Mutant Cu,Zn Superoxide Dismutase in Amyotrophic Lateral Sclerosis Molecular Mechanisms of Neurotoxicity Doctoral dissertation To be presented by permission of the Faculty of Natural and Environmental Sciences of the University of Kuopio for public examination in Auditorium, Mediteknia building, University of Kuopio, on Saturday 3 rd May 2008, at 12 noon Department of Neurobiology A.I. Virtanen Institute for Molecular Sciences University of Kuopio TONI AHTONIEMI JOKA KUOPIO 2008 KUOPION YLIOPISTON JULKAISUJA G. - A.I. VIRTANEN -INSTITUUTTI 61 KUOPIO UNIVERSITY PUBLICATIONS G. A.I. VIRTANEN INSTITUTE FOR MOLECULAR SCIENCES 61
Series Editors: Research Director Olli Gröhn, Ph.D. Department of Neurobiology A.I . Virtanen Institute for Molecular Sciences
Research Director Michael Courtney, Ph.D. Department of Neurobiology A.I . Virtanen Institute for Molecular Sciences
Author’s address: Department of Neurobiology A.I . Virtanen Institute for Molecular Sciences University of Kuopio P.O. Box 1627 FI-70211 KUOPIO FINLAND
Supervisors: Professor Jari Koistinaho, M.D., Ph.D. Department of Neurobiology A.I . Virtanen Institute for Molecular Sciences University of Kuopio
Docent Gundars Goldsteins, Ph.D. Department of Neurobiology A.I . Virtanen Institute for Molecular Sciences University of Kuopio
Reviewers: Docent Pekka Rauhala, M.D., Ph.D. Institute of Biomedicine University of Helsinki Helsinki, Finland
Professor Dan Linholm, M.D., Ph.D. Minerva Research Institute University of Helsinki Helsinki, Finland
Opponent: Professor Michael Thomas Heneka, M.D. Department of Neurology University of Münster Münster, Germany
ISBN 978-951-27-1120-8ISBN 978-951-27-1102-4 (PDF)ISSN 1458-7335
KopijyväKuopio 2008Finland
Ahtoniemi, Toni. Mutant Cu,Zn Superoxide Dismutase in Amyotrophic Lateral Sclerosis:Molecular Mechanisms of Neurotoxicity. Kuopio University Publications G. - A.I. VirtanenInstitute for Molecular Sciences 61. 200 . 107 p.ISBN 978-951-27-1120-8ISBN 978-951-27-1102-4 (PDF)ISSN 1458-7335
ABSTRACTAmyotrophic lateral sclerosis (ALS) is an untreatable and fatal neurodegenerative disease thatleads to muscle atrophy, paralysis of voluntary muscles and death. ALS is charactherized bythe selective destruction of upper and lower motor neurons in the spinal cord, brain stem andmotor cortex. The majority of ALS cases are sporadic, whereas 5–10% of cases have a geneticcomponent and are familial. The typical age of onset for both forms is between 50 and 60years and prevalence is approximately 4-6 in 100 000 individuals per year. The causes formost cases of ALS are unknown and the clinical course is highly variable, suggesting thatmultiple factors underlie the disease mechanism.
Mutations in the ubiquitously expressed protein, Cu,Zn-superoxide dismutase (SOD1),are associated with about 20% of familial ALS cases. Because the pathology and clinicalsymptoms of familial and sporadic ALS cannot be distinguished, transgenic animal modelsover-expressing mutant SOD1 offer a valuable tool for understanding the pathogenicmechanisms shared by both sporadic and familial forms of ALS. Importantly, severalpathogenic SOD1 mutations do not affect SOD1 activity significantly, and a hypothesis for 'atoxic gain of function' of the mutated protein rather than a lack of its antioxidant function, hasbeen postulated.
In this work, we used transgenic ALS rats and mice to address the role of SOD1 inALS pathogenesis by analyzing the proteomic profile in the spinal cord and by analyzingstability and oxidation state of human mutant SOD1 through the disease progression. Inaddition, we set out to investigate the role of mutant SOD1 in mitochondria.
Results showed that mutant SOD1 is oxidized and destabilized in the affected tissuesof the transgenic animals. A role of aggregation was further supported by the finding ofincreased chaperone expression in proteome profiling. Moreover, SOD1 may have adeleterious role in the intermembrane space of mitochondria, however, not because ofaggregation, but caused instead by uncontrolled activity of the enzyme that can lead toincreased production of harmful reactive oxygen species and damage to mitochondria. Inaddition, we also tested an anti-oxidant/inflammatory drug treatment with pyrrolidinedithiocarbamate for SOD1 transgenic ALS rats. However, the result of this drug treatmentwas unexpected as the treatment did not provide neuroprotection, but in opposite to thehypothesis, accelerated the disease progression. The mechanism of action showed a newtarget for the drug as the beneficial anti-oxidative and anti-inflammatory effects wereoverridden by a harmful inhibition of immunoproteasome induction.
The results of this thesis show the importance of mammalian specific proteasomecomponent in the disease pathogenesis of ALS. Moreover, our results demonstrate a newmitochondrial target and mechanism for SOD1 neurotoxicity that applies both to sporadic andfamilial ALS cases.
National Library of Medicine Classification: WL 359, WE 550, QU 58.5, QU 140, QU 350,QZ 180Medical Subject Headings: Neurodegenerative Diseases/etiology; Motor Neuron Disease;Amyotrophic Lateral Sclerosis; Superoxide Dismutase; Mutant Proteins; Proteomics; SpinalCord; Mitochondria; Oxidative Stress; Reactive Oxygen Species; Molecular Chaperones;Proteasome Endopeptidase Complex; Disease Models, Animal; Mice; Rats; Drug Therapy;Antioxidants; Pyrrolidines
8
"Nothing shocks me. I'm a scientist."Indiana Jones
ACKNOWLEDGEMENTS
This study was carried out in the Molecular Brain Research Group, Department ofNeurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, duringthe years 2002-2007.
I wish to express my sincere gratitude to my principal supervisor, Professor Jari Koistinaho,M.D., Ph.D., for his supervision, wonderful insight on neurobiology and excellent leadership.It has truly been a priviledge to work in the MBR-group for these past years. I couldn’t evenbegin to imagine a better choice for PhD-studies than the MBR-group with Jari as asupervisor. I also wish to thank my supervisor Docent Gundars Goldsteins, Ph.D., for hissupervision, unbelievable technical and scientific insight and for scrutinizing andtroubleshooting. Gundars, Gunu or Mr. Wizard, your input on my views of science has beentruly remarkable and your views and advices will be always heard on every word.
I am grateful to the official reviewers of this thesis, Professor Dan Lindholm, M.D., Ph.D., atMinerva Institute for Medical Research, University of Helsinki and Docent Pekka Rauhala,M.D., Ph.D. at Institute of Biomedicine, University of Helsinki for their valuable evaluationof this thesis.
I am thankful to my co-authors for their contiribution that is beyond any price. Velta Keksa-Goldsteine, M.Sc., and Merja Jaronen, M.Sc., thank you for your work in the protein lab -keep Gunu's lab a happy and a productive place. I want to thank from the bottom of my heartKatja Kanninen, M.Sc., and Tarja Malm, Ph.D., who have shared many memorable momentsthrough these years. Katja and Tarja, your contribution and friendship goes beyond any scaleimaginable. I am also deeply thankful to the co-authoring past members of the A.I.VirtanenInstitute, Eija Seppälä, Ph.D., Egils Arens, Ph.D., and Karl Åkerman, Ph.D., as well as to co-authors outside AIVI, Antero Salminen, Ph.D., at the Department of Neurology, SeppoAuriola, Ph.D., at the Department of Pharmaceutical Chemistry, University of Kuopio,Caterina Bendotti, Ph.D., Department of Neuroscience, Istituto di Ricerche FarmacologicheMario Negri, Milan, Italy, and Pak Chan, Ph.D., Department of Neurosurgery, StanfordUniversity School of Medicine, Stanford, California, USA.
My warm thanks goes to our lovely technicians Mirka Tikkanen and Laila Kaskela for theirhelp, contribution and friendship in and outside the lab. I would also like to thank all themembers of the MBR-group; Susanna Boman, VMD, Riikka Heikkinen, M.Sc., Georges FulKuh, M.Sc., Johanna Magga, Ph.D., Anu Muona, Ph.D., Rea Pihlaja, M.Sc., Eveliina Pollari,M.Sc., Yuriu Pomeshchick, M.D., and Taisia Rolova, M.Sc. Many thanks goes to the pastmembers of MBR-group and participants of the legendary New Orleans SFN tour of 2003;Antti “Possum Jenkins” Nurmi, Ph.D., and Suvi Leskinen, M.Sc. The previous generations ofMBR-PhDs Tiina “Tikka” Kauppinen, Ph.D., Kaisa Kurkinen, Ph.D. and Nina Vartiainen,Ph.D. are also cordially acknowledged. A special thank you goes to a past member of theMBR-group and my current boss Docent Milla Koistinaho Ph.D. for giving me a job-opportunity of a lifetime at Medeia. From AIVI staff I would also like to thank Docent RiittaKeinänen and Mrs. Sari Koskelo for their help in countless affairs and in their dedication toMBR-group.
Love and gratitude goes for my parents Tuija and Hannu, who have always encouraged me tostudy hard and reach as high as possible. I would like to thank my brother Janne for hisfriendship, views on life and for being an excellent brother for the years past and to come. Iwould also like to thank Jannes’s lovely wife Maiju, even lovelier daughter Ella and
newcomer Akseli. Lots of love and kisses to my littlesister Ellinoora. Families Nygård andJärvelä are also warmly acknowledged.
Finally, all my love goes to my wife Pauliina and sons Tomas and Leevi. Thank you for yourlove, support and generally just for putting up with me during the finalizing steps of thisthesis... In the end family is what matters the most and you are the counterbalance in my life.
This thesis is based on the following original publications, which in text are referred to by
their roman numerals.
I Ahtoniemi T, Goldsteins G, Keksa-Goldsteine V, Malm T, Kanninen K, Salminen A,
and Koistinaho J. Pyrrolidine dithiocarbamate inhibits induction of
immunoproteasome and decreases survival in a rat model of amyotrophic lateral
sclerosis. Mol Pharmacol. 2007 Jan;71(1):30-37
II Ahtoniemi T, Jaronen M, Keksa-Goldsteine V, Goldsteins G, and Koistinaho J.
(2008). Mutant SOD1 from spinal cord of G93A rats binds to inner mitochondrial
membrane and increases ROS production. Submitted.
III Goldsteins G, Keksa-Goldsteine V*, Ahtoniemi T*, Jaronen M, Egils A, Åkerman K,
Chan PH, and Koistinaho J. Deleterious role of superoxide dismutase in the
mitochondrial intermembrane space. J. Biol. Chem. 2008 Mar 28;283(13):8446-52
IV Ahtoniemi T, Seppälä E, Goldsteins G, Auriola S, Bendotti C, and Koistinaho J.
Proteomic analysis of protein expression and oxidation in a mouse model of ALS.
Manuscript.
*Contributed equally to the work
TABLE OF CONTENTS
1. INTRODUCTION ............................................................................................................ 152. REVIEW OF THE LITERATURE ................................................................................... 17
2.1 Amyotrophic lateral sclerosis (ALS) ........................................................................... 172.1.1 ALS and other motor neuron diseases................................................................... 17
2.1.1.1 Motor neuron system ......................................................................... 172.1.1.2 Motor neuron diseases ....................................................................... 18
2.1.2 Characteristics of ALS ......................................................................................... 192.1.3 Genetics of ALS ................................................................................................... 21
2.1.3.1 Sporadic and Familial ALS ................................................................ 212.1.3.2 ALS1 - SOD1 .................................................................................... 232.1.3.3 ALS2, ALS4 and ALS5 - Juvenile forms ALS ................................... 252.1.3.4 ALS3, ALS6 and ALS7 with classical late-onset phenotype ............... 262.1.3.5 ALS with dementia ............................................................................ 262.1.3.6 ALS8 and progressive lower motor neuron disease - atypical ALS ..... 272.1.3.7 VEGF and ANG ................................................................................ 27
2.1.4 Models of ALS..................................................................................................... 282.1.4.1 Transgenic SOD1 models................................................................... 282.1.4.2 Other in vivo models of motor neuron degeneration ........................... 302.1.4.3 In vitro models of ALS ...................................................................... 32
2.2 Mechanisms for motor neuron cell death ..................................................................... 332.2.1 Oxidative damage ................................................................................................ 33
2.2.2 Protein Aggregation ............................................................................................. 362.2.2.1 Aggregates ......................................................................................... 362.2.2.2 Proteasome and Immunoproteasome .................................................. 372.2.2.3 Chaperones ........................................................................................ 382.2.2.7 Neurofilaments and axonal transport .................................................. 39
2.2.3 Glutamate excitotoxicity ...................................................................................... 392.2.4 Inflammation........................................................................................................ 402.2.5 Mitochondria........................................................................................................ 412.2.6 Role of non-neuronal cells .................................................................................... 432.2.7 Pathway of motor neuron cell death in ALS ......................................................... 45
2.3 Therapeutics for ALS .................................................................................................. 472.3.1 Drug treatments .................................................................................................... 482.3.2 Growth factors ..................................................................................................... 492.3.3 Gene therapies...................................................................................................... 502.3.4 Stem cell therapies ............................................................................................... 502.3.5 Pyrrolidine dithiocarbamate (PDTC) .................................................................... 52
3. AIMS OF THE STUDY ................................................................................................... 544. MATERIALS AND METHODS ...................................................................................... 55
4.3.1 Isolation of mitochondria ..................................................................................... 574.3.2 Functional integry of the isolated mitochondria .................................................... 584.3.3 Isolation of mitoplasts .......................................................................................... 584.3.4 Exposure of mitoplasts with cytosolic homogenates of G93A-SOD1 rat tissues.... 584.3.5 Measurement of ROS production ......................................................................... 594.3.6 Isolation of intermembrane space and measurement of SOD1 activity .................. 59
4.3.7 SOD activity with zymography ............................................................................ 594.4 Western blotting (I-III)................................................................................................ 60
4.4.1 Sample preparation .............................................................................................. 604.4.2 Electrophoresis and transfer ................................................................................. 604.4.3 malPEG modification of free cysteines ................................................................. 604.4.4 Antibodies ............................................................................................................ 61
4.9.1 Protein extraction ................................................................................................. 634.9.2 Carbonyl derivatization and detection................................................................... 634.9.3 Two dimensional electrophoresis ......................................................................... 644.9.4 In-gel digestion .................................................................................................... 644.9.5 Mass spectrometry ............................................................................................... 65
4.10 Flow cytometry (III) ................................................................................................. 654.10.1 Peroxide production in mouse blood lymphocytes .............................................. 654.10.2 Antimycin A-induced apoptosis in lymphocytes ................................................. 65
4.11 Isolation and purification of human SOD1 (II,III) ..................................................... 664.12 Measurements of cytochrome c catalysed peroxidation (III) ...................................... 664.13 Statistical analysis (I-IV) ........................................................................................... 66
5. RESULTS ........................................................................................................................ 675.1 PDTC reduced survival of G93A-SOD1 ALS rats without affecting NF- B (I) ........... 675.2 Copper levels were increased in ALS rat tissues and were further increased in spinalcords by PDTC treatment (I) ............................................................................................. 685.3 PDTC inhibited immunoproteasome (I) ...................................................................... 685.4 PDTC upregulated GLT-1 (I) ...................................................................................... 695.5 PDTC prevented glial immunoproteasome induction (I) .............................................. 695.6 Mutant SOD1 was oxidized and destabilized in spinal cords of G93A-SOD1 rodentmodels (II,IV) ................................................................................................................... 705.7 PDI was upregulated and its levels inversely correlated with the levels of cysteinereduced SOD1 (II,IV) ....................................................................................................... 715.8 Mitochondrial SOD1 levels were the highest in the spinal cord of G93A rats (II) ........ 725.9 Mutant SOD1 bound to mitoplasts and enhanced ROS production (II) ........................ 725.10 Mutant SOD1 increased hydrogen peroxide production in the intermembrane space ofmitochondria (III) ............................................................................................................. 735.11 Mutant SOD1 activity and ROS production were increased in spinal cordmitochondria of G93A-SOD1 mice (III) ........................................................................... 75
6. DISCUSSION .................................................................................................................. 776.1 PDTC inhibits immunoproteasome induction resulting in reduced survival of ALS rats(I) ..................................................................................................................................... 776.2 Mutant SOD1 oxidation and destabilization precede aggregation and loss of activity (II,IV) .................................................................................................................................... 796.3 Destabilized mutant SOD1 associates with inner membrane of mitochondria andincreases ROS production (II) ........................................................................................... 816.4 Elevated SOD1 activity in the intermembrane space leads to increased hydrogenperoxide production resulting in cytochome c catalyzed oxidation (III) ............................. 826.5 Hypothesized role of destabilized mutant SOD1 toxicity in mitochondria ................... 85
7. SUMMARY AND CONCLUSIONS ................................................................................ 878. REFERENCES ................................................................................................................. 89
15
1. INTRODUCTION
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by loss of
upper and lower motor neurons in the spinal cord, brain stem and motor cortex. Loss of motor
neurons leads to muscle atrophy, paralysis of voluntary muscles and death in 3-5 years from
onset of the disease with failure of the respiratory muscles usually being the fatal event
(Kandel et al., 1991). What makes ALS a very cruel disease is the fact that although the body
becomes completely paralysed and the ability to communicate is lost, the mind and senses
remain intact as sensory neurons or autonomic neurons are not affected (Kandel et al., 1991).
