoglycerate kinase 1, PGK1) and
verified for the presence of target transcripts using PCR. Finally, quantitative PCR was
performed using sample cDNA, primers, SYBR Green Dye (Invitrogen) and the Applied
Biosystems 7500 Real-Time PCR System. Relative expression of target genes was quantified
using the ΔΔCt method (as described in Livak & Schmittgen, 2001) and normalized to PGK1 levels.
Final results were reported as a ratio of WT expression.
2.2.3 Immunohistochemistry
Mice were anesthetized and intra-cardially perfused with 4% paraformaldehyde. Brains were
removed, stored in 30% sucrose for at least 24 hours (cryoprotection) and sectioned to 50µm
coronal sections using a Leica cryostat. Striatal sections were 1) quenched using 0.5% sodium
borohydride, 2) rinsed, 3) blocked using 10% normal goat serum, 3% fish gelatin and 0.1%
Triton X-100 and 4) incubated with primary rabbit anti-TH antibody (1:500, Millipore)
overnight. Sections were then rinsed and incubated with the appropriate anti-rabbit secondary
antibody for 1 hour (IRdye 800 or AF680 1:5000, Rockland Inc.). Sections were mounted on to
slides and cover-slipped. Immunofluorescence was visualized using the LI-COR Odyssey
Imaging System (LI-COR).
2.3 Neurochemistry
2.3.1 High performance liquid chromatography (HPLC)
HPLC with electrochemical detection (HPLC-EC) was used to measure dopamine, DOPAC and
HVA levels in striatal tissue. Dissected striata were homogenized in 0.1M perchloric acid and
centrifuged (9,400 x g for 10 min at 4oC). The supernatant was filtered through a 0.22µm
membrane (Millipore). Samples were analyzed using a Hypersil Gold C18 column (150 x 3mm;
5µm; Thermo Scientific) and a LC-4C Amperometric Detector (BASi) set at an oxidizing
potential of +0.75V. The mobile phase contained 24mM Na2HPO4, 3.6mM 1-octanesulfonic
acid, 30mM citric acid, 0.14mM EDTA in 19% methanol, adjusted to pH 4.7 using concentrated
NaOH. After the column was equilibrated with the mobile phase, appropriate electrochemical
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separation of the following compounds was confirmed using standard solutions: dopamine,
DOPAC, HVA, serotonin, 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of serotonin, and
2,3-dihydroxybenzoic acid (DHBA), an internal standard that was added to every sample.
Calibration curves were generated using increasing concentrations of dopamine, DOPAC, HVA
and DHBA for quantification of these chemicals in brain tissue. Area under the curve was used
to estimate concentration. Dopamine, DOPAC and HVA tissue content was normalized to
DHBA levels. Metabolite-to-dopamine ratios were calculated by dividing metabolite tissue
concentration by the tissue levels of dopamine for each animal.
5-S-Cysteinyl-dopamine and 5-S-cysteinyl-DOPAC were measured in collaboration with the
Richardson lab. Since sensitivity has been an issue for HPLC analysis of cysteinyl adducts, we
designed two positive controls that were expected to demonstrate enhancement of cysteinyl
modified products. First, WT mice were treated with 5mg/kg of reserpine (i.p.), a VMAT2
inhibitor, and sacrificed 16 hours later when brain tissues were harvested. Other groups have
shown that this reserpine regimen increases formation of cysteinyl DA by 135% (Fornstedt and
Carlsson, 1989; Hatcher et al., 2007). Second, Caudle et al. report that VMAT2-kd mice display
increased levels of cysteinyl L-DOPA and cysteinyl DOPAC, therefore, they were used as a
second positive control (Caudle et al., 2007). Striatal tissue was dissected from positive controls,
WT and DAT-tg mice and rapidly frozen in liquid nitrogen. Frozen striatal samples were shipped
to the Richardson lab for HPLC-EC analysis that has been previously described elsewhere
(Caudle et al., 2007; Hatcher et al., 2007). Briefly, samples were sonicated in 0.1M perchloric
acid containing 347µM sodium bisulfite and 134µM EDTA. Homogenates were centrifuged,
filtered and separated on a C18 column. The electrochemical detector was set at an oxidizing
potential of +0.65V. The mobile phase was MD-TM (ESA) containing 2mM NaCl and adjusted
to pH 2.1 using concentrated HCl. Quantification of all neurochemicals was conducted by
referring to calibration curves constructed from pure standards (purity >98%; dopamine,
DOPAC, HVA and DHBA from Sigma Aldrich; 5-S-cysteinyl-dopamine and 5-S-cysteinyl-
DOPAC from NIMH Chemical Repository).
2.3.2 Fast-scan cyclic voltammetry (FSCV)
FSCV was performed in slice preparations of DAT VMAT2 mice to determine electrically-
evoked dopamine release and uptake in the dorsal striatum. These studies were conducted in
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collaboration with the Miller lab at Emory University as they possess the necessary equipment
and expertise for FSCV. We sent live animals to the Miller lab and they performed FSCV
according to previously described methods (Kile et al., 2012; Lohr et al., 2014). Briefly, mice
were anesthetized, decapitated, and coronal slices (300µm) from the striatum were cut and
maintained in cold artificial cerebral spinal fluid (pH 7.4, 95% O2 / 5% CO2). Recordings were
performed in a slice perfusion chamber at 37oC (Warner Instruments). Dopamine release was
electrically stimulated by biphasic (2 ms per phase) constant-current (350µA) pulses generated
from a tungsten-bipolar electrode on the surface of the slice. The carbon-fiber detection
microelectrode was placed 75-100µm into the slice and 100-200µm away from the stimulating
electrode. Carbon-fiber microelectrodes were calibrated with dopamine standards. For each
animal, four different sites were sampled in the dorsal striatum with 5-min intervals between
stimulations. The waveform for dopamine detection consisted of a −0.4 V holding potential
versus an Ag/AgCl reference electrode. The applied voltage ramp ranged from −0.4 V to 1.0 V.
Dopamine release and uptake measures were extracted using nonlinear regression analysis. Data
were analyzed using two redundant, yet different methods. The first simplistic method
approximates “dopamine release” using the peak amplitude and “dopamine clearance” using the
decay-time constant, tau (where lower tau, measured in seconds, implies faster clearance)
(Yorgason et al., 2011). The second method, recently proposed by Hoffman and colleagues, uses
curve modeling to determine release and uptake parameters (Hoffman et al., 2016).
2.4 Stereology
Stereological counts of dopaminergic neurons in the SNpc of DAT VMAT2 mice were
performed in collaboration with 2 different laboratories (Miller and Brotchie) in 3 independent
experiments to ascertain the results. Dopamine neurons were identified with a dopaminergic
marker, TH and a neuronal marker, NeuN or Nissl. The stereological techniques used have been
described in detail previously and are briefly summarized for each collaboration (Lohr et al.,
2014, 2015; Taylor et al., 2014).
With the Miller lab at Emory University, we sent them whole brains that were transcardially
perfused with 4% paraformaldehyde and cryoprotected in sucrose. Tissue was serially sectioned
at a thickness of 40 μm (24 μm after staining/dehydrating) using a freezing sliding microtome.
Coronal sections were stained according to Lohr et al, 2014 using rabbit-anti TH (AB152,
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Millipore) and counterstained with 0.1% cresyl violet. Of all sections that contained the SNpc
region, every 6th section was counted using the optical fractionator method (frames were 50 x 50
μm, counting grid was 120 x120 μm) in the Stereo Investigator software (MicroBrightField,
Colchester, VT). This method is not affected by changes in the volume of the structure sampled.
Boundaries of the SNpc were outlined under magnification of the 4× objective with reference to
a mouse brain atlas (Franklin and Paxinos, 2012). Stereological counts were performed under a
40× objective using guard zones of 2 μm. Immunoreactive neurons were only counted if the
recognizable profile came into focus within the counting frame. This method certifies a uniform,
random and systematic cell count (Gundersen coefficients of error were less than 0.1).
With the Brotchie lab at Toronto Western Research Institute, we sent them perfused and
sectioned tissue for staining and analysis. The mouse brain was perfused with 0.9% saline and
heparin to clear out any blood in the tissue. The brain was then fixed with 4% paraformaldehyde
and cryoprotected using serial sucrose concentration solutions (15% and 30%). Relevant tissue
from the midbrain was sectioned using the cryostat (40 μm thickness) and collected in antifreeze
solution. To perform immunohistochemical staining, endogenous peroxidase activity of the
tissue was quenched and non-specific binding sites were blocked. Primary antibodies used:
mouse anti-NeuN (Chemicon International, MAB377, 1:1000) and rabbit anti-TH (Chemicon
International, AB152, 1:2000). Appropriate secondary antibodies were used. Vector DAB was
used to stain NeuN positive neurons and Vector Blue alkaline phosphatase was used to
immunolabel TH positive cells. Sections were mounted, clarified and cover-slipped for
stereological counting using the StereoInvestigator software on equipment configured by
MicroBrightField. For each brain, every 4th section from the SNpc was counted, producing a
total of 6 sections/brain. TH and NeuN cells were counted simultaneously in brightfield. For
each mouse, SNpc boundaries were delineated by closely tracing around the region of TH+ cells
and excluding the SNpr and VTA, according to a mouse brain atlas (Franklin and Paxinos,
2012). Counting parameters were as follows: guard zone 2 μm, dissector height (Z) 20 μm,
counting frame 175 x 175 μm, sampling grid 275 x 275 μm, section evaluation interval 4. The
right SNpc was counted for every animal.
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2.5 Radioligand binding
Radioligand binding was used to assess D1 and D2 receptor levels in the striatum as described
previously (Ghisi et al., 2009). Striatal tissues were rapidly dissected and homogenized in lysis
buffer (50 mM Tris–HCl (pH 7.4), 120mMNaCl, 1mMEDTA) containing protease inhibitors.
The homogenate was centrifuged (1000 rpm for 10 min at 4 °C) to remove nuclei. The resulting
supernatant was centrifuged (40,000 g for 20 min at 4 °C), the pellet was resuspended in lysis
buffer and centrifuged again under the same conditions. The final pellet was resuspended in
assay buffer (50 mM Tris–HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM
MgCl2). Protein concentration of membranes was determined using BCA protein assay (Pierce).
For D1 receptor saturation experiments, prepared striatal membranes (1.2 μg/μl, 50 μl) were
incubated with [3H]-SCH23390, a D1 receptor antagonist (50 μl, 16 nM) and ketanserin (100
nM, 50 μl), a serotonin receptor antagonist, to prevent radioligand binding to these receptors.
This reaction was performed in assay buffer (200 μl) at room temperature for 1 hour. In parallel
reactions, nonspecific binding was measured using non-radiolabeled flupenthixol (10 μM), a
dopamine receptor antagonist. For D2 receptor saturation experiments, prepared striatal
membranes (0.5 μg/μl, 150 μl) were incubated with [3H]-spiperone, a D2 receptor antagonist (50
μl, 3 nM). This reaction was performed in assay buffer (250 μl) at room temperature for 2 hours.
In parallel reactions, nonspecific binding was measured using non-radiolabeled haloperidol (6
μM), a D2 antagonist.
All reactions were terminated by filtration over Brandel GF/C glass fiber filters and washing
with cold assay buffer. Filters were incubated overnight in high flash point scintillation cocktail
(5 ml, Lefko-Fluor). Radioactivity was counted using a liquid scintillation counter. Counts of
non-specific binding were subtracted from total binding to obtain specific [3H]-SCH23390 or
[3H]- Spiperone binding, which corresponds to D1 or D2 binding, respectively. Radioactivity
counts were converted to fmol/mg tissue for final results.
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2.6 Behavioral Assessments
2.6.1 Open field locomotor activity
Baseline motor behavior of untreated animals was assessed using open field activity chambers.
Open-field locomotor activity and stereotypy were measured using the VersaMax Animal
Activity Monitoring System (Omnitech Electronics). Mice were placed in acrylic chambers
(20cm x 20cm x 45cm) and infrared light sensors were used to track movement. Dim light is
maintained throughout testing and external noise is minimized. Locomotor activity was
measured as: distance traveled (measured in cm), number of horizontal movements (discrete
movements must be separated by at least 1 second), horizontal activity (number of beam
interruptions that occur in the vertical sensor) and vertical activity (number of beam interruptions
that occur in the vertical sensor). Stereotypic behavior is defined as repetitive movements such as
grooming, head bobbing etc. The software identifies stereotypic behavior when an animal breaks
the same beam or set of beams repeatedly. Stereotypy was measured as stereotypy count
(number of repetitive beam breaks) and stereotypy number (number of times the monitor
observes stereotypic behavior in an animal, a break of one second or more is required to separate
stereotypic episodes). These parameters are recorded in five minute intervals over a two-hour
period to assess baseline behavior. Data are presented in two ways: 1) in 5 minute increments to
display behavior over time or 2) as a sum of the 2-hour period to represent total activity.
Aside from baseline measures, open field activity is also assessed in response to drug treatment.
For these experiments, first the animal is allowed to habituate to the chamber while activity is
monitored typically for 60 minutes, unless otherwise denoted. Then, the animal is removed from
the activity chamber and injected with the drug of choice. The animal is returned to the activity
chamber immediately following injection and activity is monitored for another 90 minutes.
When data parameters are analyzed over time, the entire 150 minutes of monitoring is shown
(60-minute habituation plus 90 minutes post-injection), to gauge the animal’s behavior before
and after drug administration. However, to measure total activity (e.g. total distance traveled),
only the 90-minute period following drug injection is summed to demonstrate drug-induced
effects on behavior.
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2.6.2 Wire-hang test
The wire-hang test, an assessment of motor strength, was conducted by placing a mouse on a
wire cage lid and shaking the lid slightly to make the animal grip the wires. Then the lid was
inverted and suspended above a clean cage containing bedding. The latency of the mouse to fall
off the grid was measured. Trials were stopped if the mouse remained on the lid for over 10
minutes. Average values were calculated from two trials (at least 15 minutes apart).
Figure 2-1. Wire-hang test apparatus.
Mice are suspended on a wire lid above an open clean cage containing bedding. Latency of mice
to let go of the wire lid and fall into the cage below is recorded. Image adapted from Stanford
Medicine, 2016.
2.6.3 Challenging beam traversal task
The challenging beam traversal is a test of fine motor skill. It was conducted in bright light
according to the method described by Fleming et al., 2004. Animals were trained to traverse the
length of a Plexiglas beam consisting of four sections (25 cm each, 1 m total length) decreasing
in width from 3.5 cm to 0.5 cm by 1 cm increments. Mice were trained for two days (3 trials
each) to traverse the beam that led to the animal's home cage. On the third day (test day), a mesh
grid (1 cm squares) of corresponding width was placed over the beam surface. A space of
approximately 1 cm separated the grid from the surface of the beam. Animals were videotaped
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while traversing the grid-surfaced beam over three trials, separated by at least 5 minutes. For
each trial, animals were allowed a maximum of 5 minutes to traverse the beam. Video files were
recorded using a camcorder for later manual scoring. Scoring was performed blind. The video
was viewed frame-by-frame to detect errors defined as paw slips through the mesh-grid and paws
placed on the side rather than the top of the grid during forward motion. Other parameters
recorded include number of steps to cross the beam and latency to traverse the beam. Number of
errors, number of steps, errors per step and time to traverse the beam were quantified per trial
and averaged over three trials for each animal.
Figure 2-2. Challenging beam traversal task.
(A) Side view of apparatus on test day (Day 3) showing beam and mesh grid on top.
Representative frames from video recordings demonstrating errors (denoted by arrows) such as
(B) front paw slip, (C) rear paw placed on the side of the grid rather than the surface and (D)
both paws misplaced. Images taken by Laura Vecchio at Salahpour lab, University of Toronto.
For Project 1, the effect of L-DOPA treatment on fine motor ability was also evaluated using the
challenging beam traversal task. In this case, animals were trained for 2 days without any drug
treatment. On the test day (Day 3), animals were treated with 12.5 mg/kg of benserazide (i.p.),
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followed 20 minutes later by 25 mg/kg of L-DOPA (i.p.). In the control group, animals received
two 0.9% saline injections separated by 20 minutes. Testing on the challenging beam began 10
minutes after the second injection. This treatment regimen has previously been used to assess L-
DOPA effects on challenging beam motor behavior (Hwang et al., 2005). Data were analyzed as
mentioned before.
2.6.4 Puzzle box
The puzzle box strives to assess executive functions in rodents (Ben Abdallah et al., 2011).
Using different tasks, the puzzle box evaluates multiple aspects of cognition including problem-
solving, short- and long-term memory. The puzzle box is divided into 2 compartments: a large,
brightly-lit start box (58 x 28 x 27.5 cm) and a smaller, dark, enclosed goal box (14 x 28 x 27.5
cm), that are connected by a door and underpass.
Figure 2-3. Puzzle box apparatus.
Images showing top view of the puzzle box which is divided into 2 compartments (start box and
goal box) that are connected by an underpass. Image adapted from Ben Abdallah et al., 2011.
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Since mice are comfortable in dark, enclosed spaces, when they are first introduced to the bright
start box, they develop a preference to “escape” to the dark goal box. Over 9 trials, mice are
required to complete escape tasks of increasing difficulty to reach the goal box within a limited
amount of time. The paradigm consists of 3 tasks per day for 3 consecutive days (total of 9 tasks,
T1-9), during which mice are trained to solve a puzzle by removing obstructions at the underpass
to reach the goal box. Between each trial (on the same day), the mouse is returned to its home
cage for two minutes. A brief description of the tasks is shown in the table below.
Table 2-1. Description of tasks on the puzzle box test.
For each task, latency to escape to the goal box is recorded. The timer is stopped when the two
hind legs of the mouse have entered the goal box. Each task lasts for a maximum of 300 seconds
(5 minutes) and mice that do not complete the task within that time frame are assigned the
maximum score (300 seconds). For all tasks, shorter latency to escape indicates better
performance. Mice that cannot complete the first task on Day 1 (T1) are eliminated from the
study.
2.6.5 Elevated plus maze
The elevated plus maze is used to measure anxiety-like behavior in rodents. The maze consists of
4 arms (each 30.5 cm long, 5.0 cm wide) connected by a central zone. Two opposite arms are
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open (no walls) while the other two opposite arms are enclosed with walls (15.25 cm high).
Typically, animals prefer to remain in the dim enclosed arms, hence, exploration of open arms is
thought to represent reduced anxiety. This test was performed under 2 light conditions: 1) dim
light (15-16 lux) and 2) ambient light (210–240 lux), using different groups of mice. At the
beginning of the test, mice are placed in the center zone, facing an open arm. The test lasts for 8
minutes, during which mice explore the maze. Biobserve Viewer3 software was used to track
animal movement and the amount of time spent in each arm. Data are reported as percent of total
time spent in 1) open arms and 2) closed arms. Total time is defined as time spent in all arms as
well as the center zone. The software also estimates distance traveled during the test using video
tracking.
Figure 2-4. Schematic image of elevated plus maze.
Image shows side view of elevated plus maze (2 open and 2 closed arms). Reproduced from
Stoelting Co., 2017.
2.6.6 Abnormal Involuntary Movements Scale
The Abnormal Involuntary Movement Scale (AIMS) is used to assess drug-induced dyskinesias.
We used this scale to evaluate the effects of 2mg/kg of amphetamine in DAT VMAT2 mice.
Animals were placed in an acrylic box and allowed to habituate to the environment for 60 mins.
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Then they were removed from the box, injected with amphetamine (2 mg/kg) and returned to the
box. Their activity was monitored for 75 minutes following injection. In particular, the behavior
of each animal was video-recorded for 1 minute intervals every 15 minutes. The first recording
was taken right before drug injection to obtain a baseline measure. A total of 6 recordings (15
minutes apart) were taken of each animal for manual scoring. Scoring identified abnormal
movements that lacked purpose (rather than akinesia). A score was assigned for 4 individual
categories of abnormal movements: 1) locomotor (abnormal locomotion such as crouching low
while walking, full body tremors, backwards movement), axial (abnormal postures of the head
and trunk; head bobbing), limb (abnormal movements of paw; resting paw tremor, kicking out
back legs while walking), and orolingual (vacuous chewing, biting, tongue thrusting). A score of
1 to 4 was assigned for each category based on the duration of an abnormal movement in a 60
second recording (0-15 seconds = 1; 16-30 seconds = 2; 31-45 seconds = 3; and 46-60 seconds =
4). For each time point, the minimum aggregated score from all categories is 4 while the
maximum is 16. For an overall effect of the drug, the scores for each time point are summed
(minimum 24, maximum 96).
2.7 Drug treatment
2.7.1 MPTP
MPTP hydrochloride (Sigma Aldrich) was dissolved in phosphate buffered saline (PBS) and
administered (i.p. 0.1 ml/ 10g body weight) twice, 10 hours apart, at a dose of 15 or 30 mg/kg of
body weight. Animals were sacrificed after seven days and brains were harvested for
biochemical and neurochemical analyses.
2.7.2 Dopaminergic drugs
Several drugs that interact with the dopamine system (e.g. psychostimulants, dopamine receptor
agonists) were acutely injected into drug-naïve animals prior to conducting behavioral analyses.
The table below provides a summary of the dose, route of administration, dissolving vehicle and
source of each drug that was used.
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Table 2-2. List of dopaminergic drugs administered
All injections were administered at a concentration of 0.1ml per 10g of body weight. i.p.,
intraperitoneal; s.c., subcutaneous.
Drug Dose (mg/kg) Injection Vehicle Solution Source
Amphetamine 0.5, 1, 2 or 5 i.p. PBS R&D Systems Europe
Cocaine 20 i.p. 0.9% saline (NaCl) Medisca
Methylphenidate 5 i.p. PBS Tocris Bioscience
Apomorphine 2 s.c. 0.1% ascorbic acid in distilled H2O
Sigma Aldrich
SKF-81297 2 i.p. 0.9% saline (NaCl) Sigma Aldrich
L-DOPA (methyl ester) 25
i.p. 0.9% saline (NaCl) Sigma Aldrich
Benserazide (co-administered with L-DOPA)
12.5 i.p. 0.9% saline (NaCl) Sigma Aldrich
2.8 Statistics
All data are shown as mean ± SEM. Data were statistically analyzed by two tailed t-tests, one-
way ANOVA with Bonferroni post hoc tests, or two-way ANOVA with Bonferroni post hoc
tests, as appropriate. GraphPad Prism and SPSS software were used for graphs and statistical
analyses. Significance is reported at p<0.05.