5–10% of ALS cases are inherited (familial) whereas the majority of cases have no genetic
component (sporadic) (Kurland and Mulder, 1955). The typical age of onset for familial form
is 46 years and 56 years for sporadic with prevalence of approximately 4-6 in 100 000
individuals per year (Camu et al., 1999; Yoshida et al., 1986). This corresponds to
approximately 150 new affected individuals in Finland every year, whereas the total number
of all affected people is approximately 350 in Finland, 5000 in the United Kingdom and 30
000 in the United States or Europe. The causes for most cases of ALS are unknown and the
clinical course is highly variable, suggesting that multiple factors underlie the disease
mechanism.
Mutations in the ubiquitously expressed protein, Cu,Zn superoxide dismutase (SOD1)
are associated with about 20% of familial ALS cases (Rosen et al., 1993). Because the
pathology and clinical symptoms of familial and sporadic ALS cannot be distinguished,
transgenic animal models over-expressing mutant SOD1 (Bruijn et al., 1997b; Gurney et al.,
1994; Howland et al., 2002; Jonsson et al., 2004; Nagai et al., 2007; Ripps et al., 1995; Wang
et al., 2003; Wang et al., 2005; Wong et al., 1995) offer a valuable tool for understanding the
pathogenic mechanisms shared by both sporadic and familial forms of ALS. Importantly,
several pathogenic SOD1 mutations do not affect SOD1 activity significantly, and a
hypothesis for 'a toxic gain of function' of the mutated protein rather than a lack of its
antioxidant function, has been postulated (Gurney et al., 1994; Wong et al., 1995). The nature
of this gained toxic function is not known, even though several putative pathogenic
mechanisms have been discovered, including formation of protein aggregates, saturation of
proteasome and protein folding chaperones, mislocalization and aggregation of
neurofilaments, inflammation, increased radical generation and oxidative damage,
mitochondrial dysfunction, and pro-apoptotic alterations (Boillee et al., 2006a). Despite the
many proposed disease mechanisms, the underlying initiative cause of the mutant SOD1
toxicity is still unknown. Mutations of SOD1 may affect the stability of the enzyme making it
16
more prone to aggregate or mutations may alter the catalytical site allowing aberrant
substrates to enter the catalytical site and cause unwanted oxidative reactions (Cleveland and
Rothstein, 2001). In recent studies, mitochondrial localisation of mutant SOD1 has been
implicated in ALS pathogenesis (Bergemalm et al., 2006; Deng et al., 2006; Ferri et al., 2006;
Liu et al., 2004; Vijayvergiya et al., 2005) and increased recruitment of mutant SOD1 to
mitochondria in the spinal cord might be the basis of the specific cell death of motor neurons.
However, the exact mechanisms of the selectivity and toxicity are not entirely clear.
This study was carried out to further address the role of SOD1 in ALS pathogenesis by
analyzing the proteomic profile in the spinal cord of ALS mice and by analyzing the stability
and oxidation state of human mutant SOD1 through the disease progression in transgenic
ALS mice and rats. In addition, we set out to investigate the deleterious role of mutant SOD1
in mitochondria. Finally, as oxidative damage, inflammation and apoptosis play major roles in
the pathology of ALS, we tested whether anti-inflammatory and anti-oxidative drug treatment
with pyrrolidine dithiocarbamate might have neuroprotective effects on G93A-SOD1
transgenic rats.
17
2. REVIEW OF THE LITERATURE
2.1 Amyotrophic lateral sclerosis (ALS)
2.1.1 ALS and other motor neuron diseases
2.1.1.1 Motor neuron system
The motor neuron system gives us the means to interact with the world that surrounds us from
the very basic aspects such as breathing, chewing and moving to more complex motor
performances such as controlling the air flow and the shape of oral cavity to form sound as in
speech or singing, or to delicately control movement of finger and hand to be used in writing
or playing an instrument. The neural components of the motor system extend from the highest
reaches of the cerebral cortex as upper motor neurons of the motor cortex to innervate spinal
cord and to farthest terminals of the motor axons as lower motor neurons of spinal cord
connect to muscles. There are two types of lower motor neurons: alpha motor neurons and
gamma motor neurons. Alpha motor neurons directly trigger muscle contraction and gamma
motor neurons innervate and activate intrafusal muscle fibers, which provide information and
additional control on the force of contraction and length of the muscle (Squire et al., 2003a).
Each lower alpha motor neuron descending along the spinal cord or brain stem sends
an axon to one muscle to innervate muscle fibers (from a few to a hundred or more) forming a
motor unit. As there are two types of voluntary muscle fibers - red for aerobic long-lasting
performances and white for anaerobic explosive and fast movements, there are also different
subtypes of motor units: slow fatigue resistant motor units, fast fatiguable and fast fatigue
resistant motor units. Motor neurons of the fast units are generally bigger and have a larger
diameter, faster conducting axons and generate high frequency bursts of action potentials.
Slow units have a smaller diameter and more slowly conducting axons with relatively steady
and lower frequency activity (Squire et al., 2003a).
Motor neurons are activated by interneurons of different motor programs descending
from the forebrain and the brain stem allowing activation of motor neurons with great
precision. Interneurons and motor neurons form neuronal networks containing information for
specific motor performances such as swallowing, walking or breathing and as the
corresponding network is activated, by will or reflex, the given function of the network is
executed. These networks contain motor programs for various activities form lying
horizontally in bed, which requires little activity to more complex programs like walking,
which requires sequentially activated motorneurons/muscles to act in concert with sensory
receptors for keeping the balance. The spinal cord contains motor programs for locomotion
18
and for protective reflexes, the brain stem has the programs for swallowing, chewing,
breathing and fast saccadic eye movement, whereas motor programs for fine motor skills such
as speech and control of hands and fingers are located in the motor cortex (Squire et al.,
2003b).
2.1.1.2 Motor neuron diseases
Motor neuron diseases (MND) are a class of diseases that affect the motor system leading to
muscle atrophy and in the worst case scenario to paralysis of the muscles as the motor
neurons that give the signals to muscles are lost. Motor neuron diseases can be divided into
three different categories on the basis of which class of motor neurons (upper motor neurons,
lower motor neurons or both) the disease affects. Spinal muscular atrophy (SMA) is a pure
lower motor neuron disorder; progressive bulbar palsy (PBP) and progressive muscular
atrophy (PMA) have isolated lower motor neuron signs. Primary lateral sclerosis (PLS) has
only upper motor neuron involvement, whereas amyotrophic lateral sclerosis (ALS) affects
both upper and lower motor neurons and this is one of the key characteristics of ALS
(Donaghy, 1999; Rocha et al., 2005).
ALS is the most common form of MNDs with the prevalence of 4-6 affected
individuals per 100 000 (Yoshida et al., 1986) and also the most devastating, as ALS usually
leads to complete paralysis and death in 3-5 years from the diagnosis of the disease and there
is no known cure. What makes ALS so devastating is the fact that it only affects the motor
neurons while there is no cognitive decline nor is the autonomic system affected, leaving the
person affected by ALS capable to follow the progression of the disease and the deteriation of
the body and losing the ability speak and to swallow while the mind still remains as sane as it
is possible in that situation (Kandel et al., 1991).
Other types of progressive adult MNDs include primary lateral sclerosis (PLS),
progressive muscular atrophy (PMA), progressive bulbar palsy (PBP) and only lower motor
neurons involving spinal muscular atrophy (SMA). PLS affects only upper motor neurons
leading to spasticity starting in the legs and ascending to the arms and finally to the bulbar
muscles. PLS has an average age of onset at 50 years and slow disease progression of more
than 15 years. PLS is not fatal, but affects the quality of life and PLS may often develop into
full scale ALS (Pringle et al., 1992). PBP affects lower motor neurons, which control bulbar
muscles leading to pharyngeal muscle weakness and to almost always evident but less
prominent limb weakness with both lower and upper motor neuron signs. PMA has only
lower motor neuron signs with muscle involvement mainly in limbs but also body trunk and
19
bulbar region can be affected. Both PBP and PMA often progress to classic ALS (Rocha et
al., 2005). SMA is a pure lower motor neuron disease that can be genetic or sporadic and
generally predominate in the legs. SMA has various forms, with different ages of onset,
patterns of inheritance, and severity and progression of symptoms (Harding and Thomas,
1980).
2.1.2 Characteristics of ALS
ALS was initially characterized first by a french neurologist and physican Jean-Martin
Charcot in 1869 and was known initially as Charcot's sclerosis (Charcot and Joffory, 1869).
The fact that ALS was characterized already in 1869 makes ALS as a described disease some
37 years older than Alzheimer's disease, which was reported to the medical society in 1907 by
Alois Alzheimer (Alzheimer, 1907a; Alzheimer, 1907b). Although ALS as a disease is more
rare than AD and it was still described almost forty years sooner than Alzheimer's disease,
emphasizes the importance of the motor system. On the other hand ALS is also more visible
and leaves more evident marks. What Charcot described already 139 years ago was the
observation of a distinct "myelin pallor" in the lateral portions of the spinal cord, representing
the degeneration and loss of motor neurons as they descend from the brain to connect to the
lower motor neurons within the spinal cord.
Clinical features of ALS include progressive muscle weakness, atrophy and spasticity
resulting in the end in complete paralysis of voluntary muscles as motor neurons degenerate
(Figure 1) (Kandel et al., 1991). Muscle weakness and atrophy is mainly caused by the
degeneration of lower motor neurons whereas spasticity reflects the loss of upper motor
neurons. Respiratory failure caused by the denervation of respiratory muscles or pneumonia is
usually the fatal event. ALS is fatal and there is no known cure. The most common first
symptom is weakness of one arm or leg and clumsiness of the hands. Legs can have cramps.
Muscle weakness and atrophy spread from the distal parts of the limbs to the proximal
direction to other limbs, to muscles taking care of breathing and to the bulbar region.
Alternatively, muscle weakness starts from the bulbar region and spreads in the opposite
direction. The first symptom of the bulbar region is weakening of speech and swallowing. The
disease progresses to atrophy of the voluntary muscles and paralysis without any sensory
symptoms, nor is the autonomic nervous system affected and there is no cognitive decline
(Kandel et al., 1991). However, there is also selectivity between the destruction of motor
neurons as the motor neurons controlling the bladder and sphincters are spared and also eye
20
movement is relatively spared and is affected only in the very late stage of the disease
(Kandel et al., 1991).
Figure 1. In ALS both upper and lower motor neurons degenerate. Motor neuronslocated in the brain, brain stem, and spinal cord serve as controlling units and vitalcommunication links between the nervous system and the voluntary muscles of the body.Messages from motor neurons in the brain (upper motor neurons) are transmitted to motorneurons in the spinal cord (lower motor neurons) and from them to particular muscles. InALS, both the upper motor neurons and the lower motor neurons degenerate or die, ceasing tosend messages to muscles. Unable to function, the muscles gradually weaken, waste away(atrophy), and twitch (fasciculations). Eventually, the ability of the brain to start and controlvoluntary movement is lost. As bulbar motor neurons are lost speaking, swallowing and facialexpressions are affected. Death is usually caused by respiratory failure. Modified from:http://www.als-mda.org/publications/fa-als.html.
SOD1 (Cu,Zn superoxide dismutase). Converts superoxide to water and hydrogen peroxide.UnknownUnknownUnknown
Dominant
DominantDominantDominant
Adult. 20% of inherited cases
AdultAdultAdult
Juvenile ALSALS2
ALS4
ALS5
2q33
9q34
15q15.1- q21.1
ALS2/alsin. Guanine exchange factor for Rab5 and RAc1. Organization of actin cytoskeleton / vesicle trafficking.SETX (Senataxin). Putative DNA/RNA helicase.
Unknown
Recessive
Dominant
Recessive
Juvenile Heterogeneous disease.
Juvenile. (Recessive mutations cause ataxia-oculomotor apraxia type 2Juvenile
ALS with dementiaALS-FTD
ALS-FTDP
9q21-22
17q21.1
Unknown
MAPT (Tau) Microtubule associated protein
Dominant
Dominant
Adult. ALS with frontotemporal dementia (FTD)Adult. ALS-FTD and Parkinson's Disease
Atypical ALSALS8
Progressivelower motorneurondisease
20q13.3
2p13
VABP (VAMP-associated proteinB). May be involved in vesiculartrafficking.
DCTN1 (Dynactin 1). Axonal transport of cellular organelles and proteins.
Dominant
Dominat
Adult. Heterogenous disease. Most cases with tremor; some typical ALS; 25% is late-onset spinal muscular atrophyAdult. Vocal fold paralysis; atrophy of hands and facial muscles
Better understanding of the factors influencing inheritance such as multiple effects of
single genes (pleiotropy), the interaction of multiple genes with each other (epistasis), the
interaction of genes with environmental factors, splice variants of genes, variations in copy
number and post translational modifications (as reviewed in Simpson and Al-Chalabi, 2006),
have shown that the 'one gene, one trait' model of diseases can be regarded as too simple for
23
complete description of disease risk, though it is useful for identifying genes for Mendelian
genetics. Taking these things into consideration, it is not surprising that the identified
Mendelian genes which cause ALS account for a very small percentage of cases, despite the
use of modern gene mapping methods. High genetic heterogenity and complex interactions
between genetic and environmental factors make ALS a multifactorial complex disease.
A genetic link has been determined for about 10% of ALS cases, but the mechanism
for inheritance is far from unambiguous. The identified chromosomal loci with mutations
causing ALS or ALS-like symptoms have been defined as ALS1-8, as well as for progressive
lower motor neuron disease, ALS with frontotemporal dementia (ALS-FTD) and ALS-FTD
with Parkinson's disease (ALS-FTDP) (as reviewed in Gros-Louis et al., 2006). Out of these
listed loci, six have identified genes with Mendelian genetics, namely: ALS1, ALS2, ALS4,
ALS8, ALS-FTDP and progressive lower motor neuron disease. Also, mutations in
angiogenin (ANG), vascular endothelial growth factor (VEGF) and sequence variants in
neurofilament genes have been reported. As a common factor, some of the identified genes
seem to be involved in intracellular trafficking, axonal transport and RNA metabolism.
However, the nomenclature on the genetics of familial ALS cases can be misleading,
as only ALS1, ALS3, ALS6, ALS7 mutations in ANG and VEGF and some of the ALS8
cases have the classical ALS phenotype with late onset and degeneration of both upper and
lower motor neurons that leads to progressive paralysis. Whereas ALS2, ALS4, ALS5 may
have juvenile onset, ALS8 and progressive lower motor neuron disease have only lower
motor neuron signs and ALS-FTD and ALS-FTDP feature dementia.
2.1.3.2 ALS1 - SOD1
The most common form of inherited ALS, about 20% of familial ALS, is caused by mutations
at chromosome 21q22.1 in the gene encoding protein Cu,Zn superoxide dismutase (SOD1),
also known as ALS1. These mutations correspond to 1-2% of all ALS cases. The function of
this ubiquitously expressed, 153 amino acid residues long cytoplasmic homodimeric enzyme
is to convert superoxide anion, a free radical of reactive oxygen species, to water and
hydrogen peroxide, which is further on detoxified by catalase and glutathione peroxidase
(Fridovich, 1986). The discovery that SOD1 mutations cause ALS was published in Nature in
1993 and it was a major discovery since it was the first gene shown to dominantly cause ALS
(Rosen et al., 1993). However, it was not obvious at all how SOD1, an antioxidative enzyme
expressed in all cell types and tissues throughout the body, could cause the selective
degeneration of motor neurons.
24
Missense mutations in the SOD1 gene lead to replacement of single amino acid
residues in the protein and, with some exceptions, cause dominant inheritance of the disease.
No mutations have been found in healthy people, except for mutation D90A, in which aspartic
acid in position 90 of SOD1 protein is converted to alanine. When usually SOD1 mutations
cause dominantly inherited disease, the D90A causes ALS in the scandinavian population
only when the mutation is inherited recessively from both parents and expressed
homozygously (Andersen et al., 1995; Sjalander et al., 1995). However, in some populations
with other ethnic backgrounds, the D90A mutation causes dominantly inherited ALS.
Therefore, the D90A mutation is a quite remarkable exception to the rule. It has been
suggested that in scandinavian populations the SOD1 gene with D90A mutation is linked to a
protective gene as the mutation arises from a single founder, however, the gene and
mechanism are not known (Al-Chalabi et al., 1998; Parton et al., 2002).
On the whole, the SOD1 mutations are scattered throughout the primary and three-
dimensional structure of the protein and up to date over 100 mutations have been found
(Valentine et al., 2005). Although soon after the discovery of SOD1 mutations a decrease of
dismutase activity was reported in ALS-patients (Deng et al., 1993; Orrell et al., 1995), the
lack of dismutase activity can not be the primary cause of ALS as some of the mutations do
not affect SOD1's normal enzymatic activity. Therefore it has been hypothesized that
mutations in SOD1 cause the disease through a toxic gain of function (Wong et al., 1995). A
complete list of mutations can be found at an online database for ALS genetics at
http://alsod.iop.kcl.ac.uk/Als/index.aspx. All SOD1 mutations are dominant except for D90A,
which can be either dominant or recessive (Andersen et al., 1996). Different SOD1 mutations
can cause distinct phenotypes differing in age of onset, progression and clinical symptoms.
The A4V mutation is the most common and unfortunately it also gives rise to the most
aggressive form of familial ALS with a mean survival of only one year after onset (Deng et
al., 1993). In contrast, the H46R mutation located within the copper binding domain leads to a
mild form of ALS with an average life expectancy of 18 years after disease onset (Aoki et al.,
1993; Ratovitski et al., 1999). What makes the matter even more perplexing is that mutation
H48Q, which is adjacent to the slow progressing ALS causing H46R, leads to a severe form
of ALS with rapid disease progression (Enayat et al., 1995). Moreover, mutations G37R and
L38V are predicted to have earlier onset different from mutations associated with the
aggressive phenotype, such as A4V (Cudkowicz et al., 1997). Considering the variation of
disease progression among different mutants and the fact that D90A causes ALS either
dominantly or recessively depending on population, it is evident that the clinical phenotype is
and can be used as a disease model for ALS (Bruijn et al., 1997b; Gurney et al., 1994;
Howland et al., 2002; Jonsson et al., 2004; Nagai et al., 2001; Ripps et al., 1995; Wang et al.,
2003; Wang et al., 2005; Wong et al., 1995). Most of our knowledge on the pathological
mechanisms of ALS are based on the research done by using these models.
2.1.3.3 ALS2, ALS4 and ALS5 - Juvenile forms ALS
From the discovery of the first ALS causing gene - SOD1, it took scientists nearly ten years to
identify the second gene associated with ALS. The second gene named ALS2, located at
chromosome 2q33, was linked to a rare, recessively inherited and slowly progressing juvenile
form of ALS (Hadano et al., 2001; Hentati et al., 1994). Patients in families of Arabic origin
developed juvenile onset (from 3 to 23 years) of progressive spasticity of the limbs, facial
and pharyngeal muscles, all caused by a mutation in the ALS2 gene. Altogether ten mutations
have been reported for the ALS2 gene and eight out of ten mutations are frameshift mutations,
which lead to premature termination of the transcript and a truncated protein. One nonsense
mutation and one splice variant site mutation have also been reported (as reviewed in Gros-
Louis et al., 2006). This has lead to the conclusion that loss of function of the ALS2 encoded
proteins is causing the disease.
The ALS2 gene spans 80 kbp of human genomic DNA and is predicted to encode a
184 kDa protein, named alsin, consisting of 1657 amino acids. Alsin has multiple motifs
homologous to guanine-nucleotide exchange factors (GEF) and it has been shown to function
as a GEF for Rab5 and RAc1 GTPase through the VPS9 domain linking alsin to the
organization of the actin cytoskeleton and vesicle trafficking (Kunita et al., 2004; Otomo et
al., 2003). A common feature for all found ALS2 mutations is the loss of VPS9-associated
GEF function suggesting that alsin mutations result in a deficit in intracellular trafficking
(Kunita et al., 2004; Otomo et al., 2003). However, the loss of alsin in knockout mice does
not lead to major motor deficits consistent with ALS or other MNDs (Cai et al., 2005).
Interestingly, alsin can also bind to mutant SOD1 and give neuroprotection to motor neuronal
26
cells against mutant SOD1 toxicity; by studying this interaction the role of these mutations in
ALS pathogenesis may be clarified (Kanekura et al., 2004).
ALS4 is a rare autosomal dominant form of juvenile ALS with linkage to chromosome
9q34 (Chance et al., 1998) where different missense mutations in the senataxin gene (SETX)
were found (Chen et al., 2004). Only heterozygous missense mutations in this gene are linked
to ALS, whereas homozygous deletions including missense, nonsense and deleterious
mutations are associated to an ALS unrelated disorder called ataxia-oculomotor apraxia type 2
(AOA2) (Moreira et al., 2004). As recessive deletion mutations in SETX cause AOA2, then
ALS4 is likely be to caused by gain of function of SETX.
ALS5 linked families show a similar disease phenotype as that of ALS2, except that in
ALS5 there is no spasticity of limb, facial and tongue muscles. Genetic analyses have shown
that type ALS5 is not related to ALS2 at 2q33 but to a chromosome location 15q15.1-q21.1
making ALS5 a distinct genetic entity (Hentati et al., 1998).
2.1.3.4 ALS3, ALS6 and ALS7 with classical late-onset phenotype
Genome wide screens have identified a locus with no relation to SOD1 mutations in
chromosome 18q21 for ALS3 (Hand et al., 2002), 16p12 for ALS6 and 20p13 for ALS7
(Sapp et al., 2003). In contrast to ALS2, which causes a juvenile form of ALS, ALS3, ALS6
and ALS7 give rise to classical ALS with late-onset and progressive paralysis with both upper
and lower motor neuron involvement. In fact, ALS3 was the first reported adult-onset
dominant ALS locus since ALS1. However, the genes with causing mutations have not been
identified.
2.1.3.5 ALS with dementia
ALS with frontotemporal dementia (ALS-FTD) and ALS-FTD with Parkinson's disease
(ALS-FTDP) are cases of motor neuron degeneration that occur in patients with FTD or FTD
and Parkinson's disease. In a set of families in which persons develop both ALS and FTD, a
genetic locus that is linked to ALS with FTD was identified on human chromosome 9q21-q22
(Hosler et al., 2000), whereas families with ALS alone did not link to this locus. In ALS-
FTDP, different mutations in the chromosome 17q21.1 of microtubule associated protein tau
gene (MATP) have been identified (Hutton et al., 1998). Tau has the function of stabilizing
microtubules, promoting their assembly and regulating transport of vesicles and organelles
along the microtubules by binding to tubules and modulating their stability (Rademakers et
al., 2004). However, there is considerable variation in clinical and pathological presentations
27
of patients and not all patients with ALS-FTDP have MAPT mutations, suggesting genetic
heterogeneity as in sporadic ALS (Hutton et al., 1998).
2.1.3.6 ALS8 and progressive lower motor neuron disease - atypical ALS
A missense P56S mutation in the vesicle-associated membrane protein B gene (VABP) in
chromosome 20q13.33 gives rise to autosomal dominant late-onset atypical ALS8. The
phenotype is characterized by slow progression of the disease and late onset with lower motor
neuron symptoms (Nishimura et al., 2004). Atypical signs are tremor and absence of upper
motor neuron involvement. A few cases have a typical ALS phenotype and some 25% of
cases are late-onset spinal muscular atrophy. VABP encodes a 33 kDa protein VAMP-B,
which is a vesicle membrane protein that can associate with microtubules, suggesting that
mutations in this gene may lead to dysfunction in intracellular membrane trafficking and to
variable MNDs (Nishimura et al., 2004).
Progressive lower motor neuron disease is a rare autosomal dominant form of MND,
where some but not all symptoms overlap with ALS. This form of MND has been linked to
missense mutations of dynactin1 gene (DCTN1) in chromosome 2p13 (Puls et al., 2003). The
DCTN gene encodes dynactin, an axonal transport protein, and missense mutations in the
gene are predicted to distort the folding of dynactin's microtubule binding domain, thus
suggesting that dysfunction of dynactin-mediated transport can lead to motor neuron disease
(Puls et al., 2003).
2.1.3.7 VEGF and ANG
VEGF is a growth factor that promotes the formation of blood vessels and can function also
as a neurotrophic factor: VEGF showed an implication to ALS in an animal model, where
deletion of hypoxia response element of VEGF in mouse resulted in an ALS like phenotype,
possibly through chronic neuronal ischemia and loss of direct neurotrophic effect of VEGF
(Oosthuyse et al., 2001). It is of interest as well that crossbreeding of these mice with G93A-
SOD1 mutant mice accelerates the disease progression, indicating that VEGF may be a
modifier for motor neuron degeneration in SOD1 ALS-mouse. Screening for mutations of
VEGF gene and regions of the promoter in patients showed a 1.8-fold increased risk of
developing ALS in a Belgian, Swedish and a British population (Lambrechts et al., 2003;
Terry et al., 2004), but not in other populations (Brockington et al., 2005; Gros-Louis et al.,
2003).
28
Mutations in ANG have also been linked to ALS, which together with VEGF,
highlights the role of angiogenesis in motor neuron degeneration. However, missense
mutations of ANG in ALS patients were restricted only to Irish and Scottish populations and
it is still unclear how these mutations affect ANG and provoke MND. Moreover, it is not
known whether ANG has neurotrophic properties (Greenway et al., 2006).
2.1.4 Models of ALS
2.1.4.1 Transgenic SOD1 models
The discovery of SOD1 and the fact that SOD1 mutations cause ALS dominantly made it
possible for scientists to create transgenic mouse model for ALS. To date, overexpression of
either G37R, G85R, G86R, G93A, G127X, H46R/H48Q or H46R/H48Q/H63G/H120G
mutant SOD1 in mice (Bruijn et al., 1997b; Gurney et al., 1994; Howland et al., 2002;
Jonsson et al., 2004; Ripps et al., 1995; Wang et al., 2003; Wang et al., 2005; Wong et al.,
1995) and G93A or H46R mutant SOD1 in rats (Howland et al., 2002; Nagai et al., 2001)
have been shown to cause a neurodegenerative disease similar to human ALS. As the
mutations have distinct phenotypical features in humans, also these models with different
mutations vary in age of onset, disease progression and histopathological features, and thus
reflect human ALS. Moreover, the penetrance in diffrent populations can be mimicked by
varying the mouse strain carrying the mutation (Kunst et al., 2000). On the other hand, the
survival times of these mice vary greatly from 4 months to over a year, depending maybe not
so much on the mutation but rather on the levels of mutant SOD1 expression.
The first paper on transgenic mice expressing human mutant SOD1 with G93A
mutation was published in 1994 (Gurney et al., 1994), just one year after the initial discovery
of SOD1 mutations. The results from that paper were highly significant as it was shown that
first of all, these mice developed ALS-like symptoms and secondly, they developed the
disease despite markedly elevated SOD1 activity and therefore gave the first evidence that
SOD1-linked ALS is not caused by loss of dismutase activity. The second set of transgenic
mice expressed SOD1 with G37R mutation (Wong et al., 1995). These mice developed ALS
like symptoms as well, although G37R-SOD1 retained nearly full enzymatic activity and thus
taken into account the findings made by Gurney et al. 1994 it was concluded that the toxicity
of SOD1 mutants has to arise from the property of a toxic subunit, not from the reduction of
dismutase activity. The loss of function theory was further disproved as SOD1 knock out
mice, in which endogenous murine SOD1 gene was deleted, did not develop motor neuron
29
disease (Reaume et al., 1996). The final nail for the loss of function theory came from Bruijn
et al. 1998 showing that elimination of SOD1 activity or overexpression of human wild-type
SOD1 in the presence of mutant G85R-SOD1 with reduced dismutase activity in mice does
not protect from the disease and, surprisingly, it can even accelerate it (Deng et al., 2006;
Jaarsma et al., 2000) and thus toxicity is independent of SOD1 activity. In addition, Bruijn et
al. also found protein aggregates containing SOD1 to be a common pathological feature for
SOD1 mutations that otherwise give different phenotypical features (Bruijn et al., 1998). It is
undisputedly clear now that mutations in SOD1 do not cause ALS as a loss of dismutase
activity but through a toxic gain of function. However, the mechanism of how mutant SOD1
exerts toxicity to motor neurons is still uncertain. In addition to many different mutant SOD1
expressing strains, also SOD1 knock out mice (Reaume et al., 1996) and human wild type
SOD1 overexpressing mice (Jaarsma et al., 2000) and rats (Chan et al., 1998) are used greatly
in ALS research as they serve as good controls for the transgene over-expression and for the
role of endogenous SOD1.
SOD1 is a ubiquitously expressed protein and one of the key questions in ALS
research is how only the motor neurons are selectively destroyed while there is no pathology
in other tissues. The fact that SOD1 is ubiquitously expressed raises the possibility that the
toxicity is not coming from the motor neurons themselves but also from the non-neuronal
glial cells surrounding the motor neurons. Although mainly motor neurons are degenerating,
there is also pathology present in astrocytes already in the early phase of the disease (Bruijn et
al., 1997b). To address the role of astocytes, a line of transgenic mice were created that
expressed SOD1 only in astrocytes. These mice had high levels of mutant G86R-SOD1 in
astrocytes driven by a GFAP promoter. Although the mice had increased astrocytosis with
aging they did not develop motor neuron degeneration, thus the authors concluded that
expression of mutant SOD1 in the neurons is critical for the initiation of the disease (Gong et
al., 2000). In another set of transgenic mice the expression of mutant SOD1 was restricted to
neurons alone either by neurofilament promoter (Pramatarova et al., 2001) or by neural
specific enolase promoters (Lino et al., 2002), but the outcome of these trials was that
neuron-restricted expression of mutant SOD1 does not cause pathology or motor neuron
disease. However, some doubts remained as the neuron restricted expression of mutant SOD1
may have resulted in too low protein levels to yield disease. Recently, also neuron specific
expression of human mutant SOD1 was shown to induce motor neuron death in mice
(Jaarsma et al., 2008). Nevertheless, a more definitive answer to the contribution of different
cells to ALS pathogenesis came from a study with chimeric mice that were mixtures of
mutant SOD1 expressing cells and normal wild type cells showing that toxicity to motor
30
neurons requires damage from mutant SOD1 to surrounding non-neuronal cells (Clement et
al., 2003). In fact, expression of mutant SOD1 in motor neurons at levels which cause early
onset and rapidly progressing disease if expressed ubiquitously, do not cause cell-autonomous
degeneration or death of individual motor neurons.
The most recent development in SOD1 models of ALS has been the creation of mice
carrying a mutant SOD1 gene flanked by loxP, sites allowing the deletion of mutant SOD1
gene by Cre recombinase enzyme (Boillee et al., 2006b). Tissue specific expression of Cre
recombinase either in motor neurons or microglia, and hence the deletion of mutant SOD1 in
respective cells, has shown that mutant SOD1 expression in motor neurons and non-neuronal
neighbors have a different contribution to the disease onset and progression; Mutant SOD1
within microglial cells accelerates disease progression while mutant action within the motor
neurons determines onset and progression of early disease (Boillee et al., 2006b). The role of
mutant SOD1 toxicity within different cells of the CNS is reviewed more extensively in
chapter 2.2.6 Role of non-neuronal cells.
In addition to mouse models, there is also an invertebrate model available for studying
the toxic effect of SOD1 mutations in Caenohabditis elegans, a nematode roundworm.
Although C. elegans is only 1mm long, we must not underestimate the power of C. elegans
models or as Professor of Bioinformatics, Garry Wong from the University of Kuopio put it:"
A worm is not a mouse that is not a man." In fact, aspects of mutant SOD1 toxicity have been
modelled in C. elegans as worms expressing mutant SOD1 showed greater vulnerability to
oxidative stress, and under oxidative stress the mutant forms, but not human wild-type SOD1,
formed potent aggregates to muscles (Oeda et al., 2001). Human mutant or wt SOD1 has also
been expressed in Drosophila (fruit fly) motor neurons, where it however showed no toxic
effect but extended lifespan by 40% (Elia et al., 1999; Parkes et al., 1998).
2.1.4.2 Other in vivo models of motor neuron degeneration
Before the emergence of SOD1 transgenic rodent models, no other model could completely
replicate disease progression as thoroughly as the SOD1 models and as these models failed to
replicate the disease, treatment successes from these models were not carried to human trials
for treatment of ALS. The in vivo rodent models used in ALS research prior to the SOD1
models include axotonomy induced motor-neuron death and some naturally occurring
mutations in mice (as reviewed by Elliott, 1999).
When performed in neonatal animals, direct trauma to the motor nerve axon by
peripheral nerve transsection (axotomy) results in apoptotic cell death of all motor neurons
31
whose axons were severed. Although axotomy induced motor neuron cell death is invaluable
for studying apoptosis, its relevance to ALS is less certain as the injury caused by axotomy is
acute and the events following may differ a lot from the pathways that are activated in chronic
ALS pathology.
In addition to SOD1 transgenic ALS models also other naturally occurring genetic
rodent models are available for motor neuron degeneration research that have been used for
preclinical testing of agents for human ALS. The wobbler mouse represents a phenotype
characterized by progressive forelimb weakness beginning at about one month of age and
animals survive to the age of one year (Andrews et al., 1974; Mitsumoto and Bradley, 1982).
Pathology includes axonopathy with proximal axonal degeneration as well as neuropathy with
vacuolar changes within anterior horn cells of the spinal cord. However, opposite to ALS, the
pathology is limited only to the spinal cord with limited involvement in brain. The wobbler
phenotype has autosomal recessive inheritance and the gene responsible is Vps54 (vacuolar-
vesicular protein sorting 54) involved in vesicular trafficking (Schmitt-John et al., 2005).
The progressive motor neuronopathy (pmn) mice have a recessively inherited
mutation in tubulin chaperone E gene (Bommel et al., 2002; Martin et al., 2002) and these
mice develop pelvic and hind limb weakness and die by 7 weeks of age (Schmalbruch et al.,
1991). Pathologically the phenotype is characterized by a prominent distal motor neuron
axonopathy. However, the motor neuron soma is relatively spared and in this regard the
pathology is dissimilar to ALS.
The neuromuscular degeneration (nmd) mouse is another autosomal recessive model
of spontaneous progressive motor weakness (Cook et al., 1995). These mice develop rapidly
progressive weakness in their hindlimbs beginning at two weeks of age as motor neurons
degenerate in the lumbar spinal cord. These mice rarely survive past four weeks. The genetic
defect has been identified as a single amino acid deletion and spice donor site mutation in the
gene encoding a ubiquitously expressed ATPase/DNA helicase, also known as SMbp2 (Cox
et al., 1998).
The motor neuron disease mouse, or the mnd mouse, has dominantly inherited
autosomal motor neuron disease with late onset. The mnd mouse is characterized by onset in
the hindlimbs with stiffness, atrophy and paralysis starting at 5-11 months of age and lifespan
of 14 months (Messer and Flaherty, 1986). Pathology indicates neuronal swelling with
cytoplasmic inclusions and motor neuron degeneration in the spinal cord, hypoglossal nuclei
and motor cortex (Messer et al., 1987) but also retinal degeneration (Messer et al., 1993). The
gene is located in chromosome 8 and is a homolog for gene CLN8 encoding a putative
membrane protein with yet unknown functions (Ranta et al., 1999).
32
Although these mouse models do not mimic all the features of human ALS as
extensively as the SOD1 models, the advantage of these models is that they have generalized
and naturally occurring motor weakness over a more chronic time course with gradual
progression of the disease. However, in comparison to human ALS, the nmd has a different
temporal time course with very early onset, the mnd model has differences in spatial patterns
as also retinal neurons are degenerating and the pmn has quite many dissimilarities in
pathology as only axonopathy is occurring. Differences may suggest that these models are
different disorders from ALS altogether. The wobbler and mnd models exhibit a clinical
course and pathology more closely resembling human motor neuron disease, but it is not
known whether similar molecular or biochemical defects underlie these conditions and human
ALS.