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Chapter 3
Results
Results
Thesis results have been divided into two main sections depending on the mouse model that is
characterized: 1) DAT over-expressing transgenic mice and 2) mice that simultaneously over-
express DAT and under-express VMAT2.
3.1 Characterization of DAT over-expressing transgenic mice
The majority of the results shown in this section have been published (Masoud et al., 2015).
Since DAT-tg mice were first generated by Dr. Salahpour at Duke University before I began my
doctoral thesis, the following characteristics of these mice have already been summarized in two
previous publications: 1) expression and function of DAT, 2) response to psychostimulants and
3) post-synaptic dopamine receptor function (Salahpour et al., 2008; Ghisi et al., 2009).
Furthermore, prior to this thesis work, there were three interesting unpublished findings from
DAT-tg mice; these animals showed: 1) a 33% reduction in striatal dopamine tissue content, 2) a
72% decrease in electrically-evoked dopamine release from striatal slices and 3) 32-36% and 28-
30% loss of TH positive neurons in the SN and VTA, respectively. These results suggested that
DAT over-expression produces detrimental effects in midbrain dopamine neurons of DAT-tg
mice. Outlined below are the experiments conducted to expand on previous findings and provide
new results regarding underlying mechanisms, behavioral outcomes and response to toxicants.
3.1.1 Presynaptic dopamine homeostasis
The first set of experiments were designed to evaluate how dopamine is handled within the
presynaptic neuron of DAT-tg mice. Initially, the expression of DAT in the striatum of DAT-tg
mice was confirmed using western blots (Fig. 3.1). As expected, DAT protein levels were
significantly higher in DAT-tg mice in comparison to WT animals.
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Figure 3-1. DAT protein expression in the striatum of DAT-tg mice.
DAT western blot and densitometry analysis (N=3). DAT levels were corrected for loading using
Na/K ATPase and normalized to WT expression. Data shown are means ± SEM. ** p<0.01.
After confirming that transgenic animals over-express DAT, we investigated whether greater
DAT-mediated dopamine uptake may result in accumulation of dopamine within the presynaptic
neuron. Functional characterization of DAT had previously indicated faster dopamine uptake in
striatal slices from DAT-tg mice (Salahpour et al., 2008). This suggests that the neurotransmitter
could potentially buildup in the cytosolic space of the presynaptic neuron, which can produce
deleterious consequences for the cell. Since direct in vivo measurement of cytosolic dopamine is
not technically feasible, indirect parameters are used to gauge cytosolic dopamine levels. In
particular, the cytosolic fraction of dopamine is exposed to various metabolic reactions. While
dopamine tissue content in the striatum was previously measured in these animals, levels of
DOPAC and HVA, the major metabolites of dopamine, were unknown. Therefore, using HPLC-
EC (high performance liquid chromatography with electrochemical detection), we assessed
metabolite levels in relation to dopamine content, as an indicator of dopamine degradation.
DAT-tg animals showed a 60% increase in the DOPAC/dopamine ratio (Fig. 3.2A) and a 38%
increase in the HVA/dopamine ratio (Fig. 3.2B), suggesting a higher turnover of dopamine in
these animals. Furthermore, in collaboration with the Goldstein lab at the National Institutes of
Health, we assessed levels of DOPAL, a volatile and potentially toxic metabolite of dopamine, in
the striatum of DAT-tg mice (Goldstein et al., 2013). As illustrated in Figure 3.2C, DAT-tg mice
show a trend (p=0.05) towards a higher DOPAL-to-dopamine ratio in comparison to WT mice.
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Given the propensity of DOPAL to cause oxidative damage, a possible enhancement of DOPAL
content in these mice may contribute to dopaminergic toxicity. In summary, since intraneuronal
metabolism of dopamine occurs specifically in the cytosolic space, enhanced dopamine turnover
in DAT-tg mice implies buildup of cytosolic dopamine.
Ratio of (A) DOPAC-to-dopamine, (B) HVA-to-dopamine and (C) DOPAL-to-dopamine tissue
content (N=10-11). DOPAL-to-dopamine ratios were measured in collaboration with Dr.
Goldstein at NIH. Data shown are means ± SEM. **p<0.01.
Cytosolic dopamine levels are regulated by various mechanisms including plasma membrane
uptake via DAT, metabolic reactions and VMAT2-mediated vesicular storage. Since the majority
of intracellular dopamine is sequestered within vesicles and VMAT2 plays a crucial role in
maintaining low levels of cytosolic dopamine, VMAT2 protein levels were evaluated in the
striatum of DAT-tg mice. As shown in Figure 3.3, transgenic animals displayed 30% lower
VMAT2 protein levels than WT mice. While this decrease may reflect the concurrent loss of
dopaminergic neurons in DAT-tg mice, it also implies that reduced vesicular storage could
contribute to buildup of cytosolic dopamine in these animals.
Figure 3-2. Metabolite to dopamine ratios in the striatum of DAT-tg mice.
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VMAT2 western blot and densitometry analysis of striatal tissue from WT and DAT-tg mice
(N=4). VMAT2-knockdown (VMAT2-kd) samples were used as a negative control to identify
the specific VMAT2 band. VMAT2 levels were corrected for loading using GAPDH and
normalized to WT expression. Data shown are means ± SEM. **p<0.01.
Taken together, these data indicate possible accumulation of cytosolic dopamine in presynaptic
dopaminergic neurons of DAT-tg mice due to 1) increased expression of DAT, the protein
responsible for transporting extracellular dopamine into the cytosolic space, 2) increased
metabolite-to-dopamine ratios, suggesting presence of dopamine in the cytoplasm where it can
be degraded and 3) decreased expression of VMAT2, the protein responsible for sequestering
cytosolic dopamine into vesicles.
3.1.2 Markers of oxidative stress
According to previous findings, DAT-tg mice display reductions in dopamine tissue content and
electrically-evoked dopamine release in the striatum, despite greater uptake of dopamine
(Salahpour et al., 2008). Furthermore, stereological counts of midbrain dopamine neurons
corroborated these findings by revealing a concurrent loss of dopamine cells in DAT-tg mice.
Although these data demonstrated compromised integrity of dopamine neurons in transgenic
animals, potential mechanisms underlying this damage were unexplored. A large body of
literature suggests that cytosolic dopamine is highly reactive and can induce oxidative stress.
Figure 3-3. VMAT2 protein expression in the striatum of DAT-tg mice.
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Also, enhanced dopamine metabolism in DAT-tg mice can generate ROS as by-products.
Therefore, we investigated whether the spontaneous loss of dopaminergic neurons in DAT-tg
mice may be associated with oxidative damage (Graham, 1978; Hastings et al., 1996; Stokes et
al., 1999). Several markers of oxidative stress such as protein carbonylation, nitrosylation and
cysteinyl adducts were explored in addition to anti-oxidant mechanisms.
ROS can react with biological molecules such as proteins and modify their structure. Carbonyl
groups are often formed on protein side chains as a result of direct or indirect oxidative reactions
(Dalle-Donne et al., 2003). Since these carbonyl groups are relatively stable and easily
detectable, carbonylation is commonly used as a general marker of protein oxidation. The level
of protein carbonylation in the striatum of WT and DAT-tg mice was assessed using synaptic
plasma membrane preparations as they are enriched with mitochondrial membranes which
contain proteins that are particularly sensitive to oxidative damage. First, 3-5 month old (13-20
weeks) mice were used since dopaminergic neurodegeneration is evident at that age, however no
differences were detected between genotypes (Figure 3.4A). Then, we postulated that if
oxidative stress was causing dopaminergic cell loss, evidence of oxidative damage would be
expected to precede the onset of neurodegeneration. Therefore, protein carbonyl levels were
evaluated in younger mice (6-8 weeks), however, no changes were observed (Figure 3.4B).
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Figure 3-4. Protein carbonylation in the striatum of DAT-tg mice.
Western blots and quantification of protein carbonyls in synaptic plasma membrane fractions
from the striatum of WT and DAT-tg mice. Striata from 3-4 mice were pooled per sample.
Animals were (A) 13-20 weeks (3-5 months) old or (B) 6-8 weeks old. Data presented as mean ±
SEM.
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Aside from ROS, reactive nitrogen species such as peroxynitrite can also interact with proteins
and alter their structure. These radical species mediate the nitration of susceptible tyrosine
residues on proteins to produce 3-nitrotyrosine, another general biomarker of nitrosative and
oxidative damage in cells. Assessment of 3-nitrotyrosine levels in striatal synaptic plasma
membrane fractions showed no differences between WT and DAT-tg mice using western blots
(Figure 3.5A). In general, oxidative stress arises as a result of anti-oxidant mechanisms being
overwhelmed by the production of reactive oxygen and nitrogen species. Therefore, in addition
to evaluating markers of oxidative damage, anti-oxidant enzyme levels were also investigated to
gauge the overall redox environment. Specifically, we measured protein expression of
manganese superoxide dismutase (MnSOD), a key mitochondrial enzyme that detoxifies
superoxide radicals generated through respiration. In total striatal homogenates, no differences
were detected in MnSOD protein levels between WT and DAT-tg mice (Figure 3.5B).
(A) Western blot and quantification of 3-nitrotyrosine in striatal synaptic plasma membrane
fractions from WT and DAT-tg mice. Striata from 3-4 mice were pooled per sample. Adult mice
Figure 3-5. Protein nitrosylation and MnSOD levels in DAT-tg mice.
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(3-5 months old) were used. (B) Western blot and quantification of manganese superoxide
(MnSOD) protein levels in total striatal extracts from WT and DAT-tg mice (N=6-7). Data
presented as mean ± SEM.
While protein carbonylation, nitrosylation and MnSOD levels are used to judge the general level
of oxidative stress, these measures are not specific for dopaminergic cells. Since DAT over-
expression selectively affects dopaminergic neurons in DAT-tg mice, we needed a sensitive
method to specifically measure dopaminergic oxidative modifications. The formation of
cysteinyl adducts on dopamine and its metabolites result from reactions between volatile
dopamine-quinones and cysteine residues. Importantly, cysteinyl-modified dopamine, L-DOPA
and DOPAC are indicative of oxidative stress occurring particularly within dopaminergic
neurons, where these substrates are located (Graham, 1978; Fornstedt and Carlsson, 1989;
Hastings and Zigmond, 1994). Hence, we designed an HPLC protocol to electrochemically
separate cysteinyl adducts of dopamine, L-DOPA and DOPAC from other commonly found
neurochemicals in the brain. After optimizing this technique, we were able to achieve isolation of
9 neurochemicals including the afore-mentioned cysteinyl adducts using HPLC-EC as shown in
Figure 3.6A. Although this protocol was effective in measuring these chemicals in standard
solutions of known concentrations, due to the lack of adequate sensitivity, we were unable to
detect trace levels of cysteinyl adducts from brain tissue. Therefore, we collaborated with Dr.
Jason Richardson from Rutgers University as his laboratory is equipped with sensitive
electrochemical detectors that are optimized for HPLC analysis of cysteinyl adducts. We sent
him striatal tissue from 3-5 month old WT and DAT-tg mice, as well as reserpine-treated WT
mice as positive controls since reserpine administration has been shown to enhance formation of
cysteinyl adducts of dopamine (Fornstedt and Carlsson, 1989). DAT-tg mice exhibit a 35%
increase in 5-S-cysteinyl-dopamine (p< 0.05, Fig. 3.6B), in addition to a 62% increase in 5-S-
cysteinyl-DOPAC levels (p< 0.01, Fig. 3.6C). Elevated tissue content of cysteinyl-dopamine and
cysteinyl-DOPAC suggests that oxidative stress may underlie the dopaminergic cell loss
observed in these mice.
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(A) Representative HPLC traces showing 9 peaks corresponding to 9 individual chemicals
separated using HPLC-EC and eluted at distinct time points. The blue trace corresponds to a
solution containing all 9 chemicals while the black trace corresponds to a solution containing
cysteinyl L-DOPA, cysteinyl-DOPAC, cysteinyl-dopamine and the internal control, DHBA.
Although this method was appropriate for separation, it could not be used for detection in brain
tissue. HClO4, perchloric acid; DHBA, 2,3-dihydroxybenzoic acid (internal control); DA,
Figure 3-6. Cysteinyl adducts of dopamine and its metabolites in DAT-tg mice.
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dopamine; 5HIAA, 5-hydroxyindoleacetic acid; 5HT, serotonin. Quantification of (B) 5-S-
cysteinyl-dopamine and (C) 5-S-cysteinyl-DOPAC tissue content in the striatum of WT and
DAT-tg mice (N=9-10) was performed in collaboration with Dr. Richardson at Rutgers
University. Data shown are means ± SEM. *p<0.05, **p<0.01.
In summary, general markers of oxidative stress such as protein carbonylation, nitrosylation and
levels of the ubiquitous anti-oxidant enzyme, MnSOD, are unchanged in DAT-tg mice.
However, specific markers of dopaminergic oxidative stress (cysteinyl dopamine and cysteinyl
DOPAC) are significantly elevated in DAT over-expressing animals. These data suggest that the
oxidative damage in these mice specifically arises from dopaminergic cells instead of non-
dopaminergic sources. Previous experiments on DAT-tg mice demonstrate signs of
dopaminergic toxicity including reduced dopamine tissue content, reduced dopamine release and
decreased number of midbrain dopamine neurons. The concurrent presence of dopamine-specific
oxidative stress in these animals suggests that it may play a role in propagating dopaminergic
damage.
3.1.3 Motor behavior
Since the nigrostriatal dopamine pathway is heavily involved in controlling motor activity, we
assessed whether dopaminergic cell loss in DAT-tg mice had any influence on their baseline
motor behavior. First, open-field locomotion was measured for two hours and no changes were
detected in total distance traveled (Fig. 3.7A) or stereotypy (Fig. 3.7B) in DAT-tg mice. Second,
animals were assessed using the wire-hang test, a measure of muscle strength where rodents are
inverted on a wire grid and suspended above a cage until they fall off (Luk et al., 2012; Oaks et
al., 2013). A previous paper has reported that mice with dopaminergic degeneration display
deficits on the wire hang test (Luk et al., 2012) even though there were no differences in gross
locomotion. Since DAT-tg mice also do not display deficits in general locomotor activity, this
test may represent a more sensitive measure of motor coordination. As shown in Figure 3.7C,
DAT-tg mice showed 36% shorter latency to fall off the wire in comparison to their WT
counterparts (p< 0.05), demonstrating compromised motor strength.
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Figure 3-7. Motor behavior of DAT-tg mice.
(A) Total distance traveled and (B) stereotypy counts from WT and DAT-tg mice tested in open
field activity monitors for two hours (N=25-28). Stereotypy counts are defined as the number of
beam breaks detected on the infrared monitor during stereotypic behavior. (C) Average latency
of mice to fall off the wire in the wire-hang test (N=37-40). Data shown are means ± SEM.
*p<0.05.
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Third, the challenging beam traversal task was used to test fine motor skills of DAT-tg mice.
These experiments were performed by Laura Vecchio, Lien Nguyen and myself. In this task,
animals traverse a progressively narrow beam in order to reach their home cage. Any slips or
misplaced paws during traversal are scored as errors. Results are summarized as number of
errors, steps, errors per step and time to traverse the beam. This task is particularly responsive to
motor deficits that arise from nigrostriatal dopamine dysfunction (Drucker-Colín and García-
Hernández, 1991; Fleming et al., 2004). In addition to testing DAT-tg mice at baseline (saline
treatment), we also administered L-DOPA and benserazide prior to behavioral assessment. Since
L-DOPA is the precursor to dopamine, we evaluated whether replenishing dopaminergic tone in
DAT-tg mice can alter their outcomes on the challenging beam traversal task. Saline-treated
DAT-tg mice showed a 50% increase in number of errors (slips and misplaced paws) and a 47%
increase in errors per step while traversing the beam (p< 0.01, Fig. 3.8A and p< 0.01, Fig. 3.8C,
respectively). However, when treated with L-DOPA, DAT-tg animals performed significantly
better as demonstrated by decreased errors, fewer steps taken and lower errors per step in
comparison to saline-treated transgenic mice (p< 0.01, Fig. 3.8A; p< 0.05, Fig. 3.8B and p< 0.05,
Fig. 3.8C, respectively). Across all groups, there were no differences in time to traverse the beam
(Fig. 3.8D). Collectively, results from these behavioral tests indicate that although DAT-tg mice
do not show any changes in gross locomotion, they display significant deficits in fine motor
coordination. Moreover, L-DOPA-treatment can reverse these deficits, suggesting that the motor
deficiency in DAT-tg mice is due to loss of dopaminergic cells and reduced dopamine tone.
.
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Animals were injected with benserazide (12.5 mg/kg), followed 20 minutes later by L-DOPA (25
mg/kg). Control animals were injected with 0.9% saline separated by 20 minutes. Mice (N=8-13)
were tested on the challenging beam traversal task (3 trials) 10 minutes after the second
injection. (A) Number of errors (including slips and misplaced paws) made while traversing the
beam. (B) Number of steps taken to traverse beam. (C) Number of errors per step taken. (D)
Time to traverse the beam. Data shown are means ± SEM. *p<0.05, **p<0.01.
While conducting behavioral analyses on DAT-tg mice, some sex differences were noted
especially in the wire hang task. Qualitatively, female mice seemed capable of hanging on the
wire for much longer periods than male mice. To highlight this difference, the initial wire hang
data were stratified by sex as shown in Figure 3.9C. Additionally, baseline data for locomotor,
stereotypy and challenging beam traversal were also sex-stratified (see Figure 3.9A, B and D,
Figure 3-8. Challenging beam traversal task in DAT-tg mice with L-DOPA treatment.
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respectively) to provide a comprehensive overview of sex differences in these behaviors. Female
mice seemed to show greater locomotor activity than male mice regardless of genotype. In the
behavioral tests that showed significant differences between WT and DAT-tg mice (wire hang
and challenging beam), similar trends were also recapitulated in the sex-stratified data.
Figure 3-9. Baseline behaviors of DAT-tg mice stratified by sex.
(A) Total distance traveled and (B) stereotypy counts from WT and DAT-tg mice tested in open
field activity monitors for two hours (N=10-15 per sex per genotype). Stereotypy counts are
defined as the number of beam breaks detected on the infrared monitor during stereotypic
behavior. (C) Average latency of mice to fall off the wire in the wire-hang test (n= 18-21 per sex
per genotype). (D) Number of errors (including slips and misplaced paws) made while traversing
the challenging beam (N=11-12 per sex per genotype). Differences are denoted by lines
comparing two groups. Data shown are means ± SEM. * p<0.05, **p<0.01, ***p<0.001.
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3.1.4 Response to MPTP-induced dopaminergic damage
Sensitivity of DAT-tg mice to exogenous toxicant insult was investigated using MPTP, a
compound shown to cause selective damage to dopaminergic neurons in humans as well as
animal models. MPTP is converted to its toxic metabolite MPP+, which enters dopamine cells
through DAT and inhibits mitochondrial complex I, eventually causing cell death. Since DAT-tg
mice basally show evidence of dopaminergic damage, we investigated whether they would also
be more vulnerable to toxicant insult. Specifically, using two doses of MPTP, 15 and 30 mg/kg
of body weight, we assessed expression of TH, the synthetic enzyme for dopamine and marker of
dopaminergic cells in the striatum. TH protein expression was evaluated qualitatively by
immunohistochemistry (Fig. 3.10 A) and quantitatively using western blots (Fig. 3.10 B, C). At
15 mg/kg of MPTP, DAT-tg mice displayed lower TH immunofluorescence (Fig. 3.10 A) and
protein levels (p< 0.05, Fig. 3.10 C) than WT animals (Fig. 3.10 A, B). Indeed, in WT mice, this
dose of MPTP did not elicit any significant change in TH immunoreactivity (Fig. 3.10 A) or
protein levels (Fig. 3.10 B) when compared to saline treatment. At 30mg/kg of MPTP, TH
immunofluorescence was decreased in both WT and DAT-tg mice (Fig. 3.10 A) however, the
extent of reduction was greater in DAT-tg mice as quantified by western blot analysis (Fig. 3.10
B, C). In particular, TH levels were reduced by 65% in transgenic animals (p< 0.001, Fig. 3.10
C) in contrast to only 28% in WT animals (p< 0.01, Fig. 3.10 B), when compared to saline
treatment. These results demonstrate that DAT-tg mice are more vulnerable to MPTP treatment
and exhibit sensitivity at doses that do not significantly affect WT animals.
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Adult mice were treated with saline, 15 or 30 mg/kg of MPTP. (A) Immunohistochemical
analysis of tyrosine hydroxylase (TH) in the striatum of WT and DAT-tg mice treated with
saline, 15 or 30 mg/kg of MPTP. Representative TH-labeled (black) coronal sections are shown.
Western blot analysis of TH protein expression in the striatum of (B) WT and (C) DAT-tg mice
treated with saline, 15 or 30 mg/kg of MPTP (N=3-4). TH levels were corrected for loading
Figure 3-10. Effect of MPTP treatment on TH protein levels in DAT-tg mice.
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using α-tubulin and normalized to WT expression. Data shown are means ± SEM. Differences
are in comparison to saline-treated animals. * p<0.05, **p<0.01, ***p<0.001.
Next, striatal dopamine tissue content was measured to assess the integrity of dopaminergic
nerve terminals in MPTP-treated mice. At both 15 and 30 mg/kg of MPTP, the respective
reductions in dopamine tissue content were greater in DAT-tg mice compared to WT controls,
indicating that increased DAT levels exacerbate MPTP-induced neurotoxicity (15 mg/kg MPTP,
p< 0.05; 30 mg/kg MPTP, p< 0.01; Fig. 3.11). A difference in striatal dopamine content was also
detected between saline-treated WT and DAT-tg mice (p< 0.01, Fig. 3.11), corroborating the
basal reduction in dopamine tissue levels previously observed in untreated transgenic animals.