2.1.4.3 In vitro models of ALS
In vitro systems offer ease of manipulation of cells by direct pharmacological administration
or by gene transfections. However, preparation of pure motor neuron cultures is complex as
identification and isolation of motor neurons is difficult. In addition, the neurons used for this
preparation have to be isolated from embryonic or late neonatal time points (Hanson et al.,
1998; Martinou et al., 1992). The lifespan of motor neurons in cultures is also short allowing
better assessment of acute rather than chronic injuries. Pure cultures also do not allow
interaction of motor neurons with other neurons or glia, although, this can also be the whole
point of making pure cultures.
Maybe the best in vitro model of ALS and SOD1 mediated toxicity so far is
microinjection of mutant SOD1 to primary cultured neurons (Durham et al., 1997). The
expression of microinjected mutant SOD1 cDNA results naturally in protein expression, but
also in selective killing of motor neurons but not sensory neurons, whereas cDNA of wild-
type SOD1 does not result in any neurotoxicity. In addition, expression of mutant SOD1
cDNA, but not wild-type SOD1, results in formation of protein aggregates, which is followed
by motor neuron cell death (Bruening et al., 1999; Durham et al., 1997). These findings lead
to the initial proposition that aggregates may have a role in SOD1 mediated toxicity.
Organotypic slices of spinal cord can also be used as a model system for studying
ALS. Slices are prepared from 9 day old mice and motor neurons in these cultures can survive
for up to 3 months (Rothstein et al., 1993). The advantage of the slice model is that some of
the spinal cord structures such as dorsal and ventral horns are preserved and the slice
preserves also some of the neuron-neuron and neuron-glia cell interactions. However,
33
dissecting the slice from the animal results in multiple axotonomies, leaving motor neurons
deafferented and although many motor neurons survive preparation, it is possible that the
procedure is selective in motor neuron survival (Elliott, 1999).
For the cultures or slices to be used as a model for ALS, the cells have to be
manipulated in order to reflect the disease state. Glutamate excititoxicity is one of the mostly
used methods to cause motor neuron degeneration either by inhibition of glutamate transport
(Rothstein et al., 1993) or by causing excitotoxicity directly by application of N-methyl-D-
aspartic acid (NMDA) or NMDA agonist (Annis and Vaughn, 1998; Delfs et al., 1997).
Agents that block glutamate receptors, inhibit glutamate release or decrease glutamate
synthesis are capable of preventing this form of motor neuron death in vitro (Annis and
Vaughn, 1998; Delfs et al., 1997; Rothstein and Kuncl, 1995).
2.2 Mechanisms for motor neuron cell death
2.2.1 Oxidative damage
2.2.1.1 SOD1 activity
After the landmark discovery that mutations in SOD1 are a cause of ~20% of familial ALS
cases fourteen years ago (Rosen et al., 1993), it was shown that SOD1 activity in patient
blood is reduced (Deng et al., 1993; Orrell et al., 1995) and it was hypothesized that loss of
SOD1 activity, and thus increased levels of superoxide radicals, was central to the disease.
However, soon after it was realized that mutations in SOD1 do not cause ALS through loss of
activity for the following reasons: transgenic mice with mutant SOD1 expression developed
progressive motor neuron disease despite markedly elevated SOD1 activity levels (Gurney et
al., 1994), SOD1 knock out mice in which endogenous SOD1 is completely deleted do not
develop overt motor neuron disease (Reaume et al., 1996), some SOD1 mutants retain full
specific activity (Borchelt et al., 1994) and neither the age of onset or rapidity of disease
progression correlates with dismutase activity levels (Bowling et al., 1995; Cleveland et al.,
1995). The inevitable conclusion is that mutations in SOD1 do not cause ALS through loss of
activity but through toxic gain of function.
Although the loss of activity and increased levels of superoxide anions was not proven
to cause the disease, the oxidative damage might be caused by unwanted oxidative reactions
caused by mutant SOD1. Normally dismutation of superoxide anion is catalysed by SOD1 in
two asymmetric steps by the reactive copper atom, which is alternately reduced and oxidized
by superoxide (figure 2a) (Fridovich, 1986). Even the wild-type SOD1 can exhibit additional
34
enzymatic activities (Liochev and Fridovich, 2000) and the most obvious hypothesis of how
over 100 different mutations could mediate toxicity is that mutations in SOD1 result in a less
tightly folded protein conformation, allowing greater access of substrates to the reactive
copper and thus the possible catalysis of unwanted oxidative reactions of abnormal substrates.
Some likely abnormal substrates to give oxidative damage include hydrogen peroxide
(H2O2) and peroxynitritre (-ONOO) and the first hypothesized theories on unwanted oxidative
reactions of SOD1 (as reviewed in Cleveland and Rothstein, 2001) were the peroxidase
hypothesis (Wiedau-Pazos et al., 1996) and two peroxynitrate theories (Beckman et al., 1993;
Estevez et al., 1999). The unwanted reactions of these substrates and the dismutase activity of
SOD1 are presented in figure 2.
2.2.1.2 Aberrant SOD1 activity
The first suggested abnormal oxidative reaction of mutant SOD1 included peroxynitrite as a
substrate (Beckman et al., 1993). Peroxynitrite can be formed spontaneously from superoxide
and nitric oxide and if it is used as a substrate by SOD1 it will yield protein tyrosine nitration
(figure 2c). In the case of zinc-depleted mutant SOD1, superoxide anions are formed by
mutant SOD1 itself from O2 (Estevez et al., 1999) leading to spontaneous peroxynitrite
formation and protein nitrasylation (figure 2d). The peroxynitrite hypothesis was supported by
immnohistochemistry results from both mice (Andrus et al., 1998; Bruijn et al., 1997a;
Ferrante et al., 1997) and humans (Beal et al., 1997) showing elevated protein nitrotyrosine
levels as predicted by the hypothesis. However, in the same fashion as the loss of function
theory was disproved by showing that manipulation of SOD1 activity levels does not have a
positive effect on the disease progression applies also to the peroxynitrite theory, as increased
wild type SOD1 expression should quench the levels of superoxide and prevent them from
spontaneously forming peroxynitrite. As the toxicity of mutant SOD1 cannot be ameliorated
by increased SOD1 activity, the damage cannot be arising from superoxide or any
spontaneous reaction product of it.
Maybe the most attractive model for oxidative damage generated by aberrant substrate
is the peroxidase hypothesis. A second proposed aberrant substrate was hydrogen peroxide
(Wiedau-Pazos et al., 1996), which is interesting, as hydrogen peroxide is the normal end
product of the oxidized SOD1-Cu2+ form of the enzyme. Use of peroxide as a substrate by the
reduced SOD1-Cu1+ form might produce the extremely reactive hydroxyl radical (figure 2b).
An increase has been reported in the use of hydrogen peroxide by A4V- and G93A-SOD1
35
Figure 2. SOD1 activity (a) and proposed aberrant substrates (b-d). a) Normal activity:SOD1 dismutases superoxide into oxygen and hydrogen peroxide in two asymmetric steps bythe reactive copper atom, which is alternately reduced and oxidized. Toxic hydrogen peroxideis converted to water by glutathione peroxidase or catalase. b) Hydrogen peroxide used as asubstrate by the reduced form of SOD1 may lead to formation of hydroxyl radicals. c)OONO as a substrate may lead to protein nitration. d) Zinc-depleted SOD1 may generate
superoxide from oxygen leading to perooxynitrite formation and further on to proteinnitration. Modified from (Cleveland and Rothstein, 2001).
SOD–Cu1+ SOD–Cu2+
O2•O2
–
H2O2H+ + •O2– 2H2O2
SOD–Cu1+ SOD–Cu1+–OH + OH–
H2O2
•OH
SOD–Cu1+ SOD–Cu2+
OH– + NO2-Tyr-Protein H-Tyr-Protein
•O2– + NO• –OONO
(Zn–) SOD–Cu1+ (Zn–) SOD–Cu2+ + •O2–
(Zn–) SOD–Cu2+
NO
NO2-Tyr-Protein (Zn–) SOD–Cu2+ + ONOO–
(reduced) (oxidized)
(reduced)
O2(oxidized)
a
b
c
d
Glutathione peroxidasecatalase
36
mutants in vitro by spin trapping with 5, 5'-dimethyl-1-pyrrolline N-oxide (DMPO) (Wiedau-
Pazos et al., 1996).
However, the discovery was very soon challenged on technical grounds as it was
shown that a significant fraction of DMPO/•OH formed was derived from the incorporation of
oxygen from water due to oxidation of DMPO to DMPO/•OH, presumably via DMPO radical
cation (Singh et al., 1998). Still, products consistent with peroxidase hypothesis have been
found in the G93A-SOD1 mice strain as shown by increased lipid peroxidation (Hall et al.,
1998a) and increased protein carbonyl content (Andrus et al., 1998)
Overall, increased levels for markers of oxidative damage have been found in G93A-
SOD1 mouse strain (Andrus et al., 1998; Ferrante et al., 1997), but not in transgenic animals
with other mutations such as G37R-SOD1 (Bruijn et al., 1997a) or G85R-SOD1 (Williamson
et al., 2000). In human cases, markers of oxidative damage have been found in sporadic ALS
(Bowling et al., 1993; Ferrante et al., 1997; Shaw et al., 1995b) but not in SOD1-mediated
familial ALS (Bowling et al., 1993; Ferrante et al., 1997) and despite the elegant hypothesis
and hard work on the theories, aberrant substrate activity of SOD1 is most likely not the
primary cause of toxicity in ALS. Moreover, both of the theories and the proposed activities
require copper in the catalytic site of the enzyme and the strongest argument against the
theories comes from the discovery that an inactive SOD1 lacking all copper coordinating
histidines and also a reaction catalyzing copper ion still causes overt and progressive motor
neuron disease (Wang et al., 2003). On the other hand, this hypothesis does not account for
the possible formation of the hetrodimeric SOD1 (Witan et al., 2008) that might be formed in
the mouse models by mutant SOD1 and mouse endogenous wt SOD1, which is active and
contains copper binding sites. Furthermore, genetically decreased copper concentrations in
spinal cord of G86R-SOD1 mouse model of ALS have been shown to prolong life span by
9% (Kiaei et al., 2004), indicating a neurotoxic role for copper and active SOD1 in ALS
pathogenesis. Moreover SOD1 may affect ROS production, not just by its catabolic activity,
but by stimulating excess ROS production of NADDPH oxidase in microglia by binding to a
signaling GTPase protein Rac1 (Harraz et al., 2008).
2.2.2 Protein Aggregation
2.2.2.1 Aggregates
Protein aggregation is a common pathological hallmark of many different neurodegenerative
disorders like -amyloid and tau in Alzheimer's disease, -synuclein in Parkinsons's disease
37
and huntingtin in Huntington's disease. For ALS, intracellular, cytosolic protein aggregates in
motor neurons and also in surrounding astrocytes are observed in both sporadic and familial
cases as well as in mutant SOD1 transgenic mice, where aggregates are highly
immunoreactive against SOD1 (Bruijn et al., 1997b; Bruijn et al., 1998; Watanabe et al.,
2001). In ALS the term aggregate has been used to refer to abnormal intermediate filaments
like neurofilaments and peripherin as detected by immunostaining of spinal cord tissue
(Hirano et al., 1984b), accumulation of detergent insoluble forms of proteins, including
SOD1, detected by immunoblotting (Johnston et al., 2000) as well as small SOD1 or ubiquitin
positive fibrillar inclusions in spinal cord sections (Wang et al., 2002). However, the thing is
that in ALS, as well as in other neurodegenerative diseases with protein aggregation, it is not
known whether the aggregates are toxic and by which mechanism, or are they a beneficial end
product of sequestered harmful and toxic intermediates of protein aggregates. Several
different theories have been put forward to explain the possible toxicity of protein aggragates,
including loss of protein function through coaggregation, depletion of protein folding
chaperones, dysfunction of the proteasome overhelmed by misfolded ubiquitinated protein
and inhibition of cell organelle function through aggregation within them e.g. in mitochondria
or peroxisomes (as reviewed in Bruijn et al., 2004b).
2.2.2.2 Proteasome and Immunoproteasome
Protein degradation is necessary for maintaining cellular homeostasis as cellular structures are
continually rebuilt and misfolded proteins, formed i.e. by mutations or oxidative stress must
be degraded. The ubiquitin proteasome pathway is the major proteolytic system of eukaryotic
cells, where it catalyzes the selective hydrolysis of ubiquitin tagged proteins in an ATP-
dependent manner (DeMartino and Slaughter, 1999).
Aggregates in patients and mouse models of ALS contain ubiquitin, a marker for the
protein in question to be degraded in the proteasome pathway. Excess accumulation of
ubiquitinated proteins may adversely affect the proteasome machinery and impair normal
proteasome function. For ALS, it has been shown that already partial inhibition of the
proteasome is sufficient to cause formation of large aggregates in cultured nonneuronal cells
that express mutant SOD1 (Johnston et al., 2000). However, from there on the results on
proteasome activity and function in ALS pathogenesis have been controversial as it has been
shown that overall proteasome activity is down-regulated in lumbar spinal cord well before
disease onset (Kabashi et al., 2004), upregulated at symptomatic stage (Puttaparthi and Elliott,
2005) or remains unaltered with disease progression (Cheroni et al., 2005). Though the results
38
may seem contradictory, regulation of proteasome function may explain the differing results
on proteasome activity in ALS pathogenesis.
The proteasome is a 700 kDa cylinder shaped protease composed of four heptameric
rings of and subunits; the subunits form the outer rings and subunits form the inner
rings. In eukaryotes the subunits represent 14 gene products, seven of subunits and seven of
subunits. The proteasome is a multicatalytic protease charactherized by three specific
activities: a chrymotrypsin-like, trypsin-like and caspase-like activity and each catalytic
activity is linked with a specific subunit (as reviewed in Baumeister et al., 1998). In higher
eukaryotes each of the constitutive subunits, termed 1 or Y or ; 2 or Z; and 5 or X, has a
close homolog (LMP2, MECL-1 and LMP7, respectively) that can be selectively induced
with -interferon (Fruh et al., 1994), resulting in the replacement of their constitutive counter
parts and formation of proteasomes with altered catalytic characteristics that favor the
generation of peptides suitable for binding the MHC class I antigen presenting molecules
(Dick et al., 1996). In normal brains, the constitutive subunits are predominant, whereas the
inducible subunits are marginally expressed (Stohwasser et al., 2000).
Cheroni and colleagues showed for ALS for the first time that although overall
proteasome activity remained unaltered, the activity of constitutive proteasome decreases with
disease progression. This decrease was supplemented by induction of the immunoproteasome
so that the overall activity remained unaltered although levels of constitutive proteasome and
immunoproteasome varied (Cheroni et al., 2005). Similarly, a paper published later the same
year showed that immunoproteasome is activated in ALS, although this time changes with
constitutive proteasome levels were not observed. Hence the overall proteasome activity
increased with disease progression as immunoproteasome was induced (Puttaparthi and
Elliott, 2005). Even more importantly, Puttaparthi and Elliot showed that induction of
immunoproteasome was localized only to astrocytes and microglia, not to motor neurons.
This is interesting in the light of recent results showing that non-neuronal cells surrounding
motor neurons can either help the motor neurons to survive in the ALS pathogenesis, or to
strike a toxic cascade against them. Induction of the immunoproteasome would seem to be a
beneficial phenomenom that might help the motor neurons and non-neuronal cells to cope
with aggregating proteins.
2.2.2.3 Chaperones
Motor neurons have a high threshold for induction of the stress response which can contribute
to their selective vulnerability in ALS (Batulan et al., 2003). Keeping this is mind,
39
destabilized and misfolded SOD1 is a possible initiator of aggregation through clogging of
protein folding chaperone machinery and heat shock proteins (HSPs). Multiple recombinant
SOD1 mutants inhibit chaperone function in vitro (Bruening et al., 1999) and even more
importantly, an overall decrease of chaperone activity has been reported in spinal cord
extracts of ALS mice from presymptomatic to the end stage of the disease (Tummala et al.,
2005). However, some chaperones are upregulated with disease progression; for example,
B-crystallin and Hsp27 are elevated in the spinal cords of G37R-SOD1 and G93A-SOD1
mice. Expression of B-crystallin has increased overall and in all cell types, but Hsp27 is
predominantly present only in glial cells and at late stages of the disease (Vleminckx et al.,
2002; Wang et al., 2003).
Elevating expression of different HSPs (Hsp70, Hsp40, Hsp27) in cultured cells and
primary motor neurons decreases aggregate content and apoptosis and improves axonal
outgrowth (Bruening et al., 1999; Patel et al., 2005; Takeuchi et al., 2002). However, this
positive effect does not apply in vivo, as elevated expression of Hsp70 to a level that is
protective in mouse models of acute ischemic insult and selective neurodegenerative disorders
did not ameliorate disease or pathology in four different mutant SOD1 lines (Liu et al., 2005).
2.2.2.7 Neurofilaments and axonal transport
Neurofilaments are the most abundant structural proteins in many types of mature motor
neurons. Aberrant neurofilament accumulations are a pathological hallmark of both familial
and sporadic ALS (Hirano, 1991; Hirano et al., 1984a; Hirano et al., 1984b) and are also seen
in mice expressing mutant SOD1 (Bruijn et al., 1997a; Dal Canto and Gurney, 1994).
Neurofilaments compose of heteropolymers of neurofilament-light (NF-L), -medium (NF-M)
and -heavy (NF-H) subunits. Transgenic mice expressing a mutation in NF-L develop motor
neuron disease as axonal architecture is perturbed (Lee et al., 1994), whereas deletion of NF-L
prolongs survival of two SOD1-mouse models of ALS (Nguyen et al., 2001; Williamson and
Cleveland, 1999), possibly because of the reduction of phospohorylated neurofilament tail
domains on the axons. Taken together, neurofilament content and disorganization are
important contributors and probable risk factors for the disease.
2.2.3 Glutamate excitotoxicity
Neurotransmitter glutamate released form presynaptic terminals triggers action potentials in
motor neurons by activating calcium-permeable AMPA glutamate receptors. If glutamate is
40
not promptly cleared away from the synaptic cleft by glutamate transporters, chronic over
activation of glutamate receptors will cause repetitive neuron firing and increased intracellular
calcium. This glutamate mediated exitotoxicity can induce neuronal damage and death. Glial
glutamate transporter EAAT2 in astrocytes is one of the five subtypes of glutamate
transporters and is responsible for ~90% of the clearance of glutamate for motor neurons
(Rothstein et al., 1996; Tanaka et al., 1997). Preventing glutamate exitotoxicity within the
spinal cord is the job of astrocytes.
In ALS, glutamate excitoxicity involvement arose from the discovery of increased
glutamate levels in cerebrospinal fluid of ALS patients (Rothstein et al., 1991; Rothstein et
al., 1990; Shaw et al., 1995a) and is nowadays reported in 40% of sporadic ALS patients
(Spreux-Varoquaux et al., 2002). Measurement of functional glutamate transport in ALS
revealed an evident decrease in the affected brain regions caused by loss of glutamate
transporter protein EAAT-2 (Rothstein et al., 1995). Lowering of EAAT2 levels with an anti-
sense oligonucleotide has been shown to induce neuronal death both in vivo and in vitro
(Rothstein et al., 1996). In addition, expression of two SOD1 mutants in mice leads to
functional loss of EAAT2 (Nagano et al., 1996) and expression of G93A-SOD1 in rats
induces a focal loss of EAAT2 selectively from the astrocytes within the ventral horn of the
spinal cord (Howland et al., 2002). Motor neurons have a high sensitivity to excitotoxicity
since their AMPA glutamate receptors have a low portion of the calcium resistant GluR2
subunit, rendering them more permeable to calcium ions (Van Damme et al., 2002).
Glutamate excitotoxicity with astrocytic dysfunction is likely to be an important
contributor to neuronal death in ALS and is one of the few mechanistic links between
sporadic and mutant SOD1 mediated ALS. Moreover, the only currently FDA-approved
therapy in ALS, Riluzole, functions by decreasing glutamate toxicity (Bensimon et al., 1994;
Lacomblez et al., 1996).
2.2.4 Inflammation
Microglia are the resident immune cells of the central nervous system and are primary
mediators of neuroinflammation (Kreutzberg, 1996). In the resting state microglia have a
ramified shape characterized by a small cell body with fine processes and minimal expression
of surface antigens. They form a network of immune alert resident macrophages with a
capacity for immune surveillance and control. Upon injury to the CNS the microglia are
activated and acquire an ameboid shape. Activated microglia are mainly scavenger cells but
also perform various other functions in tissue repair and neural regeneration. Activated
41
microglia can destroy invading micro-organisms, remove potentially deleterious debris,
promote tissue repair by secreting growth factors but they can also exert neuroinflammation
through the release of oxygen radicals, NO, cytokines, glutamate and prostaglandins. The
transformation of microglia into potentially cytotoxic cells is under strict control and occurs
mainly in response to neuronal or terminal degeneration, or both (Hanisch, 2002; Kreutzberg,
1996).
Microglial activation has been described in many acute and chronic neurological
diseases, including Alzheimer's disease (Kreutzberg, 1996; McGeer and McGeer, 1999). In
ALS patients, microglial activation and proliferation has been described in the brain and
spinal cord in regions of motor neuron loss (Engelhardt and Appel, 1990; Henkel et al., 2004;
Kawamata et al., 1992; McGeer et al., 1991; Troost et al., 1993; Turner et al., 2004).
Microglial activation is also seen in the spinal cord of different mutant SOD1 mice (Hall et
al., 1998b; Henkel et al., 2006; Kriz et al., 2002) where microglial reactivity is initiated before
motor neuron loss (Henkel et al., 2006) and expression of proinflammatory mediators like
TNF , IL-1 and COX-2 produced by microglia and other inflammatory cells is also an early
event (Alexianu et al., 2001; Almer et al., 2002; Elliott, 2001; Hensley et al., 2002; Nguyen et
al., 2001).
Microglial involvement in ALS is also supported by results showing that minocycline,
a tetracycline antibiotic derivative able to block microglial activation (Yrjänheikki et al.,
1999), also delays disease progression in ALS mice and reduces microglial activation (Kriz et
al., 2002; Van Den Bosch et al., 2002; Zhu et al., 2002). When minocycline treatment is
started in young presymptomatic mice, minocycline will delay disease onset but not disease
progression after the onset (Zhu et al., 2002), whereas administration at disease onset results
in slowing of the disease after onset (Kriz et al., 2002). However, later on minocycline failed
to show benefit in a large phase III humal trial of ALS (Gordon et al., 2007) and also findings
in the mouse model have been challenged (Scott et al., 2008). Inhibition of COX-2, expressed
by neurons and astrocytes, with celecoxib prolonged survival in mice by diminishing
asrtogliosis and microglial activation (Drachman et al., 2002), indicating the importance of
gliosis to the disease pathogenesis. However, also celecobix later failed to provide benefit in
human trials (Cudkowicz et al., 2006).
2.2.5 Mitochondria
Mitochondria among other cell organelles are likely targets for mutant SOD1 toxicity.
Although SOD1 is classically considered as a cytosolic enzyme (Fridovich, 1986), it is also
42
located in the mitochondrial intermembrane space (Okado-Matsumoto and Fridovich, 2001;
Sturtz et al., 2001). Vacuolated and dilated mitochondria with disorganized cristae and
membranes have been observed in ALS early on in muscles and spinal cord of both sporadic
and familial patients (Afifi et al., 1966; Hirano et al., 1984a; Hirano et al., 1984b). Since then
it has been shown with mutant SOD1 animal models that mutant SOD1 is preferentially
imported into mitochondria in affected tissues (Bergemalm et al., 2006; Deng et al., 2006; Liu
et al., 2004; Vijayvergiya et al., 2005). If we consider the hypothesized theories of mutant
SOD1 toxicity on mechanisms leading to motor neuron cell death, including oxidative
damage, aggregation and excitotoxicity, it can be noted that mitochondria can be directly
linked to all of these harmful cascades in ALS pathogenesis. However, there is yet no specific
mechanism for the toxicity of SOD1 in mitochondria and many contradictions remain to be
resolved.
Oxidative stress is a likely cause of mitochondrial damage as mitochondria themselves
are a source of reactive oxygen species as an unavoidable byproduct of oxidative respiration.
Mutant SOD1 may interfere with the electron transport chain disrupting ATP production and,
in fact, ATP levels have been shown to be depleted in presymptomatic G93A-SOD1 mice
(Browne et al., 2006) and at the time of onset, ATP synthesis itself was defective (Mattiazzi et
al., 2002). However, this holds only for the G93A-SOD1 model as ATP synthesis has been
shown to be unaffected in aged symptomatic G85R-SOD1 mice (Damiano et al., 2006), and
also other aspects of electron transport chain have been reported as either unchanged or
altered. Activity of the mitochondrial cytochrome oxidase has been shown to be elevated
(Bowling et al., 1993), reduced (Mattiazzi et al., 2002) and unchanged (Damiano et al., 2006).
In addition, treatment with creatine extended the survival in G93A-SOD1 mice alleviating
energy deficits (Browne et al., 2006; Klivenyi et al., 1999), but did not have any beneficial
effects in human clinical trials (Groeneveld et al., 2003; Shefner et al., 2004).
One of the most convincing connections between SOD1 toxicity and mitochondria is
the disruption of mitochondrial calcium buffering capacity in SOD1 mice. Although studies
on mitochondrial energy metabolism had no consensus of the mutant SOD1 action between
different mouse models or human ALS, calcium-buffering capacity has been shown to be
impaired in spinal cord of both G93A-SOD1 and G85R mice at presymptomatic stage and
only in affected tissues (Damiano et al., 2006). Mutant SOD1-mediated disruption of calcium
homeostasis is also compatible and can be linked directly with excitotoxic mechanisms
leading to neuronal cell death as, excitotoxicity from repeated neuron firing increases
intracellular calcium levels and leads to the opening of mitochondrial membrane permeability
transition pore (PTP) and release of cytochorme c triggering the apoptotic process. Caspase
43
mediated apoptotic neuronal death and activation of caspase 3 is the final event in ALS
pathogenesis appearing in motor neurons and astrocytes (Li et al., 2000; Pasinelli et al.,
2000). Moreover, activation of caspases can be linked back to excitotoxicity as caspase 3
activation in glial cells proteolytically inactivates the glutamate transporter enhancing
excitotoxicity, calcium entry and mitochondrial damage (Boston-Howes et al., 2006).
Mutant SOD1 aggregates may also damage mitochondria and induce apoptosis by
interfering with anti-apoptotic activity of Bcl-2 and triggering the premature activation of the
apoptotic process (Pasinelli et al., 2004). Mutant SOD1 aggregating onto the mitochondrial
surface might also impede the transport of nuclear encoded proteins into mitochondria
through translocators of the outer and inner membrane (TOM and TIM, respectively) (Liu et
al., 2004). In addition to aggregates, high levels of mutant SOD1 expression and
mitochondrial abnormalities have been detected in motor neuron presynaptic terminals at the
onset of neuromuscular denervation, before accumulation of mutant SOD1 (Gould et al.,
2006).
Although SOD1 is believed to be an important part of the mitochondrial superoxide
scavenging system (Sturtz et al., 2001), the role of SOD1 in the mitochondria is somewhat
controversial. In the mitochondrial matrix superoxide is dismutated to oxygen and hydrogen
peroxide by Mn-superoxide dismutase (SOD2) (Zelko et al., 2002) and in the intermembrane
space superoxide detoxification is thought to depend on cytochrome c, which can efficiently
oxidize superoxide to oxygen acting as a true antioxidant (Pereverzev et al., 2003), whereas
SOD produces oxygen and hydrogen peroxide that further needs to be detoxified by
glutathione peroxidase or catalase, which are present in the intermembrane space only in low
levels (Martin et al., 1998). Moreover, SOD1 in the intermembrane space is mainly kept
inactive (Inarrea et al., 2005) raising questions about the suggested role in the mitochondrial
superoxide scavenging system.
2.2.6 Role of non-neuronal cells
In the previous chapters the role of microglia as the resident immune cell of the central
nervous system and the important function of astrocytes in clearing the neurotransmitter
glutamate from the synaptic cleft of motor neurons were described briefly. At this point it is
important to note that although ALS affects only motor neurons, the disease pathogenesis is
not, however, cell-autonomous. In other words, the toxicity that kills the motor neurons does
not rise solely from the mutant SOD1 expressing motor neurons themselves, but also from the
44
surrounding non-neuronal cells as astrocytes also show pathology (Bruijn et al., 1997b) and
microglia are activated in the regions of motor neuron loss (Hall et al., 1998b).
Restricted expression of mutant SOD1 in astrocytes (Gong et al., 2000) or motor
neurons alone (Lino et al., 2002; Pramatarova et al., 2001) failed to provoke overt motor
neuron disease in mice, supporting the idea of non-cell- autonomous disease pathogenesis, as
reviewed in chapter 2.1.4.1. Moreover, chimeric mice having both mutant SOD1 and normal
cells in their CNS showed that even a fraction of normal cells in the CNS can save the
animals from the disease, despite high levels of mutant SOD1 in motor neurons (Clement et
al., 2003).
The role of mutant SOD1 toxicity in microglia and in motor neurons themselves was
further analyzed by the Cre/Lox approach, where mutant SOD1 gene flanked by LoxP sites
was deleted from microglia or motor neurons by Cre-recombinase excision targeted either to
motor neurons by a motor neuron specific promoter Islet-1, or to microglia and peripheral
macrophages with a CD-11b promoter (Boillee et al., 2006b). This approach has identified the
differential contribution of mutant damage within motor neurons and microglia on disease
onset and progression. Excision of mutant SOD1 within motor neurons extended survival of
the mice by slowing onset and early phase of disease progression. Similar effects on survival
have been obtained by removal of neurofilaments (Williamson et al., 1998) or the tail domain
of the NF-M and NF-H subunits (Lobsiger et al., 2005) specifically in motor neurons. The
deletions provided extended survival but only by slowing disease onset. In contrast,
diminishing mutant SOD1 expression in microglia has little effect on the onset of disease but
slows later disease progression, resulting in remarkable extension of the overall survival as
the increase in survival was longer for Cre-CD11b animals than for Islet-1 mice. In addition
to floxing of mutant SOD1 form microglia and slowing down late phase of the disease,
similar results have been obtained in a complementary study, where the entire myeloid
lineage was replaced by transplantation of normal bone marrow cells into SOD1 mutant mice
that had a deletion of PU.1 transcription factor, making them unable to produce myeloid cells.
Transplantation had no effect on onset but slowed disease progression after the onset (Beers et
al., 2006). Moreover, transplantation of mutant G93A-SOD1 myeloid cells at birth into
G93A-SOD1 / PU.1 knock out mice produced onset and survival typical of the G93A-SOD1
line, whereas transplantation of G93A-SOD1 bone marrow cells into wild type animals did
not give rise to motor neuron disease, demonstrationg that mutant SOD1 in microglia alone is
not sufficient to cause motor neuron death, but accelerates disease progression after the onset.
Mutant SOD1 in astrocytes can also affect the survival of motor neurons in vitro.
Cultured mouse motor neurons from embryonic spinal cord or differentiated from embryonic
45
stem cells were less likely to survive when co-cultured with mutant SOD1 expressing
astrocytes (Di Giorgio et al., 2007; Nagai et al., 2007). Interestingly, in culture conditions the
survival of motor neurons was not affected by coculture with mutant SOD1 expressing
microglia or fibroblats. Moreover, astrocytes expressing mutant SOD1 did not show toxic
effects on other neuronal cells, such as GABAergic neurons or dorsal root ganglion neurons,
indicating a selective but unknown vulnerability of motor neurons (Nagai et al., 2007). But,
what is the toxic mechanism of astrocytes? Excitotoxicity through decreased glutamate uptake
by astrocytes is a central toxic mechanism implicated in ALS and glutamate transporter levels
are reduced in mutant SOD1 animals. However, extracellular glutamate was not increased in
the culture system (Nagai et al., 2007). Activated astrocytes also produce cytokines,
chemokines and other neurotoxic substances that may mediate neuronal death. However, no
abnormalities were observed in IL-1 , IL-6, INF- , TNF- or FAS ligand (Nagai et al., 2007).
The toxic mechanism remains to be elucidated, yet reactive oxygen species produced by
astrocytes might mediate motor neuron death. Secreted mutant SOD1 might be also the
damaging component directly as mutant SOD1 may be secreted by astrocytes. Extracellular
mutant SOD1 was recently shown to provoke motor neuron cell death in cultures and it is a
potent activator of microglial cells (Urushitani et al., 2006).
Summarizing the role of non-neuronal cells and motor neurons; it seems that mutant
SOD1 in glia accelerates disease progression considerably although it cannot induce disease
alone, while mutant action within motor neurons is a primary determinant of early onset
disease. It also extremely interesting to note that when ALS mice are treated with minocycline
starting from a very young age, minocycline delays disease onset (Zhu et al., 2002), while
administration of minocycline closer to the disease onset results in a slowing of disease
progression after onset (Kriz et al., 2002), suggesting that minocycline may have an effect on
both glia and neurons alleviating the toxicity. The role of non-neuronal cells has also
implications on stem cell therapies for ALS, as it may not be a good idea to transplant motor
neurons to a hostile glial environment, but rather transplant healthy astrocytes or microglia.
2.2.7 Pathway of motor neuron cell death in ALS
ALS is a multifactorial complex disease with many proposed mechanism leading to specific
cell death of motor neurons. However, instead of looking to proposed mechanisms i.e.
aggregation, excitotoxicity, oxidative damage or neurofilament disorganization as separate
causes or trying to determine which is the primary one, the hypothesized mechanisms can be
viewed as a combined pathogenic pathway (Figure 3) (as reviewed in Boillee et al., 2006a).
46
The following is a description of the pathogenic pathway from the onset to the end stage in
the inherited form of ALS composed from the results obtained form mutant SOD1 mouse
lines. Although only motor neurons are degenerating, it is important to note that damage is
not rising from the motor neurons alone but also from the surrounding non-neuronal cells. On
the other hand, one may ask why only motor neurons are degenerating despite ubiquitous
expression of mutant SOD1. The answer is because of unique functional properties of the
motor neurons; they are very large cells having axons over one meter in length in humans
reaching form spinal cord to feet. This sets high demands for energy production and
biosynthesis. Motor neurons have high rates of firing and response to glutamate inputs,
making them efficient but vulnerable to excessive glutamate and calcium.
As shown by Boillee et al. 2006b, damage within motor neurons is an important
determinant of disease onset. The earliest event in human ALS and in mutant SOD1 mice is
denervation, or in other words, withdrawal of motor neurons axons from their synapses on
muscles (Fischer et al., 2004; Frey et al., 2000; Pun et al., 2006; Schaefer et al., 2005). This
denervation causes the first symptom in ALS pathogenesis that is visible to the eye as seen by
loss of strength from the voluntary muscles that will progress to paralysis and muscle atrophy
(Cifuentes-Diaz et al., 2001). During the phase of denervation at the time of onset of the
disease, mutant SOD1 primarily acts within motor neurons as misfolded and aggregating
SOD1 damages cellular machinery, especially mitochondria, proteasome and chaperones,
inhibiting their normal functions in maintaining cellular homeostasis. Axonal transport is also
inhibited as a consequence of neurofilament disorganization, blocking the transit of cellular
components, i.e. mitochondria, to sites where energy is needed or resulting from
mitochondrial damage as energy needed for the transport is no longer supplied to the transport
machinery. The action of mutant SOD1 in motor neurons is also amplified by action within
other cell types, especially in microglia, as they respond to the initial damage through positive
feedback by activation and proliferation.