Relative striatal dopamine tissue content is shown for mice treated with saline, 15 or 30 mg/kg of
MPTP (N=7-9). Levels are represented as percent of WT saline-treated mice. Significant
differences are in comparison to WT mice at each dose. Data shown are means ± SEM. *p<0.05;
**p<0.01.
Figure 3-11. Effect of MPTP on striatal dopamine tissue content of DAT-tg mice.
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In summary, characterization of mice with increased DAT levels has revealed important changes
in presynaptic dopamine dynamics such as enhanced dopamine metabolism and reduced
VMAT2 levels, which point to the possible accumulation of cytosolic dopamine. Dopaminergic
markers of oxidative stress are also elevated in DAT-tg mice, suggesting that oxidative damage
may play a role in the loss of midbrain dopamine neurons observed in these mice. Behaviorally,
although transgenic animals display normal gross locomotion, their fine motor skills and motor
strength are compromised. Interestingly, deficits in motor coordination can be reversed with L-
DOPA treatment, implicating reduced dopaminergic tone as the culprit underlying motor
deficiencies in DAT-tg mice. Finally, transgenic animals are highly vulnerable to dopaminergic
damage induced by MPTP, indicating an important role of DAT in mediating toxicant injury.
3.2 Characterization of mice that over-express DAT and under-express VMAT2
This section encapsulates results from the second project of my doctoral thesis focusing on
transgenic mice that simultaneously over-express DAT and under-express VMAT2. These
animals were generated by crossbreeding DAT-tg and VMAT2-kd mice in a separate colony
(Caudle et al., 2007; Salahpour et al., 2008). This breeding scheme gave rise to all genotypes so
littermates can serve as controls. In this project, mice are segregated into 4 genotypes of interest:
wild-type (WT), DAT over-expression (DAT-tg), VMAT2-knockdown (VMAT2-kd) and DAT
over-expression combined with VMAT2-knockdown (DAT-tg/VMAT2-kd). Collectively, these
animals will be referred to as “DAT VMAT2” mice in this thesis. Since results are reported for
multiple genotypes and various comparisons are possible, at the end of every subsection, a
summary table is provided that outlines the specific experimental results for each genotype.
3.2.1 Confirmation of transporter levels
It should be noted that previous publications demonstrate that in DAT-tg mice, DAT over-
expression is restricted to dopaminergic neurons because expression of the transgene is guided
by the DAT promotor (Salahpour et al., 2008). On the other hand, in VMAT2-kd mice, VMAT2
expression is reduced in all monoaminergic cells (Caudle et al., 2007).
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In the first set of experiments, the intended expression of DAT and VMAT2 proteins were
assessed across the 4 genotypes of mice. DAT and VMAT2 protein levels in the striatum were
analyzed using western blots. RNA expression of the 2 transporters was determined using
quantitative reverse transcriptase PCR in the midbrain.
Striatal DAT protein levels were increased in DAT-tg and DAT-tg/VMAT2-kd mice, as
expected (Figure 3.12). The degree of DAT over-expression varies between 150-175% of WT
levels (Figure 3.12). However, in the original report on DAT-tg mice, DAT levels were increased
to 300% of WT levels (Salahpour et al., 2008). Hence, the level of DAT over-expression in DAT
VMAT2 mice is not as high as the original DAT-tg mice.
(A) Representative DAT western blot. (B) Quantification of DAT protein using densitometry.
(N=8). DAT levels were corrected for loading using Na/K ATPase and normalized to WT
expression. Data presented as mean ± SEM. Statistical comparisons are against WT mice.
*p<0.05; ***p<0.001.
Figure 3-12. DAT protein expression in the striatum.
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Striatal VMAT2 protein levels were drastically reduced in VMAT2-kd and DAT-tg/VMAT2-kd
mice, as expected (Figure 3.13). VMAT2-kd animals showed a 90% reduction in VMAT2 levels
when compared to WT, which is similar to earlier reports (Caudle et al., 2007). In the previous
characterization of DAT-tg mice, these animals showed a 25% reduction in VMAT2 protein in
comparison to WT animals (Figure 3.3), as a likely reflection of dopaminergic
neurodegeneration and/or reduced vesicular storage capacity (Masoud et al., 2015). However,
this effect was not observed in Figure 3.13 using DAT-tg mice derived from the DAT VMAT2
colony. The western blot was repeated to include a larger sample size (N=4-5, not shown) and
still no difference in VMAT2 levels was detected between WT and DAT-tg mice, indicating
disparity between original DAT-tg mice and DAT-tg animals from the DAT VMAT2 colony.
(A) VMAT2 Western blot. (B) VMAT2 protein quantification using densitometry (N=2-4).
VMAT2 levels were corrected for loading using GAPDH and normalized to WT expression.
Data presented as mean ± SEM. Statistical comparisons are against WT mice. ***p<0.001.
Next, RNA levels of these transporters were assessed in the midbrain which contains nigral
dopaminergic cell bodies. DAT mRNA was significantly reduced in the midbrain of DAT-tg and
DAT-tg/VMAT2-kd mice (Figure 3.14). Since DAT protein is over-expressed in these animals
(Figure 3.12), a reduction in mRNA levels is unexpected. Similarly, VMAT2 mRNA expression
Figure 3-13. VMAT2 protein levels in the striatum.
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is increased in VMAT2-kd and DAT-tg/VMAT2-kd mice (Figure 3.14) although these mice
show drastic reductions in VMAT2 protein levels (Figure 3.13). mRNA expression is
consistently in the opposite direction of protein levels suggesting that 1) mRNA and protein
levels do not necessarily correlate and 2) the observed changes in mRNA levels may serve as a
compensatory mechanism to normalize the genetic manipulations in these mice.
Figure 3-14. DAT and VMAT2 mRNA expression in the midbrain.
(A) DAT mRNA and (B) VMAT2 mRNA relative to wild type (N=6). qPCR results were
normalized to the housekeeping gene phosphoglycerate kinase 1 (PGK1). DAT mRNA
quantification was corroborated using 2 separate primer sets targeting different parts of the DAT
gene. Data presented as mean ± SEM. Statistical differences are in comparison to WT mice.
*p<0.05; **p<0.01; ***p<0.001.
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Table 3-1. Summary of DAT and VMAT2 expression in DAT VMAT2 mice.
Comparisons are against WT mice.
Experiment DAT-tg VMAT2-kd DAT-tg/VMAT2-kd
Transporter protein levels
DAT protein Increased 150% (not 300% like Salahpour et al, 2008)
No change Increased
VMAT2 protein No change (25% decrease Masoud et al, 2015)
Decreased Decreased
Transporter mRNA levels
DAT mRNA Decreased No change Decreased
VMAT2 mRNA No change Increased Increased
In summary, DAT-tg/VMAT2-kd mice show higher DAT and lower VMAT2 protein levels, as
expected. While protein and mRNA changes are inconsistent, proteins are the functional unit for
these transporters and they are appropriately changed in DAT-tg/VMAT2-kd mice. Of note,
DAT-tg mice do not replicate the level of DAT over-expression that was previously published,
suggesting that the phenotype of these mice may be less severe than the original DAT-tg mice.
DAT-tg mice also did not replicate decreased VMAT2 protein levels, which was a reflection of
dopamine cell loss. More results presented below support the notion of attenuated dopaminergic
toxicity in these DAT-tg mice in comparison to the original DAT-tg mice. Possible reasons for
this discrepancy include genetic background, nutrition and breeding differences which are
outlined in the Discussion.
3.2.2 Fitness
After confirming that DAT and VMAT2 protein expression was altered in DAT VMAT2 mice as
expected, we evaluated whether these animals showed any gross phenotypic changes.
Qualitatively, we had observed that DAT-tg/VMAT2-kd mice appear smaller than their
littermates and often die prematurely. Since these animals seem more fragile, they are usually
weaned at a later age and their rodent chow is routinely supplemented with peanut butter and
safflower seeds. Due to these qualitative observations, the survival and body weight of these
mice were investigated as indicators of their overall fitness.
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A survival curve was generated using retrospective data from 2012 to 2015. Data were
assimilated from 1) animals that were naturally found dead within the colony, and 2) animals that
were intentionally sacrificed to conduct experiments. The survival analysis focused on the time
frame between birth and 12 weeks of age, after which animals are usually sacrificed for
experiments. DAT-tg/VMAT2-kd mice demonstrate significantly poorer survival than the other
genotypes (Figure 3.15 A). In particular, by the end of 12 weeks, survival of DAT-tg/VMAT2-kd
mice is reduced by 46%. Furthermore, we had previously observed that more male DAT-
tg/VMAT2-kd mice were found dead within the colony, compared to their female counterparts.
Therefore, the survival data were separated by sex. Survival of male DAT-tg/VMAT2-kd mice
was significantly reduced by 67% at the end of 12 weeks (Figure 3.16 B). Female DAT-
tg/VMAT2-kd also showed a trend towards reduced survival, however, the effect did not appear
as striking as the males (Figure 3.16 C).
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Figure 3-15. Survival curve from birth to 12 weeks of age.
Kaplan-Meier curves were generated using retrospective data from animals born between 2012
and 2015. Survival curve for (A) All mice, (B) Male mice, (C) Female mice. The number of
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mice that died naturally (natural) or were sacrificed for experiments after 12 weeks (sac’d) are
shown for each genotype. In a few cases, the sex of the animal was not noted at the time of death
and therefore those cases were excluded from the sex-stratified analysis. Statistical differences
are in comparison to WT, DAT-tg and VMAT2-kd mice. ***p<0.001.
Next, body weight of 2-4 month old animals was compared across genotypes. Since male and
female mice differ in weight – these data were also stratified by sex. DAT-tg/VMAT2-kd mice
were significantly lighter than WT mice of the same sex (Figure 3.16). Male VMAT2-kd mice
were also lighter than their WT counterparts, which has been previously reported (Mooslehner et
al., 2001).
Figure 3-16. Body weight of adult mice.
(A) Male mice and (B) female mice (N=21-27 per genotype per sex). Data presented as mean ±
SEM. Statistical differences are in comparison to wild type mice unless otherwise denoted.
*p<0.05; ***p<0.001.
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Table 3-2. Summary of overall fitness of DAT VMAT2 mice.
Comparisons are against WT mice.
Experiment DAT-tg VMAT2-kd DAT-tg/VMAT2-kd
Fitness
Survival No change No change 50% reduction (67% in males)
Body weight No change Reduced in males (similar to Mooslehner et al, 2001)
Reduced in males & females
In summary, DAT over-expression combined with VMAT2 knockdown leads to impaired fitness
as evidenced by reduced survival and body weight of DAT-tg/VMAT2-kd mice. Given that
survival and body weight are complex traits and dopamine plays a variety of roles in the CNS
(e.g. locomotion, reward, motivation, lactation) as well as peripheral areas, the precise cause of
these changes is unclear. In addition, male DAT-tg/VMAT2-kd mice seem to be more
susceptible since as they depict greater decline in survival and body weight in comparison to
their female counterparts. While the fitness data were stratified by sex, biochemical,
neurochemical and behavioral results (shown below) are with both sexes combined because: 1)
typically, the sample size for these experiments are relatively low, therefore, dividing the data by
sex would diminish the power to detect differences, 2) the chance of obtaining an animal with a
particular genotype and a particular sex is 1-in-12 according to the breeding scheme for DAT
VMAT2 mice. Due to these low odds, it is not always feasible to collect enough animals and
conduct experiments in a timely manner and 3) as shown in Figure 3.15 B, male DAT-
tg/VMAT2-kd are highly susceptible to premature death, therefore, it is difficult to assess
parameters exclusively in male mice after 12 weeks of age, when most experiments are
conducted.
3.2.3 Presynaptic dopamine homeostasis
Presynaptic neurotransmitter homeostasis is maintained by various processes including release,
uptake, synthesis and metabolism. Aspects of these processes were evaluated to determine how
dopamine is handled in the presynaptic neurons of DAT VMAT2 mice. First, given that DAT
and VMAT2 are involved in packaging and transporting dopamine, levels of dopamine and its
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major metabolites, DOPAC and HVA, were assessed in the striatum of DAT VMAT2 mice using
HPLC with electrochemical detection. As shown in Figure 3.17, dopamine levels are
significantly reduced in all 3 genotypes that have altered DAT and/or VMAT2 levels in
comparison to WT mice. In particular, VMAT2-kd mice show a 95% reduction in dopamine
tissue content that is corroborated by previous characterization of these mice (Caudle et al.,
2007). Both VMAT2-kd and DAT-tg/VMAT2-kd mice show similar drastic reductions in
dopamine levels, indicating that VMAT2 is crucial in maintaining intracellular dopamine
content. In addition, DAT-tg mice show 21% reduction in dopamine levels compared to WT
mice. Previously, Dr. Salahpour had found 33% lower dopamine tissue content in DAT-tg mice
which was explained by a similar loss of dopamine neurons (Masoud et al., 2015). However, in
this case, there is no evidence for dopaminergic cell loss (see stereology results Figures 3.22-
3.24). Hence, reduced dopamine tissue content in these DAT-tg may be a reflection of terminal
changes in the striatum while cell bodies in the substantia nigra remain intact. Unlike dopamine
tissue content, metabolite levels are not as prominently altered in DAT VMAT2 mice. DOPAC
levels are increased in DAT-tg mice and slightly reduced in DAT-tg/VMAT2-kd mice while
HVA tissue content is unchanged across all 4 genotypes (Figure 3.17).
(A) Dopamine, (B) DOPAC and (C) HVA levels assessed in striatal tissue from DAT VMAT2
mice (N=6-13) using HPLC-EC. Data presented as mean ± SEM. Statistical differences are in
comparison to WT mice. *p<0.05; **p<0.01; ***p<0.001.
Figure 3-17. Striatal tissue content of dopamine and its metabolites.
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Second, metabolite-to-dopamine ratios were calculated and used as indicators of dopamine
turnover in the striatum (Figure 3.18). VMAT2-kd and DAT-tg/VMAT2-kd mice display greatly
increased DOPAC/dopamine and HVA/dopamine ratios in comparison to WT mice. DAT-tg
mice also show trends towards increased metabolite-to-dopamine ratios, as previously observed
(Masoud et al., 2015). While the increased ratios are clearly a function of reduced dopamine
tissue levels in these mice (Figure 3.17 A), they also indicate enhanced dopamine metabolism
which could serve as a mechanism to control cytosolic buildup of dopamine.
(A) DOPAC-to-dopamine (DOPAC/DA) and (B) HVA-to-dopamine (HVA/DA) ratios
calculated from striatal tissue content levels (N=6-13). Data presented as mean ± SEM.
Statistical differences are in comparison to WT mice. ***p<0.001.
Figure 3-18. . Metabolite-to-dopamine ratios in the striatum.
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Since dopamine content in the presynaptic neuron is influenced by neurotransmitter release and
uptake, these parameters were evaluated in collaboration with Dr. Miller at Emory University.
We sent them mice from the DAT VMAT2 colony and they performed fast-scan cyclic
voltammetry (FSCV) on brain slices to determine dopamine release and clearance. Dopamine
release was electrically-evoked by a stimulating electrode and the resulting current was measured
by a recording electrode. We focused on the dorsal striatum which is the major projection area
for nigral dopamine neurons. Data were analyzed using two redundant, yet different methods.
The first simplistic method approximates “dopamine release” using the peak amplitude and
“dopamine clearance” using the decay-time constant, tau (where lower tau, measured in seconds,
implies faster clearance) (Yorgason et al., 2011). The second method, recently proposed by
Hoffman and colleagues, uses curve modeling to control for the interdependence between release
and uptake and provide accurate representation of these parameters (Hoffman et al., 2016). In
this analysis, higher rate of uptake implies faster clearance. Results are shown using both these
methods in Figure 3.19. As indicated by both analyses, dopamine release in the dorsal striatum is
significantly lower in DAT-tg, VMAT2-kd and DAT-tg/VMAT2-kd mice in comparison to WT
animals (Fig 3.19 B, C). This corroborates previous data also showing decreased striatal
dopamine tissue content in the 3 genotypes mentioned (Fig. 3.17 A). In VMAT2-kd and DAT-
tg/VMAT2-kd mice, reduced vesicular storage of dopamine could lead to the observed reduction
in dopamine release. For DAT-tg mice, previous characterization also reported decreased
dopamine release due to the loss of dopamine cells (Masoud et al., 2015). However, these DAT-
tg mice do not demonstrate neurodegeneration (see Figures 3.22-3.24 in next section), suggesting
that reduced dopamine release in the striatum of these animals may indicate terminal damage in
the absence of cell loss.
With regards to dopamine clearance, DAT-tg and DAT-tg/VMAT2-kd mice were expected to
display higher uptake as a result of increased DAT expression. However, using tau
measurements (Fig 3.19 D), VMAT2-kd and DAT-tg/VMAT2-kd mice display faster dopamine
clearance compared to WT mice, while DAT-tg mice also show a non-significant trend.
Previously, using Michaelis-Menten kinetics, the clearance of evoked dopamine (Vmax) was
reported to be substantially faster in DAT-tg mice, attesting to a functional increase in DAT
(Salahpour et al., 2008). However, in this experiment, even VMAT2-kd mice, which have
normal levels of DAT, demonstrate faster dopamine clearance. Another technical consideration
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that should be noted is that tau is a simplistic measure of dopamine uptake that could have been
influenced by low dopamine release in these genotypes. If dopamine release is reduced, then
conceivably, overall neurotransmitter clearance may also require a shorter period of time. Using
the Hoffman analysis, no significant changes in dopamine uptake were observed across the
genotypes, although VMAT2-kd and DAT-tg/VMAT2-kd mice showed trends towards higher
dopamine uptake. In summary, DAT-tg mice demonstrate a trend towards increased dopamine
uptake (using tau) while DAT-tg/VMAT2-kd mice display significantly enhanced dopamine
clearance, attesting to functional over-expression of DAT in these mice.
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Figure 3-19. Electrically evoked dopamine release and uptake in the dorsal striatum.
Determined by FSCV in slice preparations. (A) Traces of dopamine currents recorded over time
following a single-pulse stimulation. The ascending curve represents dopamine release while the
descending curve represents dopamine clearance. Dopamine release is estimated by (B) peak
amplitude (N=4-5) and (C) the Hoffman parameter, r/ke (N=2-4). Dopamine uptake/clearance is
estimated by (D) the decay time constant, tau (N=3-5) and (E) the Hoffman parameter, ku (N=3-
4). For Hoffman modeling, individual data points must meet certain criteria in order to be
included in the analysis and as a result, some data were excluded. Data presented as mean ±
SEM. Statistical differences are in comparison to WT mice. *p<0.05; **p<0.01; ***p<0.001.
Another key process that regulates presynaptic dopamine levels is synthesis. Since TH is the
rate-limiting enzyme involved in dopamine production, TH protein expression and levels were
measured in the striatum of DAT VMAT2 mice. As shown in Figure 3.20, there are no
differences across the genotypes in 1) TH expression, as assessed by immunohistochemistry (A)
or 2) TH protein levels as assessed by western blots (B, C). These results suggest that dopamine
production is unlikely to be altered in mice with varying levels of DAT and VMAT2, although
other factors such as TH activity would need to be evaluated to obtain conclusive knowledge of
the synthetic pathway.
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(A) TH immunohistochemistry. Representative TH-labeled (black) coronal sections are shown
for each genotype. (B) TH western blot using GAPDH as a loading control. (B) Quantification of
TH protein using densitometry. TH levels are normalized to WT expression. (N=3-4). Data
presented as mean ± SEM
Lastly, a pilot experiment was conducted to further probe dopamine metabolism in the
presynaptic neuron. Since metabolite-to-dopamine ratios were elevated in VMAT2-kd and DAT-
tg/VMAT2-kd mice, we investigated protein expression of MAO-B, the enzyme responsible for
degrading dopamine to DOPAC, as another indicator of dopamine metabolism. Increased
DOPAC-to-dopamine ratios may suggest an up-regulation of MAO-B in these mice. In a trial
experiment, a western blot of MAO-B protein was conducted using a new antibody in total
striatal homogenates (Figure 3.21 A). Unfortunately, this antibody produced a variable signal
with high background staining. Although statistical differences between the 4 genotypes were
Figure 3-20. TH protein expression in the striatum.
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lacking, there does seem to be a trend towards higher MAO-B protein levels in DAT-tg and
DAT-tg/VMAT2-kd mice in comparison to WT animals (Figure 3.21 B). Despite multiple
attempts with various antibodies, the MAO-b western blot could not be reliably reproduced.
Figure 3-21. MAO-B protein expression in the striatum.
(A) MAO-B western blot. (B) Quantification of MAO-B protein using densitometry. MAO-B
levels were corrected for loading using GAPDH and normalized to WT expression (N=2-4). Data
presented as mean ± SEM.
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Table 3-3. Summary of presynaptic dopamine homeostasis in DAT VMAT2 mice.
Comparisons are against WT mice.
Experiment DAT-tg VMAT2-kd DAT-tg/VMAT2-kd
Striatal Neurochemistry
Dopamine 21% decrease (33% decrease Masoud et al, 2015)
96% decrease 95% decrease
DOPAC Increased No change decrease
HVA No change No change No change
Evoked dopamine release and uptake
Striatal release Decreased Decreased Decreased
Striatal uptake Trend increase (50% increase in Salahpour et al, 2008)
Increased (tau) Increased (tau)
Metabolism
DOPAC/dopamine Trend increase. Similar to Masoud et al 2015
Increased (replicate Taylor et al 2009, 2013)
Large increase
HVA/dopamine Trend increase. Similar to Masoud et al 2015
Large increase Large increase
MAO-B protein levels Unchanged (Trend increase?)
Unchanged Unchanged (Trend increase?)