The symptomatic phase is characterized by massive gliosis or activation of microglia
and astrocytes in addition to continuing damage within motor neurons. Mutant SOD1 can
itself activate microglia, promoting secretion of trophic molecules but also the secretion of
toxic proinflammatory factors like nitric oxide, TNF and IL-1 produced by activated
microglia and astrocytes, which activates neighboring cells and thus end up harming their
environment and motor neurons. Toxic mutant SOD1 may also be secreted or released
through cell leakage or lysis. Mutant SOD1 in astrocytes induces loss of the EAAT2
glutamate transporter, causing repetitive firing of glutamate receptors and excessive influx of
calcium on the motor neurons as glutamate is not rapidly cleared away from the synaptic cleft.
47
Figure 3. Mechanisms leading to motor neuron degeneration in ALS. Toxicity arises fromdamage within motor neurons and is amplified by activated microglia and astrocytes.Selective vulnerability of motor neurons to mutant SOD1 toxicity is resulting from theirunique properties (large cells with long axons, high biosynthetic load, high rates of firing andglutamate uptake) and damage to supporting non-neuronal cells. Modified from (Boillee et al.,2006a; Cleveland and Rothstein, 2001)
Ca2+
ActivatedMicroglia
ActivatedAstrocyte
Motor Neuron
Muscle
Schwann Cell
GlutamatergicNeuron
SOD1
Mitochondria
Proteasome
Chaperones
Endosomal reticulum
Caspase
Neurofilament Disorganization
EAAT2 is lostToxic factors(e.g. NO, NGF)
Glu
Toxic factors(e.g. NO, TNF )
EAAT2
Denervation
Aggregates containing SOD1
48
At the end stage, denervation has produced paralysis of voluntary muscles
accompanied by muscle atrophy. Activated microglia and astrocytes continue to produce
diffusible toxic products including nitric oxide and TNF , accelerating disease spread.
Repetive firing of glutamate receptors from lack of EAAT2 result in excitotoxicity with
excessive intracellular calcium that further damages calcium buffering mitochondria and
endosomal reticulum and triggers the caspase dependent programmed cell death pathway
within motor neurons. To summarize, the selective degeneration of motor neurons is not alone
due to their unique and possible fragile form and functions, but from a combination of factors
rising from motor neurons and the surrounding glia.
2.3 Therapeutics for ALS
2.3.1 Drug treatments
ALS is a complex multifactorial disease, making the discovery of effective drugs and
interventions challenging. On the other hand, as many different pathogenic mechanisms are
involved in ALS, intervention on any of these mechanisms may interrupt or slow down the
pathogenesis. However, no cure or effective treatment presently exists for ALS.
Many different types of drugs have been tested in rodent models of ALS and also in
clinical trials and most are based on various hypotheses of mechanisms for neuronal death
that are learned by studying the mutant SOD1 mice. The treatments have been targeted to
overcome e.g. oxidative damage, loss of trophic factor support, glutamate-mediated
excitotoxicity and chronic inflammation (Bruijn et al., 2004a). Riluzole, an inhibitor of
glutamate release and an anti-convulsant, is so far the single agent presently approved for
clinical use. It only extends survival modestly by a few months but has little or no effect on
the symptoms (Bensimon et al., 1994; Lacomblez et al., 1996). Paradoxically, it may itself
cause muscle weakness although these adverse effects are reversible after stopping the use of
the drug. A number of trophic factors, anti-inflammatory agents, and inhibitors of oxidative
stress have been reported to prolong survival in mouse models and some are now in clinical
trials (Bruijn et al., 2004a). VEGF, anti-inflammatory COX-2 inhibitor celecoxib, and
minocycline have had particularly promising results in mice (Azzouz et al., 2004; Cudkowicz
et al., 2006; Kriz et al., 2002; Storkebaum et al., 2005; Van Den Bosch et al., 2002; Zhu et al.,
2002). Immunization against mutant SOD1 may also have therapeutic effects as mutant SOD1
has secretory pathways and secreted extracellular mutant SOD1 can provoke motor neuron
cell death in culture (Urushitani et al., 2006). Passive immunization through intraventicular
49
infusion of anti-human SOD1 antibody alleviated symptoms and increased life span of G93A-
SOD1 mice (Urushitani et al., 2007), making immunization one possible candidate for the
treatment of familial ALS caused by SOD1 mutations.
Clinical trials for several of these treatment strategies are ongoing, but no
breakthroughs have yet occurred, whereas a rather dissapointing moment was faced recently
when minocycline failed to show any benefit in a large phase III humal trial of ALS (Gordon
et al., 2007). Presently it is thought that combinations of different drugs may be required to
slow the multifactorial neurodegeneration process effectively (McGeer and McGeer, 2005).
2.3.2 Growth factors
Growth factor therapies on ALS have been based on the hypothesis that whatever the disease
provoking insult is, providing increased levels of trophic factors to motor neurons would be
beneficial. However, so far clinical trials with growth factors have had only little or no
benefit. Insulin like growth factor 1 (IGF-1) slowed the progression of functional impairment
and the decline in health-related quality of life in patients with ALS in one study (Lai et al.,
1997), but failed to do so in another (Borasio et al., 1998). No beneficial effects were seen
with trials on BNDF or CNTF either (ALS CNTF Treatment Study Group 1996; BDNF Study
Group 1999). One major problem in these trials has definitely been the delivery of factors past
the blood-brain-barrier to the CNS, as growth factors were delivered subcutaneously or
directly into the spinal fluid. Direct intracerebroventicular (ICV) or intrathecal infusion of
growth factors might overcome the delivery problem. Although intrathecal administration of
BDNF was found to have no effect, ICV infusion of IGF-1 extended survival in mice (Nagano
et al., 2005a) and also showed a modest benefit to ALS patients in a clinical trial (Nagano et
al., 2005b).
In addition to direct infusion, growth factors have been delivered in mouse models
with viral vector mediated gene therapy. Adeno-associated virus (AAV) has been used to
retrogradedly transport the growth factor gene along the axon from muscle to the motor
neuron cell soma where the viral genome can express the growth factor gene and trophic
factors are secreted by the motor neuron. Delivery of IGF-1 with this strategy slowed disease
progression in mice even when the treatment was initiated after disease onset (Kaspar et al.,
2003).
VEGF gene may be a contributor to ALS, as mutations of VEGF gene and its
promoter are associated with increased risk of developing ALS (Lambrechts et al., 2003;
Terry et al., 2004). Therefore, both viral and ICV delivery of VEGF have been tested in ALS
50
models. Muscle injected with lentiviral VEGF was retrogradedly transported to motor neurons
and extended survival of ALS mice (Azzouz et al., 2004), as did also continous ICV infusion
of recombinant protein into CSF in ALS rats (Storkebaum et al., 2005).
2.3.3 Gene therapies
As mutant SOD1 causes ALS through a toxic gain of function and lack of SOD1 activity in
knock out mice is not detrimental, limiting mutant SOD1 expression either through viral
delivery of silencing RNA (siRNA) or direct infusion of antisense oligonucleotides might
offer useful treatment strategies.
In mice, lentiviral delivery of SOD1 siRNA through retrograde transport to motor
neurons reduced SOD1 expression in motor neurons, slowed disease initation and increased
survival remarkably (Ralph et al., 2005). However, disease progression from onset to end
stage was not affected at all as SOD1 was not silenced in surrounding non-neuronal cells.
A similar effect was also seen in another study using AAV as a vector (Miller et al., 2005).
Intrathecal injection of SOD1 siRNA virus to spinal cord also delayed motor neuron
degeneration near the injection site, but it had no effect on the overall disease progression
(Ralph et al., 2005).
However, when considering human clinical trials, some practical issues remain. For
example the dosage cannot be altered nor can the treatment be stopped as the virus starts the
expression of the delivered gene. An alternative to the problems of using viral vectors might
be delivery of antisense oligonucleotides. This approach has been succesful in ALS rats
resulting in lowering of SOD1 levels in brain and spinal cord and slowing of disease
progression (Smith et al., 2006).
2.3.4 Stem cell therapies
Selective cell death of motor neurons has made the idea of replacing dying motor neurons
with stem cells tempting. However, in ALS it is hard to imagine that even if transplanted stem
cells developed into motor neurons, how they can form appropriate connections with muscles
as motor axons need to extend distances for up to a meter in length to reach the target in
humans. Nevertheless, stem cell therapy for replacing motor neurons has been tried with some
success in Sindbis virus paralyzed rat spinal cords, where ES-cell -derived motor neuron
precursor cells were injected into rat spinal cord (Deshpande et al., 2006; Harper et al., 2004).
In the study by Harper et al. the cells survived and even extended axons with the help of
51
intrathecal injection of myelin repulsion alleviating molecules, but no neuromuscular
junctions were formed (Harper et al., 2004). Altered differentiation conditions, dibutyryl as an
anti-myelin repulsion factor and grafting of neural stem cells expressing glial derived
neurotrophic factor (GDNF) produced neuromuscular connections and relieved paralysis of
Sindbis virus exposed rats (Deshpande et al., 2006).
An alternative strategy to the task of replacing the long motor neurons would be to
replace surrounding non-neuronal cells, as mutant SOD1 expressing motor neurons can be
saved by surrounding wild type glial cells and extend survival of motor neurons that express
SOD1 (Boillee et al., 2006b; Clement et al., 2003). Then also other sources of stem cells
instead of motor neuron precursor cells such as bone marrow or umbilical cord blood could be
used. Human umbilical cord blood cells have also been shown to be protective (Garbuzova-
Davis et al., 2003). Bone marrow transplantations have been used with G93A-SOD1 mice in
two instances, where the first reported paper showed some extension in survival (Corti et al.,
2004) whereas in the other no protection was seen (Solomon et al., 2006). The hurdle may be
the surviving residential microglia expressing mutant SOD1. Transplantation of normal bone
marrow cells into SOD1 mutant mice that had a deletion of PU.1 transcription factor (making
them unable to produce myeloid cells) slowed disease progression after the onset (Beers et al.,
2006). Moreover, transplantation of mutant G93A-SOD1 myeloid cells at birth into G93A-
SOD1 / PU.1 knock out mice produced onset and survival typical of the G93A-SOD1 line
whereas transplantation of G93A-SOD1 bone marrow cells into wild type animals did not
give rise to motor neuron disease. These results demonstrate that mutant SOD1 in microglia
alone is not sufficient to cause motor neuron death, but expression of mutant SOD1 in
microglia rather accelerates disease progression after the onset (Beers et al., 2006).
Accordingly, bone marrow replacement might be a beneficial treatment for ALS if the
recruitment to the brain is sufficient enough to replace the residential mutant SOD1
expressing microglia possibly even if the treatment is done after the onset.
In addition to replacing cells, stimulation of the endogenous pool of stem cells to
proliferate and replace degenerating motor neurons might provide neuroprotection. In G93A-
SOD1 mice some neurogenesis has been observed in response to neurodegeneration (Chi et
al., 2006). However, the proliferating endogenous cells also express mutant SOD1 and
therefore are susceptible to the mutant SOD1 toxicity.
52
2.3.5 Pyrrolidine dithiocarbamate (PDTC)
Pyrrolidine dithiocarbamate (PDTC) belongs to a class of dithiocarbamates which have
previously been used in the treatment of bacterial and fungal infections, and have been
considered for use in the treatment of AIDS (Reisinger et al., 1990). PDTC is also known as
an inhibitor of nuclear transcription factor kappa-B (NF- B) that regulates the expression of
several proinflammatory genes and some genes related to apoptosis (Hayakawa et al., 2003;
Liu et al., 1999; Schreck et al., 1992). In addition, PDTC has been shown to be a potent
antioxidant both in vitro and in vivo (Hayakawa et al., 2003; Liu et al., 1999; Schreck et al.,
1992).
As inflammation, apoptosis and oxidative stress are implicated in a large range of
diseases including ALS, it is not surprising that PDTC has been reported to have beneficial
effects in models of diseases such as pleurisy, arthritis, colitis, liver and brain ischemia, spinal
neonatal asphyxia and autoimmune uveoretinitis (Chen et al., 2005; Cheng et al., 2006;
Cuzzocrea et al., 2002; Kitamei et al., 2006; La Rosa et al., 2004; Malm et al., 2007; Matsui et
al., 2005; Messina et al., 2006; Nurmi et al., 2006; Nurmi et al., 2004a; Parodi et al., 2005).
There is evidence that mechanisms of PDTC's beneficial effects in these models are indeed
related to it's ability to act as an antioxidant and to inhibit the expression of pro-inflammatory
genes, including COX-2, TNF and interleukin-1 (Nurmi et al., 2004a; Nurmi et al., 2004b).
In addition, PDTC may also provide protection by acting through Akt kinase -
glycogen synthase kinase (GSK) signaling (Malm et al., 2007; Nurmi et al., 2006). Activation
of GSK-3 can lead to apoptotic neuronal death and result in energy depletion in stress
conditions. In addition, GSK-3 may also inhibit the expression of transcription factors that
support cell survival (Grimes and Jope, 2001). GSK-3 is a unique enzyme in the regard that
it is activated by dephosphorylation instead of the more common way of enzyme activation
trough phosphorylation. Akt is a well known kinase able to phosphorylate GSK-3 and thus
inactivate GSK-3 (Grimes and Jope, 2001). As PDTC activates Akt, it not surprising that
PDTC was shown to be protective in models of neonatal asphyxia and Alzheimer’s disease
with GSK-3 activation (Malm et al., 2007; Nurmi et al., 2006). PDTC can act also as a metal
chelator for transitional metals transporting extracellular copper into the cell and, moreover,
the transitional metal-PDTC complex may have an inhibitory activity against the proteasome
(Kim et al., 2004).
53
PDTC has been shown to have beneficial effects in many different disease models and
it possesses capabilities for activating or inhibiting several cellular targets, making it an
interesting drug candidate for preclinical testing in ALS.
54
3. AIMS OF THE STUDY
The underlying initiative cause of the mutant SOD1 toxicity is still unknown. Mutations of
SOD1 may affect the stability of the enzyme making it more prone to aggregate, mutations
may alter the catalytical site allowing aberrant substrates to enter the catalytical site and cause
unwanted oxidative reactions. Recent results have shown that the toxic effect of mutant SOD1
may be targeted to mitochondria, but the evidence for the toxic mechanism in mitochondria is
scarce. This study was carried out to assess the stability and oxidation of human mutant SOD1
throughout the disease progression using transgenic ALS rats and mice and to analyze the role
of mutant SOD1 in mitochondria. In addition, as oxidative damage, inflammation and
apoptosis play major roles in the pathology of ALS, anti-inflammatory and anti-oxidative
drug treatment with PDTC might have neuroprotective effects, and we tested PDTC drug
treatment on G93A-SOD1 transgenic rats. The specific aims were to study:
1) Effect of an anti-inflammatory and anti-oxidative drug treatment with PDTC on G93A-
SOD1 transgenic rats and analysis of the molecular mechansims involved.
2) Stability and molecular features of mutant SOD1 in a G93A-SOD1 rat model of ALS and
the effect of mutant SOD1 from affected tissues on the mitochondria.
3) Molecular mechanism for the deleterious role of mutant SOD1 in mitochondria.
4) Proteomic analysis of differential protein expression and protein oxidation in a G93A-
SOD1 mouse model of ALS with emphasis on oxidation of mutant SOD1 through disease
progression in affected and unaffected regions of the central nervous system.
55
4. MATERIALS AND METHODS
4.1 Animals (I-IV)
All animal studies were carried out with permission of The Institutional Animal Care and Use
Committee of the University of Kuopio and the Provincial Government according to the
National Institute of Health guidelines for the care and use of laboratory animals. Rats and
mice were housed in groups of 2-3 rats in one cage in light and temperature controlled
environment with ad libitum water and standard laboratory rodent chow.
Transgenic rats expressing human mutant G93A-SOD1 [Tac:N:(SD)-
TgN(SOD1G93A)L26H, Emerging Models, sponsored by Amyotrophic Lateral Sclerosis
Association, Taconic, Hudson, NY, USA] develop motor neuron disease similar to human
ALS (Howland et al., 2002). Hemizygous rats over-express human mutant G93A-SOD1 at
levels increasing from 8-fold over endogenous SOD1 in presymptomatic rats to 16-fold in end
stage animals. Disease onset is determined by limb gait and occurs on average at 115 days (16
weeks). From the onset the disease progresses rapidly to end stage within 11 days. The end
stage of the disease was determined by righting reflex test, where the animal was placed on its
side and if the rat was not able to right itself in 30 seconds, it was scored as death and
sacrificed. Righting reflex failure typically coincides with complete paralysis of both
hindlimbs and one forelimb as a result of substantial loss of spinal cord motor neurons, as
well as marked increases in gliosis and degeneration of muscle integrity and function.
Transgenic and corresponding wild-type littermate rats were sacrificed at 8 weeks
(presymptomatic), 16 weeks (onset) and at the end stage of the disease (18 weeks of age).
Sacrificed animals were anaesthetized with 40mg/kg pentobarbital and perfused transcardially
with saline and extracted tissues were frozen in liquid nitrogen and stored at –80 C. G93A-
SOD1 rats were also treated with PDTC on a dose of 50 mg/kg/day starting at 70 days of age
(n=19). The control tg rats (n=12) received plain water. In addition, wt littermates of
corresponding age received either PDTC (n=6) or plain water (n=7). PDTC treated or
untreated animals were anaesthesized with 40mg/kg pentobarbital, perfused transcardially
with saline and extracted tissues were either frozen in liquid nitrogen and stored at –80 C or
post-fixed with 4% paraformaldehyde for 24 h, cryoprotected with 30% sucrose for three days
and then frozen in liquid nitrogen and stored at –80 C.
Transgenic G93A-SOD1 mice [B6.Cg-Tg(SOD1-G93A)1Gur/J, The Jackson
Laboratory, Bar Harbor, ME, USA] express high levels of the transgene with a 4-fold increase
in SOD activity, and exhibit a phenotype similar to ALS in humans (Gurney et al., 1994).