Synthesis
TH protein expression Unchanged Unchanged Unchanged
TH protein levels Unchanged Unchanged Unchanged
In summary, DAT-tg/VMAT2-kd mice show prominently reduced dopamine tissue content in
the striatum as well as decreased dopamine release in both the striatum and nucleus accumbens,
demonstrating mishandling of dopamine in the presynaptic neuron. Since both HPLC tissue
content and electrically-evoked FSCV parameters predominantly reflect vesicular monoamine
stores, knockdown of VMAT2 has a robust and similar effect in both VMAT2-kd and DAT-
tg/VMAT2-kd mice. Therefore, any additional contribution of DAT over-expression in DAT-
tg/VMAT2-kd mice is difficult to gauge using these techniques. Furthermore, although DAT-tg
mice display a trend towards higher dopamine uptake, it does not seem as significant as
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previously reported (Masoud et al., 2015). Similarly, while DAT-tg mice show reduced
dopamine tissue content, it is not as pronounced as previous reports (Masoud et al., 2015). This
is consistent with a lower degree of DAT over-expression in these mice as well (Figure 3.12).
Once again, collectively, these results suggest that the DAT-tg mice from the DAT VMAT2
colony do not display as severe phenotypes as the original DAT-tg line. This is corroborated by
stereology results where DAT-tg mice no longer display dopamine cell loss (see Figures 3.22 –
3.24). Regarding dopamine metabolism, DAT-tg/VMAT2-kd mice demonstrate increased
metabolite-to-dopamine ratios, suggesting that enhanced dopamine turnover may represent a
compensatory mechanism to counteract the buildup of cytosolic dopamine. Lastly, while
presynaptic dopamine dynamics are altered in DAT VMAT2 mice, TH levels appear to be
unchanged.
3.2.4 Integrity of dopamine neurons
Previously, Dr. Salahpour demonstrated a 36% reduction of nigral dopamine neurons in 3-5
month old DAT-tg mice (Masoud et al., 2015), while Caudle et al. reported a 26% loss of nigral
dopamine neurons in 22 month old VMAT2-kd mice (Caudle et al., 2007). Thus, it was
hypothesized that DAT-tg/VMAT2-kd mice will display greater cell loss than either genotype
alone, since the dual effect of DAT over-expression and VMAT2-knockdown could lead to more
accumulation of cytosolic dopamine and cause toxicity.
To test this hypothesis, we conducted stereological counts of dopaminergic neurons in the SNpc
using TH as a dopaminergic marker and NeuN or Nissl as a neuronal marker. For these
experiments, we collaborated with two research groups that possess the necessary equipment,
analytical software and expertise to routinely perform stereology on dopaminergic neurons.
Counters were blind to genotypes and 3-5 month old animals were used for stereology. At this
age, we expect DAT-tg mice to show cell loss (Masoud et al., 2015) and VMAT2-kd mice to
show no change. We repeated stereology in 3 independent experiments as outlined below.
The first attempt was a collaboration with Dr. Miller’s lab at Emory University in 2011. We sent
them perfused and fixed tissue for stereology and TH counts were performed by Carlos Lazo, a
new post-doc at the time. No differences were found between the 4 genotypes in the SNpc
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(Figure 3.22). DAT-tg mice were meant to serve as a control since we previously reported 36%
neuronal loss in these mice, however this effect was not replicated.
Figure 3-22. Stereological counts of TH+ cells in the SNpc.
Counts performed using 3-5 month old DAT VMAT2 mice (N=7-8) in collaboration with Carlos
Lazo from Miller lab, Emory University.
Second, we collaborated with Dr. Brotchie’s lab at Toronto Western Krembil Research Institute
in 2014. Initially, I started to count TH and NeuN positive cells in the SNpc using their software,
however due to high background staining, positive cells were difficult to distinguish and results
were variable. Therefore, to enhance experimental efficiency, we sent perfused, fixed and
sectioned tissue to Gabriela Reyes at the Brotchie lab for staining and stereological counting
since she regularly performs such experiments. The results for TH and NeuN are shown in
Figure 3.23. No differences were found between genotypes and the previously published cell loss
in DAT-tg mice (Masoud et al., 2015) was not replicated. For NeuN, DAT-tg mice showed a
trend towards lower counts in comparison to WT mice although this was not reflected in TH
counts (Figure 3.23 A, B).
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Figure 3-23. Stereological counts of TH+ and NeuN+ cells in SNpc.
Counts of (A) TH and (B) NeuN positive cells in the SNpc of 3-5 month old DAT VMAT2 mice.
(N=5-10). Data presented as mean ± SEM. Collaboration with Gabriela Reyes from Brotchie lab,
Krembil Institute, Toronto Western Hospital.
Third, we collaborated with Dr. Miller’s lab at Emory University again in 2015. This time,
stereological counting was performed by Amy Dunn, who is experienced at counting dopamine
neurons in genetic models. For this experiment, we also included additional controls: midbrain
sections from 18-24-month old WT and VMAT2-kd mice. At this age, VMAT2-kd mice are
expected to show approximately 20% reduction in TH+ and Nissl+ cells in comparison to WT
mice (Caudle et al., 2007). As shown in Figure 3.24, the cell loss in aged VMAT2-kd is
replicated. However, dopaminergic cell loss in DAT-tg mice is still not replicated. Similar to the
2 previous attempts, no differences were detected between any of the DAT VMAT2 genotypes.
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Figure 3-24. Stereological counts of TH+ and Nissl+ cells in SNpc.
Counts of (A) TH and (B) Nissl positive cells in the SNpc of 3-5 month old DAT VMAT2 mice
(N=6-8). Collaboration with Amy Dunn at Miller lab, Emory University. Aged (18-24 month
old) WT and VMAT2-kd mice (N=3-4) were included as controls (shown in blue). Statistical
difference between aged VMAT2-kd mice in comparison to aged wild type control animals. Data
presented as mean ± SEM. *p<0.05; **p<0.01.
Table 3-4. Summary of dopamine cell counts in SNpc of DAT VMAT2 mice.
Comparisons are against WT mice.
Experiment DAT-tg VMAT2-kd DAT-tg/VMAT2-kd
Dopamine cell counts
SNpc TH 1) Carlos (Miller lab) 2) Gaby (Brotchie lab) 3) Amy (Miller lab)
No change (does not replicate 36% loss in Masoud et al, 2015)
No change as expected (Caudle et al, 2007)
No change
SNpc Nissl/NeuN 1) Carlos (Miller lab) 2) Gaby (Brotchie lab) 3) Amy (Miller lab)
No change (does not replicate 32% loss in Masoud et al, 2015)
No change as expected (Caudle et al, 2007)
No change
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In summary, DAT-tg/VMAT2-kd did not show any change in dopamine cell number, which was
unexpected. These experiments were repeated 3 times not only to ascertain the result, but also
because of the following challenges:
1) Stereology proved to be an unreliable technique. In fact, we sent the same slides to be
counted by the same researcher (Brotchie lab) twice and obtained opposing results,
highlighting the variability of this technique. Due to these technical issues, our
confidence in the results was diminished.
2) DAT-tg mice no longer showed dopamine cell loss as previously published (Masoud et
al., 2015). The premise of the DAT VMAT2 project was based on the finding that DAT-
tg mice show dopaminergic degeneration at 3-5 months of age and we hypothesized that
by concurrently decreasing VMAT2 in these mice, the degeneration would be
exacerbated. In 3 independent experiments, DAT-tg mice failed to show dopamine cell
loss or even a trend towards reduced cell number. This result consolidated our suspicion
that DAT-tg mice from the DAT VMAT2 colony lacked the degree of dopaminergic
damage that was previously noted. Possible reasons for these differences is that original
DAT-tg mice and our current DAT-tg mice belong to separate colonies that may have
differences in genetic background/modifiers, nutrition and breeding. These issues are
further examined in the discussion.
However, keeping in mind that DAT-tg mice no longer show dopamine cell loss and VMAT2-kd
mice only demonstrate cell loss at 18 months or older, it is not surprising to find that 3-5 month
old DAT-tg/VMAT2-kd mice do not show signs of neurodegeneration.
3.2.5 Dopamine receptor levels
After assessing presynaptic dopamine homeostasis and neuron integrity, post-synaptic dopamine
receptor levels were investigated in DAT VMAT2 mice. Since DAT-tg, VMAT2-kd and DAT-
tg/VMAT2-kd show reduced dopaminergic tissue content (Figure 3.17 A) and release (Figure
3.19 B, C), it was predicted that post-synaptic dopamine receptors would be up-regulated in
these mice to compensate. It has been previously shown that DAT-tg mice have a 30% increase
in D1 receptors and a 62% increase in D2 receptors (Ghisi et al., 2009). As an additional control,
a few DAT-KO samples were also included. DAT-KO mice are hyperdopaminergic due to the
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lack of dopamine uptake and therefore, D1 and D2 receptors are downregulated by 60% and
40%, respectively (Ghisi et al., 2009).
D1 and D2 receptor binding assays were performed on striatal membranes from DAT VMAT2
mice. For D1 binding, there was noticeable variability within genotypes perhaps because frozen
striatal samples were used for these experiments. No significant differences were detected
between the 4 genotypes (Figure 3.25 A). In particular, the previously-reported increase in D1
levels of DAT-tg mice was no longer observed, consistent with other results (e.g. stereology) that
also failed to recapitulate earlier findings. However, a 67% reduction was replicated in DAT-KO
samples suggesting that the experimental conditions were suitable for detecting changes in D1
binding. Statistical comparisons between WT control and DAT-KO samples are not shown due
to small sample size (N=1-2). Regarding D2 binding, VMAT2-kd and DAT-tg/VMAT2-kd
samples show increased D2 levels in comparison to WT mice (Figure 3.25 B). However, once
again, the previously reported 62% increase in D2 levels was not replicated in DAT-tg mice
(Ghisi et al., 2009). DAT-KO mice showed 26% lower D2 binding in comparison to WT mice.
This decrease is not as large (40%) as previously reported (Ghisi et al., 2009), however with 1-2
samples, at least a similar trend was observed.
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Receptor levels determined by radioligand binding. (A) D1 and (B) D2 receptor levels (N=4-6
samples, each sample consists of striatal tissue from 3-4 mice). DAT knockout (DATKO)
samples were included as controls (N=2 samples, each sample contains striatal tissue from 4
mice, shown in blue). Statistical comparisons between WT control and DAT-KO mice were not
performed due to low sample size (N=1 for WT control sample). Differences are in comparison
to respective wild type samples. Data presented as mean ± SEM. *p<0.05.
Table 3-5. Summary of dopamine receptor levels in the striatum of DAT VMAT2 mice.
Comparisons are against WT mice.
Experiment DAT-tg VMAT2-kd DAT-tg/VMAT2-kd
Receptor levels
D1 No change – did not replicate 30% increase (Ghisi et al., 2009)
Trend increase Trend increase
D2 No change – did not replicate 62% increase (Ghisi et al., 2009)
Increased Increased
Figure 3-25. Dopamine receptor levels in the striatum.
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In summary, DAT-tg/VMAT2-kd mice show increased D2 levels and a possible trend towards
increased D1 binding. DAT-tg/VMAT2-kd mice show 1) low dopamine tissue content and 2)
decreased dopamine release, therefore, as a compensatory mechanism, upregulation of dopamine
receptors was anticipated in these animals. Since similar upregulation of dopamine receptors was
also observed in VMAT2-kd mice, enhanced receptor levels may not be sufficient to explain
some phenotypes that are unique to DAT-tg/VMAT2-kd mice such as basal hyperactivity (see
Figure 3.26 below). Once again, DAT-tg mice did not show expected increases in receptor
levels. However, since presynaptic dopaminergic cell loss was not replicated in the DAT-tg mice
from the DAT VMAT2 colony (Figures 3.22-3.24), compensatory changes in postsynaptic
dopamine receptors are also less likely to occur. Taken together, these DAT-tg mice consistently
display less severe phenotypes than the original DAT-tg mice.
3.2.6 Baseline behavior
Dopamine signaling mediates a variety of behaviors such as locomotion, cognition and reward.
Since DAT-tg/VMAT2-kd mice show significant changes in dopamine handling, we investigated
whether these changes had any effect on their baseline behaviors. In particular, the following
behaviors were examined: 1) open field locomotion, 2) fine motor skills using challenging beam
traversal, 3) executive function using puzzle box and 4) anxiety-like behavior using elevated plus
maze.
Initially, basal locomotion was assessed in open-field activity monitors and the following
parameters were measured for two hours: 1) total distance traveled, 2) number of horizontal
movements, 3) horizontal activity, defined as the number of beam interruptions that occur in the
horizontal sensor, 4) vertical activity, defined as the number of beam interruptions that occur in
the vertical sensor, 5) stereotypy count, the number of times the animal breaks the same beam(s)
repeatedly during stereotypic behavior and 6) stereotypy number, the number of times the animal
is monitored to engage in stereotypic behavior. Collectively, these parameters provide a holistic
view of an animal’s locomotor behavior and are summarized in Figure 3.26. DAT-tg/VMAT2-kd
mice are hyperactive as evidenced by a 5-fold increase in total distance traveled, higher number
of movements and greater horizontal activity in comparison to WT, DAT-tg and VMAT2-kd
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mice (Figure 3.26 B, C, D). Furthermore, when distance traveled is measured in 5 minute
intervals, DAT-tg/VMAT2-kd mice are more active than other genotypes for the first hour,
however during the second hour, their movement begins to decline and they apparently habituate
to the environment, similar to other genotypes (Figure 3.26 A). These data suggest that the
hyperlocomotion of DAT-tg/VMAT2-kd mice is a reflection of increased exploration in a novel
environment. Conversely, DAT-tg/VMAT2-kd mice show no significant changes in vertical
activity, whereas VMAT2-kd display lower vertical activity that WT mice (Figure 3.26 E).
Stereotypy refers to repetitive movements such as grooming that have been shown to involve
stimulation of striatal dopamine receptors (Delfs and Kelley, 1990). While baseline stereotypy
counts were unchanged between the 4 genotypes, DAT-tg/VMAT2-kd mice show a trend
towards increased stereotypic behavior. Also, in comparison to VMAT2-kd mice, DAT-
tg/VMAT2-kd mice display a greater number of stereotypic movements. In conclusion, the basal
locomotion of DAT-tg/VMAT2-kd mice is significantly different from other genotypes
demonstrating that the dual effects of DAT over-expression and VMAT2 knockdown produces
unique motor phenotypes in these mice.
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126
Figure 3-26. Open field locomotion and stereotypy.
Adult mice were assessed using automated VersaMax software (N=13-18). (A) Distance traveled
over time. Differences are in comparison to WT mice. Sum of (B) total distance traveled, (C)
number of horizontal movements, (D) horizontal activity (the number of beam interruptions that
occur in the horizontal sensor), (E) vertical activity (the number of beam interruptions that occur
in the vertical sensor), (F) total stereotypy count (the number of times the animal breaks the
same beam(s) repeatedly) and (G) stereotypy number (the number of times an animal is
monitored to engage in stereotypic behavior with a minimal interval of 1 second). Measures were
obtained over a two-hour period using VersaMax software. Data presented as mean ± SEM.
Statistical differences are as denoted. In the case of multiple significant differences, the top
asterisk denotes comparison to WT mice, the middle asterisk refers to comparison against DAT-
tg mice and the bottom asterisk is versus VMAT2-kd animals. *p<0.05; **p<0.01; ***p<0.001.
Given the striking and unexpected phenotype of hyperactivity in DAT-tg/VMAT2-kd mice, two
additional pilot experiments were conducted to further explore the robustness of this result. First,
the locomotor behavior of aged (12-month old) DAT VMAT2 mice was assessed. This is the
only experiment where aged mice were used. As shown in Figure 3.27, the hyperactivity of
DAT-tg/VMAT2-kd mice persists even at 12 months of age since they travel twice as much as
age-matched WT mice. However, in comparison to adult DAT-tg/VMAT2-kd animals (3-5
months old, Fig 3.26 B), the degree of hyperactivity is diminished in aged DAT-tg/VMAT2-kd
mice.
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Total distance traveled over 2 hours in open-field activity monitors (N=5-8). Data were assessed
using VersaMax software and are represented as mean ± SEM. Statistical differences are in
comparison to WT (top) and DAT-tg (bottom) mice. *p<0.05; **p<0.01.
Second, we evaluated whether the degree of VMAT2-knockdown can affect the baseline
locomotor phenotype of DAT-tg/VMAT2-kd mice. Clearly, manipulating the expression of a
single transporter, DAT or VMAT2, is not sufficient to induce hyperlocomotion since both
DAT-tg and VMAT2-kd mice show similar locomotor activity as WT mice. In DAT-tg/VMAT2-
kd mice, DAT is over-expressed by 50% while VMAT2 is simultaneously under-expressed by
95%. In this experiment, we posed the question whether a less dramatic reduction in VMAT2
levels could be coupled with DAT over-expression to recapitulate the hyperactivity of DAT-
tg/VMAT2-kd mice. Fortunately, due to the breeding scheme used in the DAT VMAT2 colony,
half of the animals produced are heterozygous for the VMAT2 locus. Hence, we had access to
VMAT2-het mice that display 50% of VMAT2 levels and have been extensively characterized
by other groups. In this pilot experiment, the locomotor activity of adult (3-5 month old) WT
Figure 3-27. Locomotor activity of 12-month old mice.
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mice was compared with DAT-tg/VMAT2-het animals, that should show 50% higher DAT and
50% lower VMAT2 expression. These mice were used as a tool to further explore the robustness
of the locomotor findings from DAT-tg/VMAT2-kd mice. Interestingly, unlike DAT-tg/
VMAT2-kd animals (95% lower VMAT2), DAT-tg/VMAT2-het mice (50% lower VMAT2) are
not hyperactive (Fig 3.28). These results suggest that on a background of DAT over-expression,
a 50% reduction in VMAT2 levels is not sufficient in producing hyperlocomotion and instead, a
95% reduction is likely necessary. Hence, a threshold effect can be observed, where a high
degree of VMAT2-knockdown is required for the locomotor phenotype to manifest.
Total distance traveled over 2 hours in open-field activity monitors (N=4-8). Data were assessed
using VersaMax software and are represented as mean ± SEM.
After evaluating gross locomotion, fine motor skill of DAT VMAT2 mice was assessed using the
challenging beam traversal task. Briefly, animals were trained to traverse a beam of narrowing
width leading to the home cage. On the test day, a mesh grid was placed on top of the beam and
any slips or misplaced paws were counted as errors. As shown in Figure 3.29A, DAT-tg mice
display significantly higher errors compared to WT, VMAT2-kd and DAT-tg/VMAT2-kd mice.
These findings corroborate previous results from Figure 3.8 where DAT-tg mice showed a 50%
Figure 3-28. Locomotor activity of DAT-tg/VMAT2-het mice.
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increase in the number of errors compared to WT mice (Masoud et al., 2015). Findings from the
challenging beam traversal task suggest that DAT over-expression negatively impacts fine motor
skill whereas concurrent VMAT2-knockdown seems to rescue this deficit in DAT-tg/VMAT2-kd
mice. No differences were detected in the latency to traverse the beam across the 4 genotypes
due to high variability (Figure 3.29 B). Although DAT-tg/VMAT2-kd mice showed increased
locomotion in open-field analyses (Fig 3.26B), they did not appear to be hyperactive during this
task and instead showed a trend towards longer latency to cross the beam compared to WT mice
(Fig 3.29 B). Perhaps the short duration of the challenging beam task (5 minutes maximum)
combined with 2 previous days of training, allows animals to become habituated to the
environment. In conclusion, despite their basal open-field hyperlocomotion, the fine motor skills
of DAT-tg/VMAT2-kd mice remain intact in the challenging beam traversal task. In fact, the
behavioral deficits in DAT-tg mice are ameliorated in DAT-tg/VMAT2-kd mice, alluding to a
beneficial effect.
Figure 3-29. Fine motor skill evaluated using the challenging beam traversal task.
DAT VMAT2 mice were trained on this task for 2 days and results were collected on the third
test day as an average of 3 trials (N=13-15). (A) Number of errors while traversing the beam.
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Errors represent slips or misplaced paws while traversing the beam. (B) Latency to cross the
beam. Data presented as mean ± SEM. For statistical differences, the top asterisk denotes a
comparison to WT mice, the middle asterisk refers to comparison against VMAT2-kd mice and
the bottom asterisk is versus DAT-tg/VMAT2-kd animals. *p<0.05; **p<0.01.
Next, executive function of these mice was examined using the puzzle box, a problem-solving
test in which mice are required to complete escape tasks of increasing difficulty within a limited
amount of time (Ben Abdallah et al., 2011). The box is divided into 2 compartments: a brightly
lit start box and a dark goal box that are connected by a door and underpass. The test consists of
3 tasks per day for 3 days producing a total of 9 tasks (T1-9). Time to solve the puzzle and
escape to the goal box is recorded for each trial, where shorter latency corresponds with better
performance. A brief description of each task is shown in Table 2.1. Collectively, the puzzle box
provides information on problem-solving, short-term and long-term memory.
The results for each task of the puzzle box test are shown in Figure 3.30. DAT-tg/VMAT2-kd
mice do not perform differently from WT mice on any of the tasks. However, interestingly,
VMAT2-kd mice are significantly worse than the other 3 genotypes on T5 and T8, when a novel
puzzle is introduced. VMAT2-kd mice also display poorer outcomes on T9 in comparison to
DAT-tg and DAT-tg/VMAT2-kd mice. Typically, T9 is a test of short term memory since the
mice are required to solve the same puzzle as T8. However, since several of the VMAT2-kd
mice failed to solve the new task in T8, the higher latency in T9 may represent their ongoing
struggle with the difficult puzzle. Taken together, problem-solving ability is diminished in
VMAT2-kd mice however, DAT-overexpression seems to reverse this deficit since DAT-
tg/VMAT2-kd mice show normal problem-solving abilities. In summary, executive function of
DAT-tg/VMAT2-kd mice is intact as assessed by the puzzle box test.
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Figure 3-30. Executive function evaluated using the puzzle box.
Latency of mice to enter the goal box is shown (N=6-13). Data presented as mean ± SEM.
Statistical differences are denoted for each task as follows: T5: ** vs WT, ** vs DAT-tg, ** vs
DAT-tg/VMAT2-kd; T8: * vs WT, *** vs DAT-tg, *** vs DAT-tg/VMAT2-kd; T9: ** vs DAT-
tg, ** vs DAT-tg/VMAT2-kd. *p<0.05; **p<0.01; ***p<0.001.
Lastly, baseline anxiety-like behavior of DAT VMAT2 mice was measured using the elevated
plus maze. The elevated plus maze consists of 2 enclosed arms, 2 open arms and a center zone.
Typically, animals prefer to remain in the closed arms and exploration of open arms is thought to
represent reduced anxiety. This test was conducted under 2 light conditions: 1) dim (15-16 lx)
and 2) bright (210-240 lx) light. Separate groups of animals were used for each condition.
Notably, depending on the amount of ambient light, the results of the elevated plus maze were
different. In dim light, there were no changes in the percent time spent in closed arms (Figure
3.31 B), however both VMAT2-kd and DAT-tg/VMAT2-kd mice spent significantly more time
exploring open arms than WT mice (Fig 3.31 A). This suggests that VMAT2-kd and DAT-
tg/VMAT2-kd mice are less anxious than their WT counterparts. During this task, the locomotor
activity of the 4 genotypes were similar, discounting any possible effect of DAT-tg/VMAT2-kd
hyperactivity in influencing these results (Fig 3.31 C). On the other hand, in bright light
conditions, there were no differences between the genotypes in time spent in the open or closed
arms (Fig 3.31 D, E). Contrary to the previous result, VMAT2-kd mice showed a trend towards
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less time spent in open arms (Fig 3.31 D), suggesting that the level of anxiety may be increased
in these mice. Interestingly, while the percent of time spent in open arms by DAT-tg/VMAT2-kd
mice was similar in dim (Fig 3.31 A) and bright conditions (Fig 3.31 D) (roughly 20%), the other
3 genotypes displayed variable behavior depending on the level of light. WT and DAT-tg mice
seemed to spend a larger percent of time in the open arms under bright light (15-18%) versus
dim light (10%) while VMAT2-kd mice displayed the opposite trend. Hence, DAT-tg/VMAT2-
kd mice appear to be relatively insensitive to ambient light conditions during the elevated plus
maze, whereas the behavior of other genotypes is altered. However, locomotor activity of
VMAT2-kd and DAT-tg/VMAT2-kd mice was reduced compared to WT mice in bright light
(Fig 3.31 F). In general, results from the elevated plus maze were dependent on the light
conditions and while reduced anxiety-like behavior was observed in VMAT2-kd and DAT-
tg/VMAT2-kd mice under dim light, these differences were no longer present under bright light.
Previously, VMAT2-kd mice were shown to display higher anxiety-like behavior on the elevated
plus maze, however there are important methodological differences between those experiments
and this study (Taylor et al., 2009). In the previous case, the light-dark cycle of the mice was
reversed for weeks prior to testing, such that they were kept in darkness during the day. Then on
the test day, when they would normally be accustomed to darkness, they were exposed to bright
light during the elevated plus maze task. Given the potent effect of light conditions on this task
as demonstrated by my experiments, it is highly likely that the difference in results obtained is
directly attributable to the cycle reversal and light conditions chosen for each particular
experiment.
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Figure 3-31. Anxiety-like behavior assessed using elevated plus maze.
This test was conducted using different groups of animals in dim (15-16 lx, N=10-14) and bright
(210-240 lx, N=5-10) light. Percent of total time spent in (A, D) open arms or (B, E) closed arms
and (C, F) distance traveled during the task are shown for each light condition (Dim: A-C,
Bright: D-F). Total time is a sum of the time spent in open arms, closed arms and the center
zone. Data are presented as mean ± SEM. Statistical differences are in comparison to WT mice.
**p<0.01.
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Table 3-6. Summary of baseline motor and non-motor behaviors in DAT VMAT2 mice.
Comparisons are against WT mice unless otherwise denoted.
Experiment DAT-tg VMAT2-kd DAT-tg/VMAT2-kd
Gross locomotion
Distance traveled Unchanged Unchanged Increased (5X)
Number of movements Unchanged Unchanged Increased
Horizontal activity Unchanged Unchanged Increased
Vertical activity Unchanged Reduced Unchanged
Aged locomotion Unchanged Unchanged Increased (2X)
Stereotypy
Stereotypy count Unchanged Unchanged Trend towards increase
Stereotypy number Unchanged Unchanged Higher than VMAT2-kd mice
Fine motor skill
Challenging beam traversal
Increased errors similar to Masoud et al, 2015.
Unchanged
Unchanged in comparison to WT. Lower than DAT-tg.
Latency to cross beam Unchanged Unchanged Trend increase
Executive function
Puzzle box Unchanged Deficits in problem solving tasks (T5, T8)
Unchanged from WT, Enhanced versus VMAT2-kd mice
Anxiety - Elevated plus maze
Dim light Unchanged Less anxious Less anxious
Bright light Unchanged Trend towards increased anxiety
Unchanged
Locomotion Unchanged Unchanged in dim light, reduced in bright light
Unchanged in dim light, reduced in bright light
With regards to motor behavior, DAT-tg/VMAT2-kd are hyperactive and show normal fine
motor skills, which were not expected. However, in light of the fact that nigral dopamine cells
are intact in these mice, it is unlikely that they would suffer from poor motor performance.
Hyperactivity is a unique and robust phenotype of DAT-tg/VMAT2-kd mice that is observed in
locomotor activity chambers, but not necessarily in the home cage (qualitative observation).
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Hence, novelty of the activity chamber may act as a stimulus to induce hyperlocomotion in these
animals. In addition, DAT-tg/VMAT2-kd mice show better executive function than VMAT2-kd
mice and improved fine motor skills compared to DAT-tg mice, suggesting that altering both
DAT and VMAT2 may be beneficial for these behaviors. Aside from voluntary movement,
dopamine plays crucial roles in attention, executive function, motivation and reward which may
be affecting these behaviors. Interestingly, DAT-tg/VMAT2-kd mice are similar to VMAT2-kd
mice in neurochemistry (Figure 3.17, 3.19) and receptor levels (Figure 3.25), however
behaviorally, these mice are unique and distinct from VMAT2-kd mice. This suggests that the
balance of DAT and VMAT2 is crucial for behavioral outputs. Lastly, DAT-tg mice show higher
errors on the challenging beam traversal task as previously reported (Masoud et al., 2015). This
suggests that fine motor skill is still affected in these DAT-tg mice, despite lack of dopamine cell
loss (Figures 3.22-3.24).
3.2.7 Response to dopaminergic drugs
In the last set of experiments, we investigated the response of DAT VMAT2 mice to different
drugs that interact with the dopamine system including 1) psychostimulants, 2) dopamine
receptor agonists and 3) the dopamine precursor L-DOPA. These drugs were used as tools to
investigate dopaminergic function of DAT VMAT2 mice.
Psychostimulants such as amphetamine, cocaine and methylphenidate have been shown to exert
their behavioral effects predominantly by altering dopamine transport mechanisms. In particular,
amphetamine increases extracellular levels of dopamine by acting on both DAT and VMAT2,
two transporters that have been altered in DAT-tg/VMAT2-kd mice. It has previously been
reported that DAT-tg mice show increased amphetamine-induced hyperactivity (Salahpour et al.,
2008) while Mooslehner and colleagues demonstrate increased stereotypic behavior in VMAT2-
kd mice treated with amphetamine (Mooslehner et al., 2001). Therefore, it was hypothesized that
DAT-tg/VMAT2-kd mice will show an even more exaggerated response to amphetamine.
Mice were habituated in the locomotor activity chamber for 60 minutes and then injected with
0.5, 1, or 2 mg/kg of amphetamine. Locomotor activity and stereotypy counts were recorded for
90 minutes post injection. At the lowest dose of 0.5 mg/kg, there was no effect of amphetamine
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on total distance traveled (Fig 3.32 A) or stereotypy counts (Fig 3.33 A) in WT, DAT-tg and
VMAT2-kd mice. However, DAT-tg/VMAT2-kd mice displayed higher locomotor (Fig 3.32 A,
D) and stereotypic (Fig 3.33 A, D) activity after drug administration. In response to 1mg/kg of
amphetamine, DAT-tg and VMAT2-Kd mice show non-significant enhancements in locomotor
activity (Fig 3.32 B) and stereotypy (Fig 3.33 B) while DAT-tg/VMAT2-kd mice display
substantial increases in comparison to WT mice (Fig 3.32 D, 3.33 D). Finally, at 2mg/kg of
amphetamine, DAT-tg and VMAT2-Kd mice show significantly more locomotor activity (Fig
3.32 C) and stereotypy (Fig 3.33 C) than WT animals, as expected. However, there is no
difference in total distance traveled or stereotypy between WT and DAT-tg/VMAT2-kd mice
(Figure 3.32 D, 3.33 D). At this dose, DAT-tg/VMAT2-kd mice displayed peculiar movements
such as tremors, jerking, backward locomotion and tongue protrusions which were measured
using the Abnormal Involuntary Movements (AIMs) scale. An AIMs score is assigned based on
the duration of abnormal movements that are divided into 4 categories: locomotor, axial, limb or
orolingual. The minimum score for each time point is 4 and the maximum score is 16 (according
to the 4 categories). As shown in Fig 3.34 A and B, only DAT-tg/VMAT2-kd mice display
severe AIMs in response to 2 mg/kg of amphetamine. The lack of locomotor activity in these
mice at 2mg/kg of amphetamine is due to their abnormal movements which do not permit normal
locomotion. Therefore, DAT-tg/VMAT2-kd mice display an inverse relationship between total
distance traveled and AIMs score, where at peak AIMs scores, locomotion is negligible and
when AIMs begin to subside about an hour post-injection, locomotor activity also begins to
resume (Fig 3.34 C).
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Figure 3-32. Amphetamine-induced locomotion.
Mice were habituated to the chamber for 60 minutes, injected with amphetamine and monitored
for an additional 90 minutes (N=8-12). Arrow denotes time of injection. Distance traveled over
time in response to (A) 0.5, (B) 1 and (C) 2 mg/kg of amphetamine. (D) Sum of distance traveled
post injection for all doses. Data are presented as mean ± SEM. Statistical differences are in
comparison to WT mice. *p<0.05, **p<0.01, p<0.001.
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Figure 3-33. Amphetamine-induced stereotypy.
Mice were habituated to the chamber for 60 minutes, injected with amphetamine and monitored
for an additional 90 minutes (N=8-12). Stereotypy counts over time in response to (A) 0.5, (B) 1
and (C) 2 mg/kg of amphetamine. Arrow denotes time of injection. (D) Sum of stereotypy counts
post injection for all doses. Data are presented as mean ± SEM. Statistical differences are in
comparison to WT mice. *p<0.05, **p<0.01, p<0.001.
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Figure 3-34. Abnormal involuntary movements (AIM) induced by 2 mg/kg of
amphetamine.
Animals were habituated to the chamber for 60 minutes and then injected with drug (N=5-7).
Arrow denotes time of injection. Behavior of animals was recorded for 1 minute immediately
before injection and in 15 minute intervals up to 75 minutes post-injection. Hence, a total of 6
recordings were scored manually on the AIMs scale on 4 categories: locomotor, orolingual, axial
and limb peculiarities. For each time point, the minimum score is 4 and the maximum is 16. A
score is assigned based on duration of abnormal movements during the test period. (A) AIMs
score over time. (B) Total AIMs score (sum of all time points). The dashed line represents the
minimum AIMs score. (C) Relationship between AIMs score and distance traveled by DAT-
tg/VMAT2-kd mice over time. Locomotion and AIMs were assessed in different cohorts of mice
due to technical limitations of videotaping animals while in the locomotor activity chamber.
Data are presented as mean ± SEM. Statistical differences are in comparison to WT mice.
*p<0.05, **p<0.01, p<0.001.
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Collectively, these results demonstrate that DAT-tg/VMAT2-kd mice are exquisitely sensitive to
the effects of amphetamine, such that they display AIMs at doses that normally evoke
hyperactivity in other genotypes. To further explore this behavior, we tested whether higher
doses of amphetamine would elicit similar abnormal movements in other genotypes. In a pilot
experiment, after injection of 5mg/kg amphetamine, WT mice were hyperactive whereas DAT-tg
mice displayed a short burst of activity followed by decreased locomotion (Fig 3.35).
Qualitatively, during the periods of reduced locomotor activity, these DAT-tg animals displayed
abnormal behavior reminiscent of DAT-tg/VMAT2-kd mice treated with 2mg/kg of
amphetamine (data not quantified). For comparison purposes, a single DAT-tg/VMAT2-kd
mouse was also treated with 5mg/kg of amphetamine and it displayed lack of locomotor activity
(Fig 3.35) and several abnormal movements, as expected. Although the abnormal movements
appeared to be more extreme in DAT-tg/VMAT2-kd mice, this experiment demonstrates that at
high enough doses, other genotypes can also develop this behavior in response to amphetamine.
Figure 3-35. Locomotor effect of 5 mg/kg amphetamine on WT and DAT-tg mice.
WT and DAT-tg mice were habituated to the chamber for 60 minutes, injected with 5mg/kg
amphetamine and monitored for an additional 90 minutes (N=4-5). A single DAT-tg/VMAT2-kd
mouse was also included for comparison (dashed red line). (A) Distance traveled over time.
Arrow denotes time of injection. (B) Sum of distance traveled post-injection. Data are presented
as mean ± SEM. Statistical differences are in comparison to WT mice. *p<0.05, **p<0.01,
p<0.001.
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After assessing the effects of amphetamine, which causes reversal of DAT, the response of DAT
VMAT2 mice to DAT inhibitors, cocaine and methylphenidate, was investigated. Both these
compounds are psychostimulants that cause behavioral hyperactivity in rodents. It was
previously shown that DAT-tg mice are similar to WT animals in their behavioral response to
cocaine, while they are hypersensitive to the effects of amphetamine (Salahpour et al., 2008).
Conversely, VMAT2-het mice displayed increased horizontal activity versus WT animals when
treated with 20 mg/kg of cocaine (Wang et al., 1997). Given these previously published findings,
it was predicted that DAT-tg mice will not display increased sensitivity to cocaine while
VMAT2-kd mice will. As shown in Figure 3.36, when treated with 20 mg/kg of cocaine, 1)
DAT-tg animals displayed a moderate increase in activity that was similar to WT mice and 2)
VMAT2-kd mice demonstrated significant hyperactivity, as expected (Fig 3.36). Furthermore,
DAT-tg/VMAT2-kd mice were also hyperactive in comparison to WT mice, however, they
behaved similarly to VMAT2-kd mice as demonstrated in Figure 3.36 B. Analogous behavioral
results were observed in response to another DAT inhibitor, methylphenidate (5 mg/kg). Similar
to cocaine, both VMAT2-kd and DAT-tg/VMAT2-kd mice were hyperactive when treated with
methylphenidate (Fig 3.36 D). DAT-tg/VMAT2-kd mice appeared to demonstrate a slightly
greater response than VMAT2-kd mice, however, the difference between the two genotypes was
not significant (Fig 3.36 D). These results suggest that unlike a DAT-reversing agent like
amphetamine which produces uniquely enhanced sensitivity in DAT-tg/VMAT2-kd mice (Fig
3.32 D, 3.33 D), DAT inhibitors induce hyperactivity to the same extent in both VMAT2-kd and
DAT-tg/VMAT2-kd mice. This suggests that knockdown of VMAT2 is particularly relevant for
locomotor response to DAT inhibition, whereas over-expression of DAT does not particularly
affect this phenotype. It is important to note that amphetamine causes release of dopamine via
DAT while cocaine and methylphenidate prevent the uptake of dopamine following activity-
dependent vesicular release. This mechanistic difference may underlie the behavioral differences
described above.
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Figure 3-36. Locomotion induced by DAT inhibitors, cocaine and methylphenidate.
Mice were habituated to the chamber for 60 minutes, injected with cocaine (N=6) or
methylphenidate (N=4-8) and monitored for an additional 90 minutes. Distance traveled over
time in response to (A) cocaine or (C) methylphenidate. Sum of total distance traveled after
administration of (B) cocaine or (D) methylphenidate. Arrow denotes time of injection. Data are
presented as mean ± SEM. Statistical differences are in comparison to WT mice. *p<0.05,
**p<0.01, p<0.001.
Next, the effect of dopamine receptor agonists on DAT VMAT2 mice was assessed as an indirect
measure of receptor function. Similar to previous studies, animals were habituated to the activity
chamber for 60 minutes, drug was injected and locomotor activity was monitored for 90 minutes.
First, the non-selective D1/D2 receptor agonist, apomorphine was used. It has previously been
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shown that in rodents, administration of apomorphine produces stereotypy, which is regarded as
a readout of striatal dopamine receptor activation (Protais et al., 1976). Hence, we assessed
stereotypic behavior (e.g. repetitive chewing/grooming) of DAT VMAT2 mice in response to 2
mg/kg of apomorphine (Fig 3.37). Qualitatively, following drug administration, some DAT-
tg/VMAT2-kd mice engaged in extreme biting behavior that results in self-injury and bleeding
which is uncommon at this dose. Quantitatively, this behavior was partially represented as
increased stereotypy counts in DAT-tg/VMAT2-kd mice compared to WT animals after
administration of apomorphine (Fig 3.37). However, other quantitative scales such as the AIM
scale may have been better suited to fully capture this behavior. VMAT2-kd mice also
demonstrate a non-significant trend towards higher stereotypy when treated with apomorphine.
In summary, increased apomorphine-induced stereotypy in DAT-tg/VMAT2-kd mice suggests
that receptor function is up-regulated, corroborating previous radioligand binding assessments of
D2 receptor levels (Fig 3.25) in these mice.
Mice were habituated to the chamber for 60 minutes, injected with 2mg/kg of apomorphine s.c.
and monitored for an additional 90 minutes (N=8-9). Sum of stereotypy counts over 90 minutes
following injection of apomorphine. Data are presented as mean ± SEM. Statistical differences
are in comparison to WT mice. *p<0.05.
Figure 3-37. Apomorphine-induced stereotypy.
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Second, we evaluated the effect of a selective D1 agonist, SKF81297, on the locomotor activity
of DAT VMAT2 mice compared to saline treatment. As shown in Figure 3.38 B, VMAT2-kd
and DAT-tg/VMAT2-kd mice seem to show higher locomotor activity than WT mice at certain
time points after SKF-81297 administration (see time course 105-135 minutes). However, when
total activity of saline-treated DAT-tg/VMAT2-kd mice is compared to SKF81297-treated DAT-
tg/VMAT2-kd mice, there are no statistical differences (Fig 3.38 D). Similarly, when the
dopamine precursor and anti-Parkinsonian drug, L-DOPA (25 mg/kg) was tested in DAT-
tg/VMAT2-kd mice, no differences were noted in total distance traveled compared to saline
treatment (Figure 3.38 D). Since DAT-tg/VMAT2-kd mice are basally hyperactive, it translates
to greater distance traveled even after saline injections (Fig 3.38 A). Due to increased locomotion
of saline-treated DAT-tg/VMAT2-kd mice, the effects of SKF-81297 and L-DOPA are masked
in these animals at the doses tested. Unlike amphetamine (Fig 3.32, 3.33), cocaine and
methylphenidate (Fig 3.36), which produce substantial increases in the locomotor activity of
DAT-tg/VMAT2-kd mice that surpasses their basal hyperactivity, the effects of SKF-81297 and
L-DOPA are more modest and therefore, difficult to distinguish from the spontaneous activity of
DAT-tg/VMAT2-kd mice in novel environments (Fig 3.38 B). It is interesting to note that in
DAT-tg/VMAT2-kd mice, the time course of activity following saline treatment (Fig 3.38 A,
downward sloping) appears different from SKF-81297 or L-DOPA treatment (Fig 3.38 B, C;
peak after injection), suggesting some behavioral effects of these drugs (versus saline) that are
not fully captured in the quantitative analysis (Fig 3.38 D).
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Figure 3-38. Effect of SKF 81297, L-DOPA and saline on locomotor activity of DAT
VMAT2 mice.
Mice were habituated to the activity chamber for 60 minutes and injected with either (A) saline
(0.9% NaCl), (B) 2 mg/kg of SKF-81297 or (C) 25 mg/kg L-DOPA with 12.5 mg/kg benserazide
(peripheral DOPA decarboxylase blocker). Distance traveled post-injection was monitored for an
additional 90 minutes. Different cohorts of mice were used for each drug (N=7-11). Figures A-C
show distance traveled over time where the arrow denotes time of injection. Data are presented
as mean ± SEM. Statistical differences are in comparison to WT mice. (D) Sum of total distance
traveled after drug administration. For each genotype, the effect of a drug is compared to saline
administration. Statistical differences are reported against saline treatment. Data are presented as
mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.
Table 3-7. Summarized effects of dopaminergic drugs on behavior of DAT VMAT2 mice.
Comparisons are against WT mice, unless otherwise denoted.