56
Hemizygous transgenic mice become paralyzed in one or more limbs and have a life span of
approximately 19-23 weeks. Paralysis is due to loss of motor neurons from the spinal cord.
Mice were sacrificed with carbon dioxide anaesthesia and decapitation at the age of eight
weeks and tissues (spinal cord, brain and liver) were collected for mitochondria isolation.
Samples from G93A-SOD1 mice at ages of 8-, 14- and 18-weeks and corresponding wt
control mice and mice expressing human wt SOD1 age of 12 weeks were kindly provided by
Dr. Catherine Bendotti from the Department of Neuroscience, Istituto di Ricerche
Farmacologiche Mario Negri, Milan, Italy.
SOD1-/- knockout mice (Reaume et al., 1996) and corresponding wild types (n = 5)
were kindly provided by Dr. Pak Chan from the Department of Neurosurgery, Stanford
University School of Medicine, Stanford, California, USA. Knockout mice were sacrificed
with carbon dioxide anaesthesia and decapitation at the age of eight weeks and tissues (spinal
cord, brain and liver) were collected for mitochondria isolation. Prior to decapitation blood
was collected with heart puncture for lymphocyte isolation.
Wistar rats (8 week old males, National Laboratory Animal Center, University of
Kuopio, Kuopio, Finland) were used as source of liver mitochondria. Wistar rats were
sacrificed with carbon dioxide anaesthesia and decapitation.
4.2 PDTC treatment (I)
G93A-SOD1 rats were treated with PDTC on a dose of 50 mg/kg/day starting at 70 days of
age (n=19). Fresh PDTC (Sigma, St. Louis, MO, USA) was administered into drinking water
every other day. The control tg rats (n=12) received plain water. In addition, wt littermates of
corresponding age received either PDTC (n=6) or plain water (n=7). Neurological signs of
disease, such as overall locomotor activity, ataxia, extension reflex of hind limbs, limb gait,
paralysis of hind limb or paralysis of fore limb and weight gain of the animals were followed
daily. Appearance of limb gait was determined as the onset of the disease. A new dose of
PDTC was calculated weekly according to animal weight and water consumption, and the
treatment was continued until the end stage of the disease when the animals were sacrificed.
The end stage of the disease was determined by rightening reflex test where animal was
placed on its side and if it was not able to righten itself in 30 seconds it was scored as death.
Sacrificed animals were anaesthesized with 40 mg/kg pentobarbital, perfused transcardially
with saline and extracted tissues were either frozen in liquid nitrogen and stored at –80 C, or
post-fixed with 4% paraformaldehyde for 24 h and cryoprotected with 30% sucrose for three
days and then frozen in liquid nitrogen and stored at –80 C.
57
4.3 Mitochondria (II,III)
4.3.1 Isolation of mitochondria
Mitochondria were isolated from liver, brain or spinal cord. Tissue was homogenized with all-
glass dounce homogenizator (Kontes, Vineland, NJ, USA) in 10 × volume of ice-cold
isolation buffer (320 mM sucrose, 1 mM EGTA, 10 mM Tris-HCl pH 7.4). The homogenate
was centrifuged at 2000 g for 3 min at 4 C, and the supernatant was transferred to a new
tube and centrifuged 10000 × g for 10 min. The supernatant was discarded and the pellet
containing the crude mitochondrial fraction was washed once with wash buffer (0.2 M
sucrose, 20 mM HEPES pH 7.2, 0.1 mM EGTA, 4 mM KH2PO4) and resuspended in the
same buffer to a concentration of 10 mg/ml. The protein concentration was determined by
protein assay dye (Bio-Rad, Hercules, CA, USA).
In order to obtain pure mitochondria from CNS tissue, the crude mitochondrial
fraction was transferred on top of a 19 % percoll solution (Sigma) in isolation buffer and
centrifuged at 30700 g and at 4 C for 10 min. The mitochondrial fraction accumulating to
the bottom was collected, diluted 1:3 with wash buffer, centrifuged at 17000 × g for 10 min at
4 C and resuspended back to wash buffer. The purity of the mitochondrial fraction was
determined with Western blotting as a presence of mitochondrial marker COX4 and absence
of cytosolic marker actin (figure 4).
Figure 4. Purity of mitochondrial fraction was determined with Western blotting as apresence of mitochondrial markers COX4 and SOD2 and absence of cytosolic marker actin.Subcellular fractions and markers from mouse cortex: Cyt = Cytosolic soluble fraction; Tot =Total homogenate; P2 = Crude mitochondrial fraction; Mito = Purified mitochondrial fraction.
MWkDa Cyt Tot P2 Mito
actin
SOD2
COX4
175836248
33
25
17
58
4.3.2 Functional integry of the isolated mitochondria
Mitochondrial membrane potential ( ) was measured using JC-1 (5,5',6,6'-tetrachloro
1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) dye. The dye undergoes a reversible
change in fluorescence emission from green to red as mitochondrial membrane potential
increases. JC-1 accumulates as aggregates in the mitochondria, resulting in red fluorescence.
The brightness of red fluorescence is proportional to . Succinate addition caused the
expected rise in , whereas uncoupling by CCCP (carbonyl cyanide 3-
chlorophenylhydrazone) led to depolarisation, indicating normal function of the mitochondrial
membrane.
Oxygen consumption was measured with an oxygraph on mitochondria respiring on
succinate. Addition of 2.5 M ADP causes increased oxygen consumption rate, coupled to
oxidative phosphorylation and yielding a mean respiratory control index (RCI) of 5.38,
indicating normal tightness (RCI between 3 and 10) of the coupling between oxidative
phosphorylation and respiration.
4.3.3 Isolation of mitoplasts
In order to obtain mitoplasts (mitochondria devoid of outer membrane), mitochondria were
incubated with a 5 × volume of cold hypotonic buffer (10 mM Tris, pH 7,4, 1 mM EDTA and
1 mM DTT ) for 10 min on ice. After 10 min, 150 mM NaCl was added and mitoplasts were
incubated 10 min on ice and centrifuged at 18000 g and at 4 C for 10 min. Mitoplasts were
resuspended into standard medium consisting of 0.3 M mannitol, 10 mM KCl, 10 mM
KH2PO4, 5 mM MgCl2, 1 mg/ml BSA, pH 7.4 to a concentration of 10 mg/ml.
4.3.4 Exposure of mitoplasts with cytosolic homogenates of G93A-SOD1 rat tissues
Five hundred micrograms of mitoplasts isolated from wt rat liver were exposed to 100 µg of
cytosolic spinal cord or cortex homogenate form 8 and 16 week old and end stage rats with
equalized amonts of human SOD1 at RT for 1 h. After the exposure, the mitoplasts were
centrifuged at 10000 × g for 1 min and rinsed three times with standard medium to wash away
unbound SOD1. An aliquot of each sample was resuspended in 1 × Laemmli sample buffer
and analyzed with anti-SOD1 Western blotting for the bound SOD1. The remaining mitoplast
suspension was analyzed for ROS production.
59
4.3.5 Measurement of ROS production
ROS measurements were carried out in 96-well plates by mixing 200 µg (20 µl) of mitoplast
or mitochondria suspension with 140 µl standard medium with final concentrations of 1.3 mM
Initial light microscopy imaging of the lumbar spinal cord sections for constitutive (20S X)
and immunoproteasome (20S LMP7) subunits showed that the constitutive proteasome was
expressed in cells throughout the gray matter in the ventral horn of the lumbar spinal cord in
both PDTC treated tg and wt rats, and in untreated tg and wt rats. However, the
immunoproteasome was expressed only in untreated tg rats at the end stage. From the
appearance, the cells showing immunoproteasome staining looked non-neuronal. Double
labeling immunohistochemistry with confocal imaging for cell markers for neurons (NeuN),
astrocytes (GFAP) and microglia (CD68), and for constitutive proteasome (20S X) and
immunoproteasome (20S LMP7), showed that the immunoproteasome 20S LMP7 was
expressed in astrocytes and microglia, whereas proteasome 20S X was also expressed in
neurons. The results indicate that immunoproteasome indution occurs at the end stage of the
disease in microglia and astrocytes, and that PDTC is able to inhibit glial immunoproteasome
induction with devastating effects on survival of the rats.
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5.6 Mutant SOD1 was oxidized and destabilized in spinal cords of G93A-SOD1 rodent
models (II,IV)
Proteomic analysis of protein expression and oxidation during pathogenesis in G93A-SOD1
mice from presymptomatic, symptomatic and end stage of the disease revealed that mutant
SOD1 is one of the oxidized proteins in the spinal cord of transgenic G93A-SOD1 mouse
model of ALS. Moreover, the oxidation of G93A-SOD1 increases significantly with disease
progression specifically in spinal cord as the level of oxidation of G93A-SOD1 was higher in
spinal cord when compared to oxidation in less affected regions of the CNS, such as the
cerebellum and hippocampus. In addition, with two-dimensional electrophoresis followed by
mass spectrometric identification we detected a fragment of human SOD1 that was oxidized.
Moreover, the mouse endogenous SOD1 was not oxidized at all, or its oxidized form is
promptly degraded, implicating a possible toxic property for the oxidized form. However, also
the human wt SOD1 is oxidized to the same extent as the as the G93A-SOD1 in the spinal
cord as shown by 2D anti-DNP immunoblotting of spinal cord from human wt SOD1
expressing mice. Analysis of protein expression also revealed that expression of PDI, a
molecular chaperone which rearranges inter- and intramolecular disulphide bonds between
thiol groups of cysteine residues on unfolded proteins (Turano et al., 2002), was upregulated
early in the disease progression, before any symptoms were seen. This suggest that thiol-
related oxidation is altered in ALS, and that the status of the disulphide bonds in SOD1 may
greatly affect the stability of the SOD1 protein.
The stability of SOD1 was analyzed by employing modified immunoblotting, which
allows distinguishing the degree of denaturation and loss of quaternary structure by binding to
a hydrophobic PVDF membrane in non-reducing conditions. Native SOD1 is an exceptionally
stabile protein dimer and in non-reducing electrophoretic conditions SOD1 should appear as a
dimer. The non-reducing PAGE analysis of purified G93A-SOD1, SOD1 from the spinal
cord, cortex and liver samples of G93A-SOD1 rats revealed that purified G93A-SOD1 and
G93A-SOD1 from the liver were present as a dimer, indicating that protein stability of the
purified mutant SOD1 or mutant SOD1 in peripheral tissues, as assessed by electrophoretical
behavior, is not altered. However, SOD1 seemed to be less stable in the cortex and
cerebellum, since the SOD1 dimer from the cortex and cerebellum had partially broken down
to monomers. In the spinal cord, the quaternary structure of SOD1 was destabilized the most
as SOD1 in the spinal cord had less of both dimeric and monomeric conformations compared
to the cortex, and the majority of SOD1 in the spinal cord was present as a misfolded
monomer having a molecular weight of approximately 20-30 kDa. The monomeric
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destabilized SOD1 with a molecular weight of 20-30 kDa was present in the spinal cord
throughout the disease from the early presymptomatic time point of 8-weeks until the end
stage of the disease, whereas non-reducing PAGE analysis of SOD1 from the cortex,
crebellum or liver showed no destabilization or misfolded monomers with disease
progression.
Surprisingly, purified human wt SOD1 did not bind to Western blotting membrane in
the non-reducing conditions at all, possibly due to the fact that native wt SOD1 is an
exceptionally stable and hydrophilic protein dimer and therefore may not bind to the
hydrophobic blotting membrane under the non-reducing conditions. It is also important to
note that the differences seen in electrophoretical mobility of mutant SOD1 from various
tissues are not due to incorporation of insoluble aggregates to the sample, as native gradient
PAGE did not show any high molecular weight bands for the samples. This indicates that
aggregation and misfolded monomeric species are originating from the soluble biologically
stable dimeric SOD1 detected in the native immunoblot.
5.7 PDI was upregulated and its levels inversely correlated with the levels of cysteine
reduced SOD1 (II,IV)
As PDI was upregulated early in the disease progression as shown by proteomic analysis, and
as mutant SOD1 was clearly destabilized over disease progression in affected tissues, the
nature of the destabilization was further investigated by malPEG assay, where the oxidation
state of the SOD1 cysteine residues were analyzed using malPEG modification and anti-
SOD1 Western blotting. Human SOD1 has four cysteines and only the reduced and exposed
cysteine residues are expected to react with malPEG. The cytosolic spinal cord and cortex
samples were modified with 3 mM malPEG, which will react and bind to free reduced
cysteine residues, causing a 5 kDa increase in molecular weight that can be detected in anti-
SOD1 Western blot. MalPEG modification showed a significant increase in the levels of free
reduced cysteine residues of SOD1 in the spinal cord at the end stage of the disease when
compared to the cortex. In the spinal cord, malPEG modification produced size shifts of both
5 kDa and 10 kDa, indicating a modification of one and two cysteine residues, respectively.
As levels of the disulphide-reduced SOD1 had indeed increased in tg mice, we
confirmed our results of the proteomics data, showing increased PDI expression in G93A-
SOD1 mice, also in tg rats with anti-PDI western blotting over disease course in G93A-SOD1
rats. PDI is a molecular chaperone, which functions to rearrange inter- and intramolecular
72
disulphide bonds between cysteine residues of unfolded proteins (Turano et al., 2002). Human
SOD1 has four cysteine residues (C6, C57, C111 and C146). Two of these, C57 and C146,
form an intrasubunit disulphide bond. Expression of PDI was upregulated before the disease
onset at 8 weeks and at the end stage was restored back to levels similar to the cortex of
mutant SOD1 expressing rats or the spinal cord of wt rats. These results are in line with the
proteomics data showing PDI upregulation at presymptomatic stage in G93A-SOD1 mice.
PDI upregulation in ALS models has also been shown by others (Atkin et al., 2006). These
data suggest that early upregulation of PDI in the disease progression may be an attempt to
cope with accumulation of destabilized SOD1 but is eventually overwhelmed by aggregation
of destabilized SOD1 with disease progression.
5.8 Mitochondrial SOD1 levels were the highest in the spinal cord of G93A rats (II)
Mitochondria are a likely target of mutant SOD1 toxicity and as SOD1 is also expressed in
the intermembrane space of mitochondria, we analyzed the levels of mutant SOD1 localized
to mitochondria from isolated and purified mitochondria with anti-SOD1 Western blotting.
Mitochondria were isolated from transgenic G93A-SOD1 rat cortex and spinal cord tissues
form different stages of the disease: 8-weeks - presymptomatic, 16-weeks - onset, and from
the end stage. The purity of the mitochondrial fraction was determined with Western blotting
as a presence of mitochondrial markers COX4, SOD2 and absence of cytosolic stuctural
component actin. SOD1 amounts were quantified from anti-SOD1 immunoblots and
normalized against COX4. SOD1 levels in mitochondria were 40-100% higher in the spinal
cord when compared to the cortex at presymptomatic stage and at the end stage of the disease.
5.9 Mutant SOD1 bound to mitoplasts and enhanced ROS production (II)
As the association of mutant SOD1 to mitochondria may also be related to the over-
expression of the transgene (Bergemalm et al., 2006), we set up a model system for mutant
SOD1 toxicity in mitochondria where liver mitoplasts (mitochondria devoid of outer
membrane) from wt rats were exposed to cytosolic tissue homogenates of G93A-SOD1 rats
from the spinal cord and cortex, with or without destabilized SOD1, respectively. The purity
of the mitoplast fraction was determined with Western blotting as a presence of mitochondrial
marker SOD2 and absence of cytosolic stuctural component actin. In addition, rat endogenous
73
SOD1 resided in the mitoplast fraction. The binding of destabilized mutant SOD1 from spinal
cord cytosol to the mitoplasts, isolated from wt rat liver, was increased after exposure when
compared to binding of SOD1 deriving from the cortex. In parallel, ROS production was
significantly elevated in mitoplasts exposed to the cytosolic spinal cord homogenate of an 8-
week-old G93A-SOD1 rat.
5.10 Mutant SOD1 increased hydrogen peroxide production in the intermembrane space
of mitochondria (III)
The role of SOD1 in mitochondria is somewhat controversial as dismutase activity is not
required in the intermembrane space. Instead of SOD1, cytochrome c can quench superoxide
efficiently in the intermembrane space by oxidizing superoxide directly to oxygen. Therefore,
cytochrome c acts as a true antioxidant (Pereverzev et al., 2003), whereas SOD1 will produce
hydrogen peroxide in addition to oxygen. We hypothesized that upon mitochondrial stress
SOD1 might compete with cytochrome c for superoxide in the intermembrane space and
generate hydrogen peroxide, which then could react with cytochrome c and oxidize the
cytochrome c molecule to oxoferryl heme. Oxoferryl heme is a highly reactive oxidant that is
able to react with a number of intracellular targets including proteins, nucleic acids and lipids
(Lawrence et al., 2003), eventually leading to a paradoxical increase in ROS production and
cellular injury.
The mechanism for increased mitochondrial ROS production and the role of SOD1
was further studied by isolated wt liver mitochondria challenged with antimycin A, an
inhibitor of complex III, resulting in a break in the electron transport chain of the oxidative
phosphorylation and a prompt superoxide production as shown by electron paramagnetic
resonance (EPR) (Han et al., 2003). The SOD activity in an intermembrane space preparation
was rapidly increased as a function of time in response to antimycin A. Mitochondrial
respiration also resulted in increased production of hydrogen peroxide as determined in
parallel by measurement of the fluorescence of 2,7-dichlorodihydrofluorescein diacetate
(DCF), a widely used probe sensitive for hydrogen peroxide (Hempel et al., 1999). The
response remarkably coincided with the maximal increase in SOD activity. SOD1 was
essential for the increased hydrogen peroxide production, because adding SOD1 inhibitors
ammonium tetrathiomolybdate or ammonium diethyldithiocarbamate, significantly and dose-
dependently reduced the DCF fluorescence in isolated mitochondria. Moreover, the
mitochondria isolated from SOD1 deficient knock out mice (SOD1-/-) mice produced
74
substantially less DCF fluorescence and did not show any response to inhibition of complex
III by antimycin A. Thus, the elevated SOD1 activity is responsible for the increased
hydrogen peroxide production in the intermembrane space, resulting in cytochrome c-
catalysed DCF oxidation.