Experiment DAT-tg VMAT2-kd DAT-tg/VMAT2-kd
Saline Locomotor
No change No change Increased (basal hyperactivity)
Psychostimulants
Amphetamine Locomotor Stereotypy AIMs
Increased locomotion and stereotypy at 2mg/kg (similar to Salahpour et al, 2008)
Increased locomotion and stereotypy at 2mg/kg
Increased locomotion and stereotypy at 0.5 and 1 mg/kg. AIMs at 2mg/kg
Cocaine Locomotor
No change (similar to (Salahpour et al., 2008)
Increased
Increased (similar to VMAT2-kd mice)
Methylphenidate No change (similar to (Salahpour et al., 2008)
Increased Increased (similar to VMAT2-kd mice)
Dopamine receptor agonists
Apomorphine Stereotypy
No change No change Increased
SKF-81297 Locomotor
No change Increased Similar increase as saline
Dopamine precursor
LDOPA Locomotor
No change No change Similar increase as saline
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In summary, VMAT2/DAT-tg mice are exquisitely sensitive to the effects of amphetamine at
very low doses which do not affect other genotypes (e.g. 0.5mg/kg). While we expected these
animals to show a left-shifted dose response curve, the development of abnormal involuntary
movements (jerking, tremor, orolingual) at a dose of 2mg/kg of amphetamine was not
anticipated. DAT-tg and VMAT2-kd mice also showed enhanced locomotor and stereotypic
response to amphetamine in comparison to WT mice, as expected, however the effect in DAT-
tg/VMAT2-kd mice was synergistic. Unlike amphetamine, DAT inhibitors like cocaine and
methylphenidate, produced significant hyperactivity in both VMAT2-kd and DAT-tg/VMAT2-
kd mice. Both of these genotypes demonstrate higher dopamine receptor levels which may
explain their locomotor response to DAT inhibitors. The non-selective dopamine receptor
agonist, apomorphine, produced increased stereotypy in DAT-tg/VMAT2-kd mice, also
indicating enhanced receptor function in these mice. However, locomotor effects of the D1
agonist, SKF-81297 and the dopamine precursor, L-DOPA, could not be distinguished from
saline treatment in DAT-tg/VMAT2-kd mice due to their basal hyperactivity. It should be noted
that qualitatively, the time courses for SKF-81297 and L-DOPA induced locomotion in DAT-
tg/VMAT2-kd mice show distinct patterns (Fig 3.38 B -C, peak after injection) in comparison to
saline injection (Fig. 3.38 A, continuous downward trend). Collectively, DAT-tg/VMAT2-kd
mice display a selective and robust response to amphetamine, indicating intense behavioral
sensitivity to DAT reversal.
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Chapter 4 Discussion
Discussion
4.1 Project 1: Characterization of DAT-tg mice
In this study, we report that over-expression of DAT is capable of triggering oxidative stress,
dopamine neuron loss and L-DOPA reversible motor deficits in DAT-tg mice. Previously,
ectopic expression of DAT was shown to cause death of non-dopaminergic cells, presumably due
to their inability to properly handle cytotoxic dopamine (Chen et al., 2008). However, we
demonstrate that even in dopamine cells that are inherently equipped with the molecular
machinery to properly store, metabolize and release dopamine, an increase in DAT expression
can lead to higher dopamine uptake and damaging consequences. Aside from our work, previous
studies using plasmid and lentiviral techniques have also reported that DAT over-expression can
increase dopamine uptake and alter downstream behaviors (Martres et al., 1998; Adriani et al.,
2009). Transgenic mice expressing DAT under the TH promoter showed higher DAT levels,
greater dopamine uptake and modest, but significant reductions in striatal dopamine tissue
content (Donovan et al., 1999), similar to DAT-tg mice. In comparison to these studies, our BAC
transgenic approach to over-express DAT has several important advantages including: 1) robust,
long-term DAT expression, 2) selectivity for dopaminergic neurons using the DAT promoter and
3) lack of injection and transfection-related complications. Collectively, this body of work shows
that increased DAT activity can significantly impact and change dopamine homeostasis.
The dopamine system is notoriously sensitive to endogenous and exogenous challenges
(Langston et al., 1983; Hastings et al., 1996; Mosharov et al., 2009). Therefore, 46% higher
dopamine uptake in DAT-tg mice (Salahpour et al., 2008) produces dramatic effects on
dopamine homeostasis, cell survival, oxidative stress and motor behaviors, as noted in our study
(Masoud et al., 2015). These results highlight the physiological importance of tightly regulating
cytosolic dopamine levels since moderate deviations in dopamine compartmentalization can
directly impact neuronal survivability. Another example of this is the VMAT2-kd mice. These
animals express only 5% of normal VMAT2 protein and display decreased dopamine tissue
content, nigrostriatal neurodegeneration and increased levels of cysteinyl-catechols (Caudle et
al., 2007), similar to DAT-tg mice. Physiologically, VMAT2-kd mice are deficient in
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sequestering intracellular dopamine into vesicles while DAT-tg mice have excess dopamine
uptake (Caudle et al., 2007; Salahpour et al., 2008). In addition to higher uptake, DAT-tg mice
also display reduced VMAT2 expression, suggesting that vesicular storage of dopamine could
also be compromised. Taken together, the genetic manipulations in VMAT2-kd and DAT-tg
mice effectively act to increase the cytosolic pool of dopamine. This buildup of cytosolic
dopamine could be a common pathway that is responsible for the basal loss of dopamine neurons
and oxidative stress evident in both VMAT2-kd and DAT-tg mice.
There are several observations supporting the hypothesis that accumulation of cytosolic
dopamine results in loss of dopaminergic neurons in DAT-tg mice. First, results from DAT-KO
animals highlight the critical role of DAT in loading the presynaptic neuron with dopamine
(Giros et al., 1996; Jones et al., 1998a) In DAT-KO mice, lack of uptake leads to 5-times higher
extracellular dopamine levels and extremely low dopamine tissue content (5%), indicating
depleted intracellular stores. Conversely, in DAT-tg mice, higher levels of functional DAT lead
to a 46% increase in dopamine uptake and a 40% decrease in extracellular dopamine, suggesting
that the neurotransmitter is accumulating in the presynaptic neuron (Salahpour et al., 2008).
However, despite the likely buildup of dopamine within each dopaminergic cell, DAT-tg mice
display a 33% reduction in overall dopamine tissue content as a direct consequence of 30-36%
loss of dopamine neurons. Secondly, we report higher metabolite-to-dopamine ratios in DAT-tg
mice. Since DOPAC is a direct product of cytosolic dopamine metabolism, a 60% increase in
the DOPAC/dopamine ratio could indicate that a greater proportion of dopamine is present in the
cytosol and not sequestered into vesicles (Di Monte et al., 1996). Elevated metabolite-to-
dopamine ratios also imply enhanced dopamine turnover that could be a compensatory
mechanism to tackle the buildup of intracellular dopamine (Zigmond et al., 2002). Thirdly,
increased levels of 5-S-cysteinyl-dopamine and 5-S-cysteinyl-DOPAC were detected in the
striatum of DAT-tg mice. These cysteinyl-modified adducts have been suggested to arise from
the oxidation of cytosolic dopamine and its metabolites (Graham, 1978; Fornstedt and Carlsson,
1989; Hastings and Zigmond, 1994). Not only are cysteinyl adducts a direct consequence of
cytosolic dopamine reactivity, they are also capable of independently inducing further neuronal
damage (Spencer et al., 2002). Next, lower VMAT2 protein expression in DAT-tg mice also
suggests potential buildup of cytosolic dopamine. Although this decrease may be a reflection of
dopaminergic cell loss per se, nonetheless, reduced VMAT2 levels can negatively impact
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vesicular storage and disable these mice from handling increased dopamine uptake caused by
DAT over-expression. Lastly, accumulation of cytosolic dopamine has been suggested to have
deleterious effects on cell survival (Caudle et al., 2007; Chen et al., 2008; Mosharov et al., 2009)
that is clearly reflected in the loss of dopamine neurons in DAT-tg mice (Masoud et al., 2015).
Collectively, these observations suggest that DAT over-expression most likely leads to high
cytosolic levels of dopamine, thereby producing the downstream detrimental effects observed in
DAT-tg mice.
We also demonstrated that DAT-tg mice are highly sensitive to MPTP-induced neurotoxicity.
Indeed, when treated with MPTP, DAT-tg mice showed greater reductions in striatal TH levels
and dopamine tissue content compared to WT animals. MPP+, the toxic metabolite of MPTP, is
a substrate for DAT and therefore, causes selective damage to dopaminergic cells (Langston et
al., 1984; Chiba et al., 1985; Ramsay et al., 1986; Gainetdinov et al., 1997; Schober, 2004).
While the dependence of MPTP neurotoxicity on DAT function has previously been
demonstrated (Gainetdinov et al., 1997; Bezard et al., 1999; Miller et al., 1999b; Schober, 2004),
our results indicate a synergistic interaction between environmental and genetic risk factors that
could have broader implications for complex pathological conditions such as Parkinson’s disease
(Cannon and Greenamyre, 2013). In Parkinson’s disease, both genetic mutations and
environmental conditions have been documented to increase disease risk (Priyadarshi et al.,
2000; Hardy et al., 2006; Martin et al., 2011; Cannon and Greenamyre, 2013). Moreover, animal
models that depend on a single type of insult seldom recapitulate the full spectrum of the
disorder (Beal, 2010). Although genes such as PINK1, DJ1 and PARK2 (parkin) have been
implicated in familial forms of Parkinson’s disease, mutating or knocking-out these genes in
most animal models does not reproduce dopaminergic cell loss (Goldberg et al., 2003;
Yamaguchi and Shen, 2007; Gispert et al., 2009). Conversely, while acute toxicant treatment
(e.g. MPTP or 6-hydroxydopamine) can produce abrupt neurodegeneration, it does not address
the underlying disease mechanism of a chronic and progressive disorder like Parkinson’s disease
(Schober, 2004). Given the shortcomings of these individual approaches, the convergence of
genetic as well as environmental insults may be more representative of idiopathic Parkinson’s
disease that is hypothesized to arise from multiple hits (Sulzer, 2007; Cannon and Greenamyre,
2013). Our results lend support to this idea by showing that genetic over-expression of DAT
combined with exogenous exposure to MPTP, aggravates toxicity to dopamine neurons.
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Although the effect of genetic mutations on DAT expression is unclear in humans, a correlation
study reports that DAT genetic variants in combination with exposure to exogenous compounds
(e.g. pesticides) can potentiate the risk of developing Parkinson’s disease by 3- or 4-fold (Ritz et
al., 2009). This highlights the significance of genetic and environmental interactions in the
pathology of Parkinson’s disease.
The cellular, neurochemical and behavioral changes observed in DAT-tg mice recapitulate
important features of Parkinson’s disease. Firstly, loss of midbrain dopamine neurons and
reduced dopamine tissue content in the striatum of DAT-tg mice capture the major pathological
characteristics of Parkinson’s disease (Dauer and Przedborski, 2003). However, it should be
noted that Parkinson’s disease is characterized by selective nigrostriatal degeneration, whereas
DAT-tg mice also demonstrate loss of VTA dopamine neurons. This is probably due to
transgenic over-expression of DAT in the VTA, which enhances the vulnerability of this region
in DAT-tg mice. Physiologically, VTA neurons do not express as much DAT as SNpc neurons
and therefore, the VTA is relatively spared from damage in Parkinson’s disease (Blanchard et al.,
1994). The relationship between DAT expression and neurodegeneration is supported by a study
in Parkinson’s disease patients showing that brain regions containing the highest levels of DAT
protein – the caudate and putamen – are also the most sensitive to damage (Miller et al., 1997).
In addition, a recent meta-analysis has identified the DAT gene as a risk factor for Parkinson’s
disease in certain populations (Zhai et al., 2014). Secondly, oxidative stress has long been
postulated to be involved in the development of Parkinson’s disease (Fahn and Cohen, 1992) and
we report that DAT-tg mice display increased levels of cysteinyl-dopamine and cysteinyl-
DOPAC, two markers that are also elevated in the SN of Parkinson’s disease patients (Spencer et
al., 1998). Thirdly, increased dopamine turnover in the transgenic mice mirrors elevated
metabolite-to-dopamine ratios that have been reported in Parkinson’s disease patients
(Birkmayer and Hornykiewicz, 1962; Zigmond et al., 2002). In addition, both DAT-tg mice and
Parkinson’s disease patients show reductions in VMAT2 protein expression in comparison to
control samples (Miller et al., 1999a). Behaviorally, DAT-tg mice do not exhibit any deficits in
gross locomotion, probably because the level of cell loss in these animals is not sufficient to
cause major motor disturbances. In Parkinson’s disease patients, motor deficits are only evident
when greater than 70% of dopaminergic tone is lost in the striatum (Bernheimer et al., 1973).
However, results from the wire-hang test and challenging beam traversal task clearly
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demonstrate that fine motor coordination, balance and strength are compromised in DAT-tg mice
similar to Parkinson’s disease patients. Other studies on dopaminergic dysfunction have shown
that these two tests are sensitive to motor impairment even in the absence of gross locomotor
changes (Hwang et al., 2005; Luk et al., 2012). Furthermore, not only do DAT-tg mice display
motor disturbances on the challenging beam traversal; these deficits are also reversed by L-
DOPA, the principal treatment for motor symptoms of Parkinson’s disease. This suggests that
dopamine neuronal loss in DAT-tg mice leads to motor deficits that can be reversed by restoring
dopaminergic tone. Hence, parallel to Parkinson’s disease patients, DAT-tg mice also
demonstrate motor behaviors that are responsive to L-DOPA treatment. Given these overlapping
results, we postulate that the mishandling of cytosolic dopamine exhibited by DAT-tg mice could
provide important insights on the unique vulnerability of dopamine cells in Parkinson’s disease.
In conclusion, in Project 1, we used transgenic mice that selectively over-express DAT in
dopaminergic neurons to investigate the effects of cytosolic dopamine accumulation in vivo. As
shown by our results, moderate increases in DAT function cause spontaneous dopaminergic cell
loss, oxidative stress and fine motor impairment that is reversed by L-DOPA treatment. These
results suggest that the integrity of dopamine neurons depends heavily on the ability of DAT to
maintain proper homeostatic control of presynaptic dopamine. Since dopaminergic cells are
selectively damaged by a broad variety of genetic and environmental insults, it demonstrates that
these cells are inherently at risk. Our results imply that buildup of cytosolic dopamine, a highly
reactive and potentially toxic molecule, may underlie the cell-specific vulnerability of
dopaminergic neurons to damage. We propose that dopamine uptake through DAT, maintains a
constant cytosolic pool of this neurotransmitter that can propagate oxidative stress in dopamine
cells. This type of chronic damage may render these neurons vulnerable to degeneration,
especially if coupled with other genetic or environmental insults that are linked with the
pathogenesis of Parkinson’s disease. Since DAT-tg mice display spontaneous neuronal loss and
heightened toxicity in response to MPTP, these mice provide a useful tool to study the effects of
endogenous and exogenous challenges on dopamine cells.
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4.2 Project 2: Characterization of DAT-tg/VMAT2-kd mice
The goal of this project was to investigate the dual effect of DAT over-expression and VMAT2
knockdown in genetically modified mice. As an extension of Project 1, the rationale for this
study also arose from a body of literature showing that cytosolic dopamine is highly reactive and
can produce neurotoxicity by triggering oxidative stress (Graham, 1978; Chen et al., 2008;
Mosharov et al., 2009). In particular, as demonstrated in Project 1, over-expression of DAT and
greater dopamine uptake produces damaging outcomes in DAT-tg mice including oxidative
stress, spontaneous loss of midbrain dopamine neurons and fine motor deficits (Masoud et al.,
2015). In addition, VMAT2-kd mice from the Miller lab at Emory University, exhibit reduced
vesicular storage of dopamine which also translates to dopaminergic damage as assessed by
diminished striatal dopamine, evidence of oxidative stress and loss of nigrostriatal dopamine
neurons in aged mice (Caudle et al., 2007). In both these mouse models, potential accumulation
of cytosolic dopamine due to increased uptake or decreased vesicular packaging, leads to
deleterious consequences. However, the level of dopaminergic cell loss in these mouse models is
moderate (around 30% for each genotype) and does not reach the extent of damage that is
typically observed in Parkinson’s disease patients (around 70%) (Caudle et al., 2007; Sulzer,
2007; Kordower et al., 2013; Masoud et al., 2015). Therefore, stemming from the results of
Project 1 (DAT-tg mice) and in collaboration with the Miller lab (VMAT2-kd mice), we
interbred DAT-tg and VMAT2-kd mice to generate double transgenic animals (DAT-
tg/VMAT2-kd mice) that were hypothesized to have greater accumulation of cytosolic dopamine
and consequently, demonstrate exacerbated symptoms of dopaminergic damage that may better
resemble the pathophysiology of Parkinson’s disease.
We systematically characterized the dopamine system of DAT-tg/VMAT2-kd mice by
evaluating presynaptic dopamine homeostasis, survival of dopamine neurons, post-synaptic
dopamine receptors, basal dopamine-mediated behaviors and behavioral response to
dopaminergic drugs. We hypothesized that DAT-tg/VMAT2-kd mice will show phenotypes
associated with dopamine toxicity such as loss of dopamine cells, reduced dopamine tissue
content, upregulation of dopamine receptors and poor motor behavior. While dopaminergic cell
loss was not observed, some phenotypes of dopamine dysregulation were recapitulated in DAT-
tg/VMAT2-kd mice. For instance, in comparison to WT animals, DAT-tg/VMAT2-kd mice
display: 1) 95% reduction in striatal dopamine tissue content, 2) 85% reduction in evoked-
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dopamine release, 3) 15- to 20-fold increase in DOPAC/dopamine and HVA/dopamine ratios,
suggesting higher dopamine turnover, 4) 33% increase in striatal D2 receptor levels, indicating
compensatory up-regulation of this receptor to tackle reduced dopaminergic tone, 5) 46%
reduction in survival of 12-week old mice and 6) decreased adult body weight. Taken together,
these findings illustrate that DAT-tg/VMAT2-kd mice have considerable impairments as a result
of mishandling dopamine. However, some of these changes were not as pronounced as
hypothesized, and more importantly, DAT-tg/VMAT2-kd mice did not show any signs of
dopaminergic cell loss or compromised motor ability, two hallmarks of Parkinson’s disease. The
lack of Parkinsonian phenotypes in DAT-tg/VMAT2-kd mice is related to a complication that
arose during this study where DAT-tg mice (that were used to breed DAT-tg/VMAT2-kd mice),
no longer displayed some previously-observed phenotypes of dopaminergic toxicity. This issue
is discussed in detail in the next section. The following discussion focuses on current findings
from DAT-tg/VMAT2-kd mice.
It is interesting to note that for most biochemical and neurochemical assessments, DAT-
tg/VMAT2-kd mice displayed similar results as VMAT2-kd mice. Indeed, on pre- and post-
synaptic measures of the dopamine system (such as dopamine tissue content, release, uptake,
metabolite-to-dopamine ratios and receptor binding), VMAT2-kd and DAT-tg/VMAT2-kd mice
were generally indistinguishable. In animals with VMAT2 knockdown (regardless of DAT
expression), the 95% reduction in VMAT2 levels severely compromised their vesicular storage
capacity as illustrated by drastic reductions in dopamine tissue content and evoked-dopamine
release. Due to the diminished dopaminergic tone, dopamine receptors were also up-regulated in
VMAT2-kd and DAT-tg/VMAT2-kd mice to compensate for reduced dopamine release. In
addition, VMAT2-kd and DAT-tg/VMAT2-kd mice demonstrated increased metabolite-to-
dopamine ratios, suggesting that dopamine was being metabolized in the cytosolic space as a
consequence of reduced vesicular storage. The impact of VMAT2 knockdown on dopamine
tissue content, evoked dopamine release and metabolite-to-dopamine ratios were so robust, that
they masked any additional contribution of DAT over-expression in DAT-tg/VMAT2-kd mice,
probably due to ceiling or basement effects. For instance, DOPAC/dopamine and
HVA/dopamine ratios are enhanced by 18 to 23-fold in VMAT2-kd mice, potentially reaching a
ceiling effect beyond which further increase in dopamine metabolism may not be physiologically
feasible or technically detectable in DAT-tg/VMAT2-kd mice. In comparison to WT animals,
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DAT-tg mice also showed moderate reductions in dopamine tissue content and release, and
trends towards faster dopamine uptake, suggesting that over-expression of DAT translates to
functional changes in the dopamine system of DAT-tg mice. However, the consequences of DAT
over-expression in animals with reduced VMAT2 levels were difficult to perceive perhaps due to
more modest effects. Between the two genetic manipulations, DAT over-expression and VMAT2
knockdown, the latter produced a stronger impact due to multiple reasons: 1) VMAT2 is
essential for survival since ablation of this gene results in post-natal death whereas DAT-KO
mice survive into adulthood, 2) VMAT2 levels were drastically reduced to 5% of normal WT
levels, whereas DAT expression was enhanced by 50-75% and 3) VMAT2 is expressed in all
monoaminergic cells and therefore, VMAT2-knockdown affected multiple neurotransmitter
systems whereas DAT over-expression was confined to the dopaminergic system. Hence, on
measures that are heavily influenced by vesicular storage capacity (such as striatal dopamine
tissue content and electrically-evoked dopamine release), knockdown of VMAT2 produced
similar effects in both VMAT2-kd and DAT-tg/VMAT2-kd mice, regardless of their DAT
expression.
However, DAT-tg/VMAT2-kd mice also display unique behavioral phenotypes that distinguish
them from other mice. First, they are hyperactive in open-field locomotion as evidenced by ~5-
fold greater distance traveled, higher horizontal activity and number of horizontal movements
than WT, DAT-tg and VMAT2-kd mice. Interestingly, genetic ablation of DAT also produces
hyperactivity in mice (Giros et al., 1996). However, DAT-KO mice show impaired habituation
which is not evident in DAT-tg/VMAT2-kd mice. Furthermore, during other short tests such as
the elevated plus maze, puzzle box or challenging beam traversal, DAT-tg/VMAT2-kd mice do
not appear hyperactive and can solve the task at hand, suggesting that their hyper- exploratory
behavior is context-dependent. Second, although DAT-tg mice display fine motor deficits on the
challenging beam traversal task, these impairments are reversed in DAT-tg/VMAT2-kd mice.
This suggests that reducing VMAT2 levels in DAT-tg mice improves their fine motor skills.
Third, in the puzzle box, while VMAT2-kd mice perform worse than all other genotypes during
problem-solving tasks (T5, T8), these deficits are rescued in DAT-tg/VMAT2-kd mice. This
suggests that increasing DAT expression in VMAT2-kd mice ameliorates their cognitive deficits.
Collectively, judging from these behavioral results, it seems that altering the balance of DAT and
VMAT2 produces hyperactivity in novel environments and improves fine-motor skill and
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executive function of DAT-tg/VMAT2-kd mice compared to their counterparts. While these
striking results were not expected, they highlight the complexity of the dopamine system in
determining behavioral outputs. Previous assessments of dopamine tissue content, release,
uptake and receptor levels suggested that DAT-tg/VMAT2-kd mice closely resemble VMAT2-
kd animals, however, their behavioral outcomes are distinctly unique. While up-regulated striatal
dopamine receptors in DAT-tg/VMAT2-kd mice could partially explain their hyperactivity,
VMAT2-kd mice also express similar neurochemical and receptor changes but do not exhibit
increased locomotion, indicating a paradox. This disconnect between neurochemical and
behavioral analyses raises an important issue in the study of mouse models. Although
neurochemical analyses offer significant value in understanding aspects of the underlying
system, they are limited by several factors: 1) they are conducted in specific tissues, brain
regions or slices, 2) they are post-mortem examinations, and 3) typically, they capture a snapshot
of the system at a particular time point. Conversely, behavioral assessments occur in intact,
living organisms and measure outcomes that are integrated from multiple neural systems. Hence,
isolated biochemical/neurochemical assessments may not adequately explain behavioral changes
that arise from complex interactions between several pathways. Indeed, dopamine plays
important roles in a variety of functions including motor control, attention, cognition, motivation
and reward; all of which can contribute to the behaviors tested.
In addition to changes in baseline behaviors, DAT-tg/VMAT2-kd mice also display differential
locomotor responses to drugs that interact with the dopamine system. Most notably, DAT-
tg/VMAT2-kd mice are exquisitely sensitive to the effects of amphetamine, a psychostimulant
that reverses the activity of DAT, ultimately causing release of dopamine. At very low doses of
amphetamine (0.5 mg/kg), DAT-tg/VMAT2-kd mice display significantly increased locomotion
and stereotypy while at higher doses (2 mg/kg), these animals demonstrate abnormal involuntary
movements – giving rise to a left-shifted dose-response curve. Both DAT-tg and VMAT2-kd
mice also depict heightened sensitivity to the stimulant effects of amphetamine, albeit to a lesser
extent than DAT-tg/VMAT2-kd mice. Hence, animals with concurrent DAT over-expression and
VMAT2 knockdown exhibit a truly synergistic and robust response to amphetamine. Conversely,
DAT inhibitors, such as cocaine or methylphenidate, produce increased locomotor responses in
both DAT-tg/VMAT2-kd and VMAT2-kd mice, indicating a lack of selectivity for the double
transgenic animals. Cocaine-induced behavioral effects cannot be explained by dopamine
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release, as indicated by preliminary FSCV experiments conducted in the presence of cocaine
(Appendix 1). However, the differential response of DAT VMAT2 mice to amphetamine versus
cocaine/methylphenidate may be explained by the drugs’ mechanisms of action. In the case of
DAT inhibitors, dopamine is released through normal activity-dependent vesicular mechanisms
but its uptake is blocked, allowing the extracellular neurotransmitter to interact with post-
synaptic receptors. Since basal evoked-dopamine release and dopamine receptor levels are
similarly altered in VMAT2-kd and DAT-tg/VMAT2-kd mice, this could explain their similar
behavioral responses to cocaine and methylphenidate. In addition, previous characterization of
DAT-tg mice revealed that they show no differences in comparison to WT mice when treated
with DAT blockers (Salahpour et al., 2008). This suggests that drug-induced blockade of
dopamine uptake is not affected by DAT over-expression, therefore, DAT-tg/VMAT2-kd mice
also effectively behave as VMAT2-kd animals when treated with DAT blockers such as cocaine
or methylphenidate. Amphetamine, on the other hand, acts by dissipating the vesicular proton
gradient which forces dopamine to accumulate in the cytosolic space. This buildup of cytosolic
dopamine along with the actions of amphetamine, reverse the activity of DAT, causing non-
vesicular dopamine efflux (Sulzer et al., 1995, 2005). Physiologically, DAT-tg/VMAT2-kd mice
are anticipated to accumulate more dopamine in the cytosolic space than DAT-tg or VMAT2-kd
mice alone, due to the dual effect of greater dopamine uptake combined with reduced vesicular
storage. If indeed cytosolic dopamine levels are higher in DAT-tg/VMAT2-kd mice, then
amphetamine-induced reversal of DAT would also release larger amounts of the neurotransmitter
in the extracellular space in comparison to other genotypes. This enhanced DAT-mediated
dopamine release in DAT-tg/VMAT2-kd mice could explain their robust locomotor response to
amphetamine.
In Project 2, by simultaneously altering DAT and VMAT2 levels in DAT-tg/VMAT2-kd mice,
we created an imbalance in dopamine compartmentalization that produced unique and
unexpected phenotypes. As mentioned, DAT-tg/VMAT2-kd mice are hyperactive, perform better
than other genotypes on fine motor skill and executive function tasks, and are highly responsive
to amphetamine treatment. Although we measured several aspects of pre and post-synaptic
dopamine dynamics including dopamine tissue content, dopamine release and uptake, dopamine
receptor levels, number of midbrain dopaminergic neurons and metabolite-to-dopamine ratios,
these parameters cannot fully explain the behavioral results obtained. One possible explanation
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for these results is that simultaneously increasing DAT and reducing VMAT2 levels produces a
buildup of cytosolic dopamine that eventually reverses DAT activity, causing dopamine to leak
out of the cell. This idea is supported by previous studies that provide evidence for DAT-
mediated reverse transport of dopamine (Leviel, 2001). First, as discussed, amphetamine’s
mechanism of action relies on its ability to promote non-exocytotic release of dopamine by
reversing the activity of DAT (Sulzer et al., 1995; Leviel, 2001). This reveals that as a
transporter, DAT is capable of moving dopamine in the opposite direction, at least in the
presence of amphetamine. Second, when a human DAT coding variant, Ala559Val, was
introduced in cells, it was shown to exhibit spontaneous DAT-mediated outward efflux of
dopamine (Mazei-Robison et al., 2008). Furthermore, knock-in mice generated from this DAT
variant displayed higher extracellular dopamine levels, consistent with DAT-mediated leakage of
dopamine (Mergy et al., 2014). These results suggest that structural modification of DAT can
give rise to a transporter that constitutively releases dopamine into the extracellular space. Third,
using the giant dopamine neuron of the pond snail Planorbis corneus, it was illustrated that
injection of dopamine within the cytosol leads to neurotransmitter efflux (Sulzer et al., 1995).
Specifically, this efflux was blocked by the DAT inhibitor, nomifensine, indicating that the
release of dopamine was DAT-dependent. Hence, in this system, increased cytosolic dopamine
concentrations were sufficient to induce DAT-mediated dopamine release. Collectively, these
findings lend support to the hypothesis that DAT-tg/VMAT2-kd mice may endogenously leak
dopamine via DAT due to buildup of cytosolic levels of the neurotransmitter. Reversal of DAT
would lead to increased extracellular dopamine levels that can explain novelty-induced
hyperlocomotion of DAT-tg/VMAT2-kd mice as well as their enhanced performance on fine
motor and cognitive tests compared to other genotypes. Furthermore, enhanced response of
DAT-tg/VMAT2-kd mice to amphetamine may reflect their basal sensitivity towards DAT
reversal. From our current FSCV results, dopamine release is diminished in DAT-tg/VMAT2-kd
mice, rather than increased, which would be expected with DAT reversal. However, it is
important to note that electrical stimulation produces vesicular, exocytotic dopamine release
which was assessed by FSCV, whereas DAT reversal would lead to non-vesicular, non-
exocytotic, transporter-mediated dopamine leakage, which could not be measured with this
method. While DAT reversal is an intriguing hypothesis to explain the unique behaviors of
DAT-tg/VMAT2-kd mice, there are also some caveats to this theory that should be considered.
For instance, unlike DAT-tg/VMAT2-kd mice, knock-in DAT Val559 mice, which display
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DAT-mediated dopamine efflux, do not exhibit overt hyperactivity. Instead, they display a
context-dependent darting phenotype (Mergy et al., 2014). Also, DAT Val 559 mice are less
sensitive to the effects of amphetamine whereas DAT-tg/VMAT2-kd mice show robust
behavioral responses to this drug. Differences between DAT Val 559 and DAT-tg/VMAT2-kd
mice are possible because in the former model, dopamine efflux is caused by a rare DAT variant,
whereas in the latter, the transporter is functioning normally however an imbalance in dopamine
compartmentalization may lead to efflux (Mergy et al., 2014). In summary, other parameters
such as extracellular dopamine levels need to be evaluated in DAT-tg/VMAT2-kd mice in order
to test the hypothesis of DAT reversal.
Another possible hypothesis to explain the unexpected phenotypes of DAT-tg/VMAT2-kd mice
relates to the modulation of dopamine’s signal-to-noise ratio. Signal is defined as the action of
dopamine on its intended synaptic receptors while noise is defined as extra-synaptic effects of
the neurotransmitter once released. In DAT-tg/VMAT2-kd mice, due to reduced vesicular
storage, the quantal release of dopamine is decreased. Conversely, due to enhanced uptake,
dopamine is quickly removed from the peri-synaptic space. We initially hypothesized that these
genetic manipulations will reduce the absolute quantity of dopamine signaling and cause
deleterious downstream effects. However, it is also possible that aside from absolute differences,
the relative imbalance of DAT and VMAT2 acts to focus the dopamine signal by releasing a
finite amount of neurotransmitter that acts preferentially on synaptic receptors and is efficiently
taken up before it diffuses and acts off-target. Hence, DAT-tg/VMAT2-kd mice show
improvements in fine motor skill, problem solving and exploration that are not observed in other
genotypes. This is another possible explanation for the results obtained however, several other
parameters need to be tested to confirm this hypothesis.
4.2.1 Discrepancy between original DAT-tg mice and DAT-tg mice from the DAT-tg/VMAT2-kd colony
The most important challenge encountered during Project 2 was that DAT-tg mice from the DAT
VMAT2 breeding colony did not fully replicate some findings obtained from the original DAT-
tg mice (Salahpour et al., 2008; Ghisi et al., 2009; Masoud et al., 2015). Some phenotypes were
present but attenuated: such as DAT over-expression (1.75-fold vs 3-fold), reduced dopamine
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tissue content (21% lower vs 33%), enhanced dopamine uptake (39% higher trend vs 60%),
reduced electrically-evoked dopamine release (62% lower vs 72%), and increased errors on the
challenging beam traversal task (45% increase vs 50%). Other phenotypes were completely
absent: such as loss of nigrostriatal dopamine neurons, decreased striatal VMAT2 protein levels
and upregulation of striatal D1 and D2 receptors. Indeed, stereological counts of midbrain
dopamine neurons were repeated in three independent experiments to ascertain the result that
DAT-tg mice no longer demonstrated dopaminergic cell loss. Taken together, these data suggest
that DAT-tg mice from the DAT VMAT2 colony consistently demonstrate less dopaminergic
toxicity than the original DAT-tg mice. Since these animals no longer show dopaminergic cell
loss, many of its downstream and compensatory changes are also not replicated, such as
decreased VMAT2 protein and upregulated dopamine receptors. However, even in the absence
of dopaminergic neurodegeneration, these DAT-tg mice still display reduced striatal dopamine
tissue content, reduced striatal dopamine release and increased fine motor errors, providing
evidence of modest dopaminergic damage at the terminals that may not have affected the cell
bodies. It is possible that the level of DAT over-expression and functional dopamine uptake in
these mice is not high enough to reach the threshold for cellular toxicity (as indicated by 1.75-
fold more DAT protein in these mice instead of 3-fold more DAT protein in the original DAT-tg
mice). Given that these DAT-tg mice are from the DAT VMAT2 line and previous DAT-tg mice
belonged to a separate colony, there can be many possible reasons behind this discrepancy:
1. Genetic background: Original DAT-tg mice were purely on a C57BL/6 background.
DAT-tg mice were then crossed with VMAT2-kd mice that were on a mixed C57BL/6
and 129SV background. Through successive generations of breeding, DAT-tg/VMAT2-
kd mice were backcrossed to C57BL/6. However, it is possible that by interbreeding from
different genetic backgrounds, some genetic modifiers may have been introduced that
dampened the phenotypes of these mice. An example of this is illustrated by results from
our own laboratory, where we found differences in cocaine response depending on
whether C57BL/6 mice were obtained from Jackson or Charles River. This highlights the
potent effect of specific genetic backgrounds on dopaminergic phenotypes.
2. Nutrition: Since DAT-tg/VMAT2-kd mice are fragile and die prematurely, we
supplement these mice (and their cage littermates) with peanut butter and safflower seeds
from birth. The original DAT-tg mice did not receive any dietary supplementation. Given
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that there is a bidirectional relationship between dopamine signaling and feeding
behavior/reward, it is possible that nutritional differences may contribute towards
phenotypic outcomes.
3. Breeding: Original DAT-tg mice were generated by breeding DAT-nTg (WT) mice with
DAT-Tg mice which produced 50% DAT-nTg and 50% DAT-Tg mice. In the DAT
VMAT2 colony, DAT-tg mice are generated by breeding +/- VMAT2-kd/ DAT-tg mice
with +/- VMAT2-kd/DAT-nTg mice which produces 6 possible genotypes. Hence DAT-
tg mice from the DAT VMAT2 colony are conceived and reared by parents that are
heterozygotes for VMAT2, a gene essential for monoamine storage. In comparison,
parents of the original DAT-tg mice were normal for the VMAT2 locus. Difference in the
breeding pairs may impact prenatal and postnatal development of the progeny,
accounting for differences in their phenotypes.
4.2.2 Hypothesis revisited
Keeping in mind that DAT-tg mice have lost their neurodegenerative phenotype, it is not
surprising that DAT-tg/VMAT2-kd mice also did not show evidence of prominent dopaminergic
toxicity such as loss of dopamine cells or poor motor ability. Our initial hypothesis hinged on the
finding that DAT-tg mice demonstrate dopaminergic cell loss (Masoud et al., 2015) and since
that effect was lost, our hypothesis regarding Parkinsonian effects in DAT-tg/VMAT2-kd mice
could no longer be supported. Due to dampened effects of DAT over-expression in these DAT-tg
mice, many features of DAT-tg/VMAT2-kd mice closely mimicked VMAT2-kd animals,
instead. Nonetheless, we discovered several unique and robust phenotypes in DAT-tg/VMAT2-
kd mice (such as reduced survival, basal hyperactivity, supersensitivity to amphetamine) that
provide novel information regarding dopamine function. In fact, in models that show
neurodegeneration, most of the changes observed are symptoms or compensations of the cell
loss. By studying animals on a non-neurodegenerative background, we can objectively evaluate
the changes in dopamine function as a result of DAT and VMAT2 expression, without the
complication of altered dopamine neuron numbers across genotypes.
In summary, the goal for Project 2 was to generate mice that simultaneously over-express DAT
and under-express VMAT2. Numerous experiments were conducted to characterize the
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dopamine system and its behavioral output in these mice. Our initial hypothesis regarding
enhanced dopaminergic toxicity in DAT-tg/VMAT2-kd mice was not supported by the results
mainly because DAT-tg mice no longer demonstrated dopaminergic cell loss. Loss of
previously-observed phenotypes in DAT-tg mice posed a major obstacle in interpreting the
results from this project, however, we executed our plan of study and uncovered interesting
phenotypes in DAT-tg/VMAT2-kd mice that shed light on the complex functioning of the
dopamine system.
4.3 Conclusion
The overall aim of this thesis was to investigate the effects of altered dopamine
compartmentalization on the function of the dopamine system and related behaviors in
genetically modified mice. In particular, we aspired to amplify cytosolic compartmentalization of
dopamine by enhancing dopamine uptake through DAT over-expression and reducing dopamine
vesicular storage through VMAT2 knockdown.
Project 1 focused on DAT over-expressing transgenic mice that displayed detrimental outcomes
including loss of midbrain dopamine neurons, oxidative stress, L-DOPA reversible motor
deficits and enhanced vulnerability to MPTP-induced toxicity. These results clearly demonstrate
that increasing dopamine uptake and probable accumulation of cytosolic dopamine is harmful for
dopaminergic cells. Although these neurons are equipped to store, metabolize and release
dopamine, a modest modification in dopamine transport produces damaging consequences.
Hence, Project 1 provided evidence that enhancing cytosolic dopamine is sufficient to cause
Parkinson’s disease-like damage in mice. This implies that constantly handling a reactive
neurotransmitter like dopamine can render dopaminergic cells inherently vulnerable, which may
contribute to their heightened susceptibility to insult in Parkinson’s disease.
Project 2 focused on mice that simultaneously over-express DAT and under-express VMAT2.
This project was meant to carry forward the findings of Project 1 by further amplifying cytosolic
dopamine levels, however, due to reasons discussed above, DAT-tg/VMAT2-kd mice did not
show Parkinsonian features like dopamine cell loss or motor deficits. First, this suggests that
there exists a threshold of toxicity that must be breached in order to achieve dopamine cell loss.
Original DAT-tg mice showed higher protein expression of DAT and greater dopamine uptake,
which translated to loss of dopamine cells (Masoud et al., 2015), whereas DAT-tg mice from the
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DAT VMAT2 colony displayed comparatively lower DAT over-expression and less pronounced
increase in dopamine uptake, which translated to dampened phenotypes and intact nigrostriatal
dopamine neurons. This phenomenon regarding the extent of DAT over-expression has
previously been observed as well. Original DAT-tg mice were estimated to contain 6 copies of
the DAT gene (2 endogenous and 4 from the BAC) based on DAT Southern blot analysis and
they demonstrated dopamine cell loss (Salahpour et al., 2008; Masoud et al., 2015). Interestingly,
when these DAT-tg animals (6 copies of DAT) were crossed with DAT-KO mice (0 copies of
DAT), their progeny lost 1 functional copy of the DAT gene due to incorporation of the DAT-
KO allele. Hence, these mice were expected to display 5 copies of the DAT gene (1 endogenous
and 4 from the BAC) and remarkably, they no longer displayed dopamine cell loss (Salahpour
unpublished). Once again, these results demonstrate a threshold effect where a certain level of
DAT over-expression is required to manifest changes in dopamine cell survival. Comparison of
Project 1 and 2 also provides similar information, suggesting that loss of dopamine cells only
occurs once the degree of cytosolic dopamine accumulation exceeds a threshold beyond which
the cell can no longer combat dopamine-induced toxicity. Second, gathering parallel data from
DAT-tg mice with or without dopaminergic neurodegeneration, allows us to directly differentiate
between phenotypes that are dependent on dopamine cell loss (such as upregulation of dopamine
receptors, only observed in original DAT-tg mice) and those that are not (such as presence of
fine motor deficits, observed in both original DAT-tg mice and those from the DAT VMAT2
colony).
Moreover, results from Project 2 also offered novel and unexpected insight regarding the role of
dopamine compartmentalization in behavioral outcomes. Despite a 95% reduction in striatal
dopamine tissue content and 62% decrease in evoked dopamine release, DAT-tg/VMAT2-kd
mice were hyperactive and performed better than their counterparts in fine-motor and problem-
solving tasks. The mice were also extremely sensitive to amphetamine-induced locomotion,
showing abnormal involuntary movements at a dose of 2mg/kg of amphetamine. These findings
highlight the immense reserve capacity of the dopamine system in sustaining behavior despite
drastic alterations in dopamine homeostasis. In one sense, these findings also offer hope, of
being able to modulate and enhance motor activity in a system that was designed to be
hypodopaminergic. Evidently, the relationship between dopamine neurotransmission and
behavior is far more complex than we had previously anticipated, giving rise to new hypotheses
164
that could potentially explain these results (e.g. DAT reversal). Thus, characteristics of DAT-
tg/VMAT2-kd mice reveal untapped potential of the dopamine system to withstand and adapt to
modifications in dopamine compartmentalization, while “improving” behavioral outcomes. This
not only broadens our understanding of the dopamine system, but also provokes new thoughts
about ways to target this system therapeutically.
Collectively, the results of this thesis have confirmed our predictions regarding cytosolic
dopamine-induced toxicity (Project 1) and also enriched our existing knowledge of the dopamine
system by revealing novel behavioral findings in mice with altered dopamine
compartmentalization (Project 2).
4.4 Technical Challenges
During the course of Projects 1 and 2, a few technical challenges were faced that complicated
our interpretation of the results obtained. The first obstacle we encountered was regarding BAC
transgenic over-expression of the DAT gene in mice. We discovered that there was a
spontaneous loss of genomic DAT copy numbers in transgenic mice (see Appendix 2). This
problem was first found in the DAT-tg colony (Project 1) and then independently discovered in
the DAT-tg/VMAT2-kd colony (Project 2). During successive rounds of breeding, extra copies
of the DAT transgene can be lost producing “low” copy DAT-tg mice. Typically, DAT-tg mice
possess 6-8 copies of the DAT gene as determined by genomic quantitative PCR of the DAT
locus. However, “low” copy DAT-tg and DAT-tg/VMAT2-kd mice only possess one extra copy
of the DAT gene (3 copies total). This dampens the effects of DAT over-expression and
produces phenotypic variability. To control this, we routinely performed genomic qPCR to check
for DAT copy number. All results shown in this thesis are from “high” copy DAT-tg mice
(except in Appendix 2 which shows data from “low” copy DAT-tg mice).
Secondly, in Project 2, the technique of stereology was found to be unreliable. We conducted
stereological counts of midbrain dopamine neurons in collaboration with other laboratories that
routinely perform these experiments. We repeated stereology in 3 independent experiments to
ascertain the results. In one case, we sent the same slides to be counted by the same researcher
(blind to the genotypes) twice - and opposing results were obtained. Thus, even when controlling
for the user and tissue staining, this technique showed high variability. Since stereology was a
165
central part of this project, challenges with this technique, compounded with altered phenotypes
in DAT-tg mice, created a major hurdle in Project 2.