In order to be sure that the seen effects were not due to damage caused to
mitochondria by isolation, the functional integrity of the mitochondria was ensured by
measuring membrane potential with JC-1 dye, and the respiration rate of the isolated
mitochondria was measured as oxygen consumption with an oxygraph. The membrane
potential increased with addition of succinate, allowing respiration, and decreased after
uncoupling with CCCP, which leads to depolarisation, indicating normal function of the
mitochondrial membrane. The respiration rate increased after addition of ADP and was
coupled to oxidative phosphorylation, indicating normally respiring mitochondria measured
as oxygen consumption with an oxygraph. Also L-NNA (N -Nitro-L-arginine), an inhibitor of
nitric oxide synthesis (NOS), had no effect on the hydrogen peroxide production, indicating
that the peroxide measure with DCF is not preoxynitrite (-OONO) formed by reaction with
superoxide and NO produced by the NOS.
In order to further test the hypothesis that SOD1 activity is responsible for the
increased hydrogen peroxide production in the intermembrane space, we isolated mitoplasts,
i.e. mitochondria devoid of outer membrane and the intermembrane space, to reconstitute
conditions for hydrogen peroxide production. No DCF oxidation could be detected in
respiring mitoplasts even after inhibiting complex III by antimycin A, indicating that the auto-
oxidation rate of DCF without peroxidase is low. Addition of cytochrome c caused a slight
increase in DCF fluorescence, possibly due to hydrogen peroxide escaping from the
mitochondrial matrix. Importantly, addition of SOD1 at 100 nM concentration more than
doubled the rate of DCF oxidation in the presence of cytochrome c. To confirm the role of
peroxide in the DCF oxidation, addition of horseradish peroxidase (HRP) to mitoplasts was
shown also to increase the DCF oxidation. Addition of SOD1 together with HRP increased
the oxidation even more, demonstrating also the contribution of superoxide dismutation.
To model the interaction of superoxide, cytochrome c and SOD1 in the intermembrane
space, we reconstituted a reaction where superoxide was generated through xantine
oxidase/xantine (XO/X). A slow oxidation of DCF occurred in the presence of this enzyme
substrate pair. The rate of DCF oxidation was slightly elevated by 5 M cytochrome c.
However, adding increasing concentrations of SOD1 in the reaction mixture strongly and
dose-dependently increased the rate of DCF oxidation, indicating that SOD1 significantly
enhances cytochrome c-catalyzed peroxidation.
75
To investigate whether SOD1 controls the production of hydrogen peroxide in the
mitochondrial intermembrane space also in intact cells, we isolated lymphocytes from wt and
SOD1-/- mouse blood by differential centrifugation, and loaded them with DCF before adding
antimycin A. Flow cytometric analysis showed that antimycin A-induced DCF oxidation was
attenuated in SOD1-/- lymphocytes at 90 min of incubation as compared to the hydrogen
peroxide production in wt lymphocytes. In addition to increased superoxide production in
mitochondria, inhibition of complex III by antimycin A has also been shown to induce
apoptosis (Wolvetang et al., 1994). To test whether SOD1 activity is important for apoptosis,
SOD1-/- and wt lymphocytes were challenged with antimycin A for 90 min and apotosis was
determined by Annexin V binding using flow cytometry. SOD1-/- lymphocytes showed
significantly less apoptosis than wt lymphocytes.
5.11 Mutant SOD1 activity and ROS production were increased in spinal cord
mitochondria of G93A-SOD1 mice (III)
According to these data, the increased amount of SOD1 associated with the mitochondrial
intermembrane space would be expected to cause enhanced ROS production. We studied
mitochondrial SOD1 activity in mutant G93A-SOD1 mice, and in fact, the SOD1 activity of
intermembrane space preparations from the spinal cord was 6-fold greater in G93A-SOD1
mice than in wt mice. In addition, SOD1 activity in the mitochondrial intermembrane space in
spinal cord was somewhat higher in comparison with non-affected brain tissues of the same
animals.
The SOD1 activity of the mitoplast preparations isolated form the spinal cord was
twice as high as from the brain of G93A-SOD1 mice, whereas the SOD2 activity was equal in
mitoplasts between spinal cord and brain. To investigate whether the elevated activity and
accumulation of mutant G93A-SOD1 in mitoplasts results in increased ROS production, we
measured DCF oxidation in the mitoplast preparations in the presence of cytochrome c. The
mitoplasts isolated from the spinal cord of G93A-SOD1 mice produced significantly more
DCF fluorescence compared to the mitoplasts that derived from the cortex of G93A-SOD1
mice, indicating that the increased SOD1 amount in the intermembrane space is also
associated with an increase in hydrogen peroxide production. The main findings reported in
the results are summarized in table 2.
76
Table 2. Main findings of the thesis.
Originalpublication Main findings
Referenceto chapter
IPDTC reduced survival of ALS rats by inhibiting induction ofALS pathogenesis specific immunoproteasome in glial cells andby increasing copper concentration in the CNS.
5.1 – 5.25.4
IImmunoproteasome inhibition lead to faster disease progressiondespite PDTC upregulated levels of glutamate transporter GLT-1 in the CNS of ALS rats.
5.3
II, IVMutant SOD1 was oxidized and destabilized specifically inspinal cord of ALS rats and mice as shown by carbonyl levelsand stability of SOD1 dimer to mild denaturation.
5.5
II, IVPDI was upregulated in spinal cord and levels of PDI inverselycorrelated with reduced disulphide bonds of mutant SOD1. 5.6
IIMutant destabilized SOD1 from spinal cord associated tomitoplasts (mitochondria devoid of outer membrane) andincreased ROS production.
5.7 – 5.8
IIIIncreased ROS production may be due to elevated SOD1activity in the intermembrane space of mitochondria leading toincreased hydrogen peroxide production and oxidation ofcytochrome c to oxoferryl heme.
5.9 – 5.10
77
6. DISCUSSION
This study was carried out to investigate the stability and oxidation of human mutant SOD1
through the disease progression using tg G93A-SOD1 ALS rats and mice and to analyze the
role of mutant SOD1 in mitochondria. In addition, as inflammation and oxidative damage are
implicated greatly in ALS pathogenesis, we treated G93A-SOD1 rats with PDTC, a drug
known for its anti-inflammatory and antioxidative properties.
6.1 PDTC inhibits immunoproteasome induction resulting in reduced survival of ALS
rats (I)
Previous studies have shown that PDTC provides protection in a number of CNS and
peripheral disease models by inhibiting activation of transcription factor NF- B, serving as a
strong antioxidant, or by activating the protective Akt-GSK3 pathway (Cheng et al., 2006;
Cuzzocrea et al., 2002; La Rosa et al., 2004; Matsui et al., 2005; Messina et al., 2006; Nurmi
et al., 2006; Nurmi et al., 2004a). Even though several antioxidants (Gurney et al., 1996; Wu
et al., 2003) and inhibitors of NF- B-driven genes, such as COX-2, TNF and IL-1
(Drachman et al., 2002; Kiaei et al., 2006) prolong survival of tg ALS mice, and activation of
the Akt-GSK3 pathway reduced mutant SOD1-mediated motor neuron cell death in vitro
(Koh et al., 2005), we found that PDTC treatment does not provide protection, but instead,
significantly decreases the survival of G93A-SOD1 rats. The fact that PDTC treatment
prevented the reduction in levels of glutamate transporter GLT-1, a potential therapeutic
target verified in numerous animal studies of ALS (Gurney et al., 1996; Howland et al.,
2002), and that PDTC treatment did not induce toxic side effects in the tg or wt rats, the
negative finding is of interest. It implies that PDTC treatment caused some harmful alterations
in cellular functions which were able to override the potentially beneficial effects of the drug.
In agreement with the previous studies on mouse ALS models (Cheroni et al., 2005;
Puttaparthi and Elliott, 2005), we observed that both an increase in proteasome activity and an
induction of immunoproteasome selectively occur in the affected spinal cord tissue of a
G93A-SOD1 rat model. Importantly, we found that PDTC treatment strongly inhibited these
ALS-model specific changes in the proteasome. On the other hand, in line with previous
reports showing that PDTC is a metal chelator and transports copper from the extracellular
medium into the cell, we found that PDTC treatment increased the copper concentration in the
spinal cord of both G93A-SOD1 tg and wt rats. Considering that PDTC is also known as a
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proteasome inhibitor (Kim et al., 2004), and that increased copper concentration may be
needed for the proteasome inhibitory activity of PDTC, it is likely that the detrimental effect
of PDTC on G93A-SOD1 rats is mediated by inhibition of the immunoproteasome. This
inhibition of the immunoproteasome was also evidenced as increased levels of ubiquitinated
proteins in PDTC-treated G93A-SOD1 rats. Because 20S X, a marker of the constitutive
proteasome, was not affected in G93A-SOD1 tg rats alone or in G93A-SOD1 by PDTC
treatment, whereas 20S LMP7, an inducible subunit of immunoproteasome, was induced
selectively in astrocytes and microglia, our results suggest that the immunoproteasome in
non-neuronal cells plays a protective role in G93A-SOD1 rats.
In addition to its effect on proteasome acitivity, an increased copper concentration
may well trigger other cellular processes, which in animals overexpressing G93A-SOD1
could accelerate neurodegeneration. An oral, 15-day PDTC treatment at millimolar doses has
been reported to increase the copper concentration and levels of lipid peroxidation products in
rat peripheral nerves (Calviello et al., 2005), implicating that at least in some rodent tissues a
long-term PDTC treatment may cause oxidative stress associated with increased copper
concentrations. On the other hand, a 7-month PDTC treatment with the same protocol as in
the present study significantly increases cortical copper concentrations in a mouse model of
Alzheimer's disease and prevents the decline in cognition without increasing oxidative stress
in the brain (Malm et al., 2007). Even though induction of the immunoproteasome was
observed selectively in G93A-SOD1 rat spinal cords and PDTC prevented this induction
without causing changes in other known targets of PDTC, we cannot exclude the possibility
that the increased copper concentration enhances motor neuron degeneration in G93A-SOD1
rats also by other mechanisms in parallel with immunoproteasome inhibition. However, it
should be noted that the -lactam antibiotic ceftriaxone, which has been reported to be
neuroprotective in models of ALS by increasing expression of glutamate transporter GLT-1
(Rothstein et al., 2005), is also a metal chelator. PDTC resembles ceftriaxone as it
significantly increases expression of GLT-1 and copper concentration in the spinal cord. We
hypothesize that even though -lactams and PDTC might both be able to modulate GLT-1
and copper concentration, only PDTC but not -lactams also inhibits immunoproteasome. As
induction of immunoproteasome may be a rather selective characteristic for ALS (models)
compared to models of ischemia, trauma and amyloid accumulating diseases, inhibition of the
immunoproteasome alone in ALS models could increase the accumulation of ubiquitinated
proteins, including ubiquitinated SOD1, which has been suggested to gain neurotoxic
functions such as increased peroxidase activity.
79
PDTC is an established inhibitor of NF- B. Even though inhibition of NF- B has
frequently been associated with tissue and cellular protection, inhibition of NF- B may also
accelerate neurodegeneration because of down-regulation of survival-supporting NF- B-
regulated genes, such as manganese SOD and Bcl-2 (Mattson and Camandola, 2001). Even
though we detected a trend for increased NF- B binding activity in the spinal cords of G93A-
SOD1 rats, no statistically significant differences between any of the untreated/PDTC treated
rats were observed. It is possible that in a chronic disease such as ALS, NF- B activity even
though being induced is not maintained at such high levels. On the other hand, oral PDTC
administration may not allow achieving as high tissue concentrations as intraperitoneal
administration does, and thereby does not result in efficient inhibition of NF- B. Also, we
cannot exclude the possibility that NF-kB binding to DNA is increased at time points other
than the presymptomatic (100 days) and end-stage time points studied here. Nevertheless, our
experiments do not provide evidence for a central role of NF- B in ALS. In a previous study,
NF- B immunoreactivity was found to be increased in astrocytes surrounding degenerating
motor neurons (Migheli et al., 1997). Therefore, the possible antiapoptotic effects of NF- B
may not be, at least, directly involved in neuronal survival in tg ALS models.
Nevertheless, PDTC inhibited the immunoproteasome induction and resulted in
decreased survival despite upregulated GLT-1 and other possible beneficial effects, including
anti-inflammatory and anti-oxidative mechanisms. These results indicate that
immunoproteasome induction in the disease pathogenesis may be an important mechanism for
coping with the toxic consequences of mutant SOD1 in tissues affected in ALS.
6.2 Mutant SOD1 oxidation and destabilization precede aggregation and loss of activity
(II, IV)
Mutant SOD1 is one of the oxidized proteins in the spinal cord of G93A-SOD1 mice and rats
and the level of oxidation increases with disease progression. However, while the mouse
endogenous SOD1 remains intact, the human mutant and wild-type SOD1 are oxidized to the
same extent in the spinal cord. Therefore, the oxidative response to the presence of abnormal
proteins is different in the spinal cord compared to the CNS regions which are not affected in
ALS. This altered oxidative response may reflect a lower capacity of the spinal motor neurons
to clear/degrade abnormal proteins. This hypothesis is also supported by our identification of
a partially degraded and oxidized form of human mutant SOD1. Oxidation of SOD1 in
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general does not result in the toxicity that leads to motor neuron death. This is due to the fact
that mutant SOD1 and human wt SOD1 with same levels of expression as the mutant are
oxidized to the same extent, and moreover, human wt SOD1 over-expression does not
provoke motor neuron disease. However, our data does not completely exclude the possibility
that oxidation renders the mutant SOD1 toxic, because the resulting forms of oxidized wt and
mutant SOD1 may be functionally different. Proteome analysis revealed the upregulation of
PDI, paralleled with increased oxidation of PDI, early in the disease progression that
coincides with accumulation of inactive SOD1 having reduced intrasubunit disulphide bonds.
These results suggest that formation of disulphide bonds and possible thiol-related oxidation
may be altered in familial ALS and may have implications to the SOD1 stability.
SOD1 was destabilized specifically in the spinal cord, which is the most affected
region in ALS pathogenesis. The destabilization, shown as loss of SOD1-dimer stability to
mild denaturaton, was evident throughout the period from late presymptomatic stage to the
end stage. As the redox state of SOD1 may determine the dismutase activity of mutant SOD1,
the nature of the destabilization was further on analyzed by malPEG assay, where malPEG
binds to free cysteine residues and reduced disulphide bonds can be detected. As a positive
control, analysis of the purified human SOD1, when reduced overnight by incubating with
2mM DTT, showed that the reduced, inactive SOD1 had a similar malPEG binding pattern as
the malPEG binding of the mutant SOD1 from the end stage spinal cord. While no changes in
the oxidation state of SOD1 cysteine residues were observed in the SOD1 derived from the
cortices, malPEG reactivity of SOD1 in the spinal cord increased over disease progression,
reaching a statistically significant difference at the end stage of the disease when compared to
SOD1 malPEG reactivity in cortices of the same age. This supports the notion that SOD1
dismutase activity and potential for increased ROS production in the intermembrane space
may be gradually lost, possibly making mutant SOD1 more hydrophobic and more prone to
aggregate, thus shifting the toxic insult from presymptomatic oxidative damage to aggregation
at the symptomatic stages of the disease. Human SOD1 has four cysteine residues and Ferri
and coworkers showed that removal of SOD1 cysteine residues C6 and C111 may suppress
oligomerization of mutant SOD1 by reducing SOD1's ability to form intermolecular
disulphide bonds (Ferri et al., 2006). This is in line with our results showing destabilization
and increased levels of reactive cysteine groups at the symptomatic stages of the disease
progression.
As levels of cysteine reduced SOD1 had increased, we analyzed the levels of protein
disulphide isomerase (PDI) also with Western blotting. PDI is a molecular chaperone, which
functions to rearrange inter- and intramolecular disulphide bonds between cysteine residues of
81
unfolded proteins (Turano et al., 2002). Human SOD1 has an intramolecular disulphide bond
between cysteine residues C57 and C146 that is required for SOD1 to be active, and thus PDI
may be a key enzyme in controlling SOD1 activity. PDI may activate SOD1 through redox
state of the disulphide bonds, similarly as thioredoxin reductase (Inarrea et al., 2007; Inarrea
et al., 2005). In addition, PDI has been shown to localize to the mitochondria (Rigobello et al.,
2001). PDI was upregulated early in the disease progression and the expression was sharply
decreased towards the end stage, correlating conversely with increased levels of cysteine
reduced SOD1 as shown by malPEG reactivity. PDI upregulation in ALS models has also
been shown by others (Atkin et al., 2006).
Oxidation and destabilization of the mutant SOD1 and reduction of disulphide bonds
within SOD1 combined with upregulation of PDI, a disulphide bond rearranging chaperone,
were specific for the affected spinal cord tissue in rat and mouse models of ALS, indicating a
structural modification that may have an impact on cellular localization, activation and
aggregation of the protein.
6.3 Destabilized mutant SOD1 associates with inner membrane of mitochondria and
increases ROS production (II)
Mutant SOD1 has been shown to be imported preferentially into mitochondria in affected
tissues of SOD1 mouse models (Bergemalm et al., 2006; Deng et al., 2006; Liu et al., 2004;
Vijayvergiya et al., 2005). However, in order for SOD1 to be imported into mitochondria it
must be in a demetallinated state lacking both Cu2+ and Zn2+, and it has to have its
intramolecular disulphide bond reduced (Field et al., 2003). After being imported in to the
intermembrane space of mitochondria, SOD1 is loaded with zinc