4.5 Future Directions
The results from this thesis, in particular Project 2, have raised several interesting questions that
warrant further exploration. With regards to Project 1, DAT-tg mice displayed dopaminergic
damage that is postulated to be caused by accumulation of cytosolic dopamine. However, since
cytosolic dopamine cannot be measured in vivo, it would be interesting to generate primary
neuronal cultures of dopaminergic neurons from DAT-tg mice and use intracellular patch
electrochemistry to measure dopamine levels in the cytosolic compartment (Mosharov et al.,
2003, 2009). This experiment would answer the question of how much cytosolic dopamine
accumulation occurs in DAT-tg mice. In addition, DAT-tg mice display enhanced sensitivity to
MPTP-induced toxicity. MPP+ is a substrate for DAT, therefore heightened toxicity in DAT-tg
mice may simply reflect increased access of the toxicant into dopaminergic neurons. To further
explore the interaction between genetic and environmental insults in disease pathogenesis, it is
important to test the effect of a toxicant in DAT-tg mice that is independent of DAT in its
activity. We attempted these experiments with the pesticide, rotenone, however these studies
were not successful due to 1) technical complications in administering a highly lipophilic drug
and 2) relative insensitivity of mice to rotenone-induced damage. Nonetheless, it would be useful
to investigate the response of DAT-tg mice to other environmental toxicants that have been
implicated in Parkinson’s disease, such as maneb. This would answer the question whether
mishandling dopamine can predispose DAT-tg mice to environmental insults and shed light on
the multiple hit hypothesis of Parkinson’s disease (Sulzer, 2007).
With regards to Project 2, there are several avenues that can be pursued to further elaborate on
the current results. First, function of the dopamine system can be further explored in DAT-
tg/VMAT2-kd mice. Although dopamine release, uptake, tissue content, cell number and
receptor levels have been evaluated in DAT-tg/VMAT2-kd mice, extracellular dopamine levels
and firing pattern of dopamine neurons should also be evaluated. These parameters are
instrumental in explaining the unique behavioral phenotypes of DAT-tg/VMAT2-kd mice, such
as motor hyperactivity, improved fine motor skill and problem solving ability compared to other
genotypes. Furthermore, if the theory of DAT reversal in DAT-tg/VMAT2-kd mice is true, then
166
we would expect extracellular dopamine levels to be increased as a result of DAT-mediated
dopamine efflux. Midbrain dopamine neurons fire spontaneously in pacemaking and/or burst
modes (Grace and Bunney, 1984). The endogenous firing pattern is important as it leads to
dopamine release and contributes to dopamine-related behaviors, such as reward-based learning
(Schultz et al., 1997; Reynolds et al., 2001). In fact, dopamine itself can regulate the firing
activity of dopamine neurons via feedback mechanisms (Paladini et al., 2003). Since the
dynamics of evoked dopamine release and uptake are drastically altered in DAT-tg/VMAT2-kd
mice, this could affect the firing pattern of dopamine cells. Hence, it would be informative to
investigate dopamine neuronal firing activity and extracellular dopamine levels in DAT-
tg/VMAT2-kd mice to explain some of their behavioral phenotypes.
Second, a robust finding of DAT-tg/VMAT2-kd that warrants examination is their poor survival.
Adult (12-week old) DAT-tg/VMAT2-kd mice demonstrate a 40-50% reduction in survival
when compared to age-matched WT, DAT-tg and VMAT2-kd mice. In addition, DAT-
tg/VMAT2-kd mice are also significantly smaller in size than WT animals. The dramatic
impairment in survival selectively affects animals that concurrently over-express DAT and
under-express VMAT2, indicating a synergistic negative effect of these two manipulations on
fitness. Although survival and body weight are complex traits that are influenced by several
factors including feeding, nutrition and development, it is evident that altering the balance of
dopamine compartmentalization produces detrimental outcomes in DAT-tg/VMAT2-kd mice.
Thus, further work unraveling the impact of both neuronal and peripheral dopamine signaling on
overall fitness is warranted.
Third, in comparison to female mice, survival and body weight measures seem to be more
drastically diminished in male DAT-tg/VMAT2-kd mice, indicating a significant role of sex in
determining response. In this thesis, neurochemical and behavioral data could not be stratified by
sex due to relatively smaller sample sizes and technical constraints including: 1) reduced survival
of male mice, 2) low probability (1/16) of obtaining an animal of a particular genotype and sex
and 3) small litter sizes (presumably because of dopamine and prolactin dysregulation).
However, it would be useful to investigate if other dopaminergic measures also show a sex bias
using larger sample sizes that are powered to detect those differences. Moreover, uncovering the
mechanisms that underlie the vulnerability of male DAT-tg/VMAT2-kd mice (and protection of
females), is particularly relevant because in humans, the likelihood of developing Parkinson’s
167
disease is also higher in men, suggesting a specific vulnerability of males to dopaminergic
malfunction (Van Den Eeden et al., 2003; Wooten et al., 2004).
Fourth, premature death of DAT-tg/VMAT2-kd mice adds a layer of complication in studying
these animals, since most experiments are conducted at or after 12 weeks of age when 46% of
DAT-tg/VMAT2-kd mice have already died. As a result, characterization of these mice
inadvertently occurs on the remaining animals that have survived to adulthood. It is conceivable
that those animals that naturally died beforehand may have expressed more extreme changes that
led to their demise in comparison to the mice that survived. This could also explain why the
phenotypes observed in DAT-tg/VMAT2-kd mice were less severe than hypothesized. We
anticipated drastic modifications in dopamine cell number, tissue content and receptor binding
beyond the levels observed in DAT-tg and VMAT2-kd mice alone. Perhaps these synergistic
changes were not observed because our studies were focused on DAT-tg/VMAT2-kd mice that
survived into adulthood and may have successfully compensated for their genetic shortcomings.
Thus, it would be useful to examine animals at earlier time points using non-invasive techniques
(e.g. behavior, imaging) and track their survival to determine whether those animals that die by
12 weeks of age, indicate signs of toxicity during early development. Also, closely tracking
mouse survival may allow for collection of viable tissue as soon as an animal is found dead. This
way, dopaminergic markers can be assessed in specific brain regions and compared to age-
matched mice.
Lastly, our experiments in Project 2 have focused primarily on the dopamine system. However,
reduction of VMAT2 levels in VMAT2-kd and DAT-tg/VMAT2-kd mice should affect all
monoaminergic systems where the protein is normally expressed. Furthermore, in addition to
dopaminergic neurodegeneration, VMAT2-kd mice are also reported to display 1) loss of
noradrenergic cells in the LC and 2) disruption of serotonin signaling (Taylor et al., 2014; Alter
et al., 2016). Some of the behavioral tests conducted on DAT VMAT2 mice focused on
attention, anxiety and cognitive phenotypes that are likely to be influenced by changes in
noradrenergic and serotonergic signaling. Therefore, it would be interesting to gauge the function
of these other monoamine systems in DAT-tg/VMAT2-kd mice. Although DAT over-expression
is confined to dopaminergic neurons, one can imagine that crippling the dopamine system of
DAT-tg/VMAT2-kd mice may unveil compensatory changes in noradrenergic or serotonergic
systems of these mice.
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Appendix 1
Project 2: Additional Experiments
A) In addition to the dorsal striatum, evoked-dopamine release and uptake were also assessed in
the nucleus accumbens of DAT VMAT2 mice using FSCV (slice preparations). Similar to
the dorsal striatum, dopamine release in the nucleus accumbens was significantly lower in
DAT-tg, VMAT2-kd and DAT-tg/VMAT2-kd mice in comparison to WT animals
(Appendix Fig 1.1B). However, in the nucleus accumbens (Appendix Figure 1.1A), overall
dopamine levels across all genotypes appeared lower than the dorsal striatum (Fig 3.19 A),
which is expected given the size and dopaminergic innervation of these regions. No
differences were observed in dopamine clearance using tau (Appendix Fig 1.1C). Hoffman
analysis of release and uptake were not performed due to multiple reasons: 1) lower evoked-
dopamine levels combined with quick uptake yields trace shapes that are not ideal for
Hoffman modeling in the nucleus accumbens, and 2) a caveat of using Hoffman modeling is
that individual data points must meet certain criteria in order to be included in the analysis
and as a result, some data had to be excluded yielding a sample size that is too low for
statistical analysis.
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Appendix Figure 1.1. Electrically-evoked dopamine release and uptake in the nucleus
accumbens determined by FSCV in slice preparations. (A) Traces of dopamine currents recorded
over time following a single-pulse stimulation. The ascending curve represents dopamine release
while the descending curve represents dopamine clearance. Dopamine release is estimated by (B)
peak amplitude (N=4-5). Dopamine clearance is estimated by (C) the decay time constant, tau
(N=4-5). Data presented as mean ± SEM. Statistical differences are in comparison to WT mice.
*p<0.05; **p<0.01.
197
B) For most dopaminergic drugs, the response of DAT VMAT2 mice was measured
behaviorally using motor outputs. However, in the case of cocaine, we also performed FSCV
measures of dopamine release and uptake in the dorsal striatum from brain slices. This
experiment was performed in collaboration with Dr. Miller at Emory University. As before,
redundant measures are used to estimate dopamine release (peak amplitude and Hoffman
analysis of release) and uptake (tau and Hoffman parameter for clearance). Appendix Figure
1.2 shows evoked-dopamine release and uptake in response to 3 ascending concentrations of
cocaine. These concentrations in slice preparations roughly correlate with 5-20 mg/kg of
cocaine i.p. in mice (Johnson et al., 2006; John and Jones, 2007; Yorgason et al., 2011). In
general, higher concentrations of cocaine tend to increase dopamine release (Appendix Fig
1.2 E, F) and decrease uptake (Appendix Fig 1.2 G, H) as a result of DAT inhibition. Since
baseline parameters are vastly different across the 4 genotypes (see 0 µM cocaine races in
Appendix Fig 1.2 A-D), release and uptake data were normalized to 0 µM cocaine and
expressed as fold change (Appendix Fig 1.2 E-H). In the normalized data, dopamine release
in DAT-tg mice was significantly more responsive to cocaine (Appendix Fig 1.2 E) than
other genotypes. This result may reflect the fact that cocaine acts on the transporter that is
over-expressed in DAT-tg mice. However, interestingly, DAT-tg mice are not different from
WT animals in their behavioral response to cocaine as previously shown (Fig 3.36 B),
suggesting that other factors aside from evoked-dopamine release may contribute to cocaine-
induced locomotor response (such as up-regulation of dopamine receptors).
198
199
Appendix Figure 1.2. Effects of cocaine on electrically-evoked dopamine release and uptake in
the dorsal striatum. These experiments were performed on brain slices using FSCV. Traces of
dopamine currents in the presence of ascending doses of cocaine (1, 3 and 10 µM) were recorded
over time following electrical stimulation in (A) WT, (B) DAT-tg, (C) VMAT2-kd and (D)
DAT-tg/VMAT2-kd mice. The ascending curve represents dopamine release while the
descending curve represents dopamine clearance. Dopamine release is estimated by (E) peak
amplitude (N=4-5) and (F) the Hoffman parameter, r/ke (N=2-4). Dopamine uptake/clearance is
estimated by (G) the decay time constant, tau (N=3-5) and (H) the Hoffman parameter, ku (N=3-
4). Data were normalized to 0 µM cocaine for each genotype. Results are presented as mean ±
SEM. Statistical differences are in comparison to WT mice. ***p<0.001.
C) In the last experiment of this section, we conducted a pilot study to evaluate whether the
behavioral effects of genotypic DAT over-expression and VMAT2 under-expression could
be mimicked pharmacologically. DAT-tg/VMAT2-kd mice display several unique
behaviors, the most striking of which is their basal hyperactivity. Typically, VMAT2
inhibitors such as reserpine or tetrabenazine, suppress locomotor activity in WT mice due to
diminished neurotransmitter packaging which depletes vesicular release (Colpaert, 1987).
However, our results show that, on a background of DAT over-expression, genetically
reducing VMAT2 function produces the opposite effect - increasing locomotor activity.
Therefore, in this preliminary experiment, tetrabenazine was used to assess whether
pharmacological reduction of VMAT2 activity on a background of DAT over-expression
(DAT-tg mice) would replicate the genetic condition of DAT-tg/VMAT2-kd mice. In
particular, only WT and DAT-tg mice were treated with tetrabenazine since VMAT2-kd
mice genetically express low levels of VMAT2. As shown in Appendix Figure 1.3, WT and
DAT-tg mice behave similarly in response to tetrabenazine or vehicle. The VMAT2
inhibitor certainly does not seem to produce the extent of hyperactivity witnessed in DAT-
tg/VMAT2-kd mice (Fig. 3.26). Another dose or preparation of tetrabenazine is needed to
differentiate between drug and vehicle treatment. Nonetheless, these preliminary data
suggest that long-term, genetic VMAT2-knockdown is required to produce basal
hyperactivity in DAT-tg/VMAT2-kd mice that cannot be recapitulated with a single, acute
dose of a VMAT2 inhibitor in DAT-tg mice.
200
Appendix Figure 1.3. Effect of tetrabenazine on locomotor activity of WT and DAT-tg mice.
WT and DAT-tg mice were habituated to the activity chamber for 30 minutes, injected with the
VMAT2 inhibitor, tetrabenazine (2 mg/kg i.p.) or vehicle solution consisting of 20% DMSO in
PBS, and monitored for an additional 90 minutes (N=6-8). (A) Distance traveled over time.
Arrow denotes time of injection. (B) Sum of total distance traveled after drug administration.
Data are presented as mean ± SEM.
201
Appendix 2
Project 1: DAT-tg mice with low number of DAT copies
Midway through Project 1, we discovered that our mouse colony was housing two strains of
DAT-tg mice: one with an expected “high” number of DAT copies and one with an unexpected
“low” number of transgenic DAT copies. DAT copy number was determined using genomic
quantitative PCR (qPCR) as shown below.
Appendix Figure 2.1. DAT copy number in WT and DAT-tg mice as assessed by genomic
qPCR. All results were normalized to WT mice (shown in blue) which contain 2 endogenous
copies of DAT. Number of DAT copies in DAT-tg mice was highly variable - ranging from 3-10
total copies. Based on copy number, DAT-tg mice were divided into 2 groups: 1) high copy
number (shown in green, 6 or more total copies) and 2) low copy number (shown in red,
typically containing 3 total copies). Experiment performed by Wendy Horsfall.
Initially, differences in DAT copy number were not detected because our routine mouse
genotyping was performed using PCR (instead of qPCR), which is unable to differentiate
between copy number. However, once qPCR revealed the presence of low copy DAT-tg mice,
we retroactively tested genomic samples from all the mice that were in our colony since it was
started. We discovered that one of the four founders that was brought to the University of
202
Toronto from Duke University (where DAT-tg mice were first generated), was a low copy DAT-
tg mouse. Breeding that low copy DAT-tg mouse gave rise to more such mice in the colony.
Thus far, all the results presented in this thesis used “high” copy number DAT-tg mice (between
6-10 total DAT copies as assessed by qPCR). However, some experiments were also
inadvertently conducted on “low” copy number DAT-tg mice (3 total copies: 2 endogenous and
1 BAC) before they were discovered in the colony. Results from these experiments are shown in
Appendix 2. Importantly, the number of DAT copies had significant impact on the phenotypes
observed.
First, the effect of MPTP administration on dopamine tissue content was assessed in the striatum
of low copy DAT-tg mice (Appendix Figure 2.2). No differences were observed between WT
and DAT-tg mice at any dose, unlike previous results from high copy DAT-tg mice (Figure
3.11). Notably, saline-treated high copy DAT-tg mice showed 25% reduction in dopamine tissue
levels compared to saline-treated WT mice (Fig 3.11) – an effect that is no longer observed in
low-copy DAT-tg mice (Appendix Figure 2.2). This confirms that low copy DAT-tg mice do not
show baseline changes in dopamine tissue content or enhanced sensitivity to MPTP-induced
toxicity (unlike high copy DAT-tg mice).
Appendix Figure 2.2. Striatal dopamine tissue content is shown for WT and low copy DAT-tg
mice treated with saline, 15 or 30 mg/kg of MPTP (n = 6-8). Data shown are means ± SEM.
203
Second, low copy DAT-tg mice were treated with rotenone, a pesticide associated with increased
risk of Parkinson’s disease. Osmotic minipumps containing 7mg/kg/day of rotenone were
surgically implanted in the subcutaneous cavity of WT and low copy DAT-tg mice. Drug
infusion was continued for 28 days after which animals were sacrificed and brains were
harvested to examine markers of dopaminergic damage in the striatum. Similar to MPTP results,
no differences were observed between WT and low copy DAT-tg mice in dopamine tissue
content (Appendix Figure 2.3 A) or TH immunofluorescence (Appendix Figure 2.3 C), when
treated with rotenone or vehicle. In general, this regiment of rotenone administration seemed to
be ineffective in producing toxicity since rotenone-treated WT mice were indistinguishable from
their vehicle-treated counterparts. However, once again, low-copy DAT-tg mice lacked the
reduction in dopamine tissue content (Appendix Figure 2.3 A) that was previously observed in
high copy DAT-tg animals (see saline treatment in Figure 3.11).
Appendix Figure 2.3. Effect of rotenone treatment (7 mg/kg, 28-day infusion) on low copy
DAT-tg mice. Rotenone is highly lipophilic therefore is was dissolved in a vehicle solution
consisting of polyethylene glycol and DMSO in a 1:1 ratio. (A) Striatal dopamine tissue content
after drug treatment (N=10-18). (B) Representative TH-labeled coronal images of the striatum
204
(one half) and (C) quantification of TH immunofluorescence (N=5-7), following drug treatment.
Data presented as mean ± SEM.
Third, protein carbonylation was evaluated in untreated low-copy DAT-tg mice as a general
marker of oxidative stress. Unlike the previous experiments where low copy DAT-tg mice did
not demonstrate evidence of toxicity (Appendix Figures 2.2-2.3), in this experiment, low copy
DAT-tg mice showed elevated levels of protein carbonylation, suggesting presence of general
oxidative stress (Appendix Figure 2.4). Interestingly, high copy DAT-tg mice did not display any
changes in protein carbonylation (Figure 3.4) when previously tested.
Appendix Figure 2.4. Protein carbonyl levels assessed in the striatum of low copy DAT-tg
mice. (A) Western blot and (B) quantification of protein carbonyls in synaptic plasma membrane
fractions from the striatum of WT and DAT-tg mice. Striata from 3-4 mice were pooled per
sample. Data presented as mean ± SEM. *p<0.05.
In summary, DAT-tg mice with low number of DAT copies are significantly different from the
original (high copy number) DAT-tg mice. Although low copy DAT-tg mice do not demonstrate
reduced dopamine tissue content or enhanced sensitivity to MPTP insult, they show evidence of
general oxidative damage. This suggests that one extra copy of DAT may lead to moderate
oxidative stress in the absence of dopaminergic damage.
205
Project 2: DAT-tg mice with low number of DAT copies
In the DAT VMAT2 colony, routine screening of genomic DAT copy number independently
identified low copy DAT-tg mice once again. This was a separate occurrence to the low copy
DAT-tg mice found in Project 1. In this case, when we retroactively traced the emergence of the
first low copy DAT-tg mouse in the DAT VMAT2 colony, it was found to have a high copy
DAT-tg parent and littermates, suggesting that the loss of copy number occurred spontaneously.
The variability in DAT copy number is shown below.
Appendix Figure 2.5. DAT copy number in DAT-tg mice from the DAT VMAT2 colony as
determined by genomic qPCR. All results were normalized to WT mice (shown in blue) which
contain 2 endogenous copies of DAT. Low copy DAT-tg mice from Project 1 (shown in orange,
3 total copies) were used as controls. DAT-tg mice from the DAT VMAT2 colony (shown in
green) demonstrate variability in the number of DAT copies. In particular, 2 mice were found to
have only 3 DAT copies (low copy mice, circled in red). Data presented as mean ± SD.
When baseline dopamine tissue content was assessed in the striatum of DAT VMAT2 mice, a
mixture of 8 high and 2 low copy DAT-tg mice were used unintentionally. In this initial
206
experiment, DAT-tg mice showed a 17% reduction in dopamine tissue content compared to WT
animals (Appendix Figure 2.6 A). However, once the 2 low copy DAT-tg mice were removed
from the analysis, the difference rose to 21% (Appendix Figure 2.6 B). This suggests that
including low copy DAT-tg mice dampened the effects of DAT over-expression on dopamine
tissue content.
Appendix Figure 2.6. Comparison of striatal dopamine tissue content in DAT-tg mice with (A)
mixed high and low DAT copy numbers versus (B) only high DAT copy numbers. A few
VMAT2-kd mice were included as experimental controls. Data presented as mean ± SD.
Statistical difference assessed between WT and DAT-tg mice. ***p<0.001.
Noting the apparent effect of DAT copy number on dopamine tissue content, a linear correlation
between these two parameters was evaluated using WT, high-copy DAT-tg and low copy DAT-
tg mice from the DAT VMAT2 colony (Appendix Figure 2.7). A significant negative correlation
was found: higher DAT copy number was associated with lower dopamine tissue content. This
suggests that greater DAT over-expression (high copy number) leads to greater dopaminergic
damage (loss of dopamine tissue content) in mice, possibly due to the deleterious effects of
cytosolic dopamine accumulation.
207
Appendix Figure 2.7. Linear regression of DAT copy number and dopamine tissue content
assessed in WT, high-copy DAT-tg and low copy DAT-tg mice from the DAT VMAT2 colony.
The regression analysis yielded a significant, negative slope (***p<0.001), indicating that the
two variables are inversely correlated. R2 = 0.55.
In summary, the independent emergence of low copy DAT-tg mice in both Projects 1 and 2
indicates that BAC transgenic mouse models that incorporate multiple copies of a gene are
sensitive to spontaneous loss of copy number over successive generations of breeding (Chandler
et al., 2007). Furthermore, alterations in DAT copy number produce differences in dopaminergic
phenotypes. This suggests that the level of DAT over-expression (or the number of DAT gene
copies) is integral in determining dopaminergic outcomes.
208
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WILEY OPEN ACCESS TERMS AND CONDITIONS
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v1.10 Last updated September 2015
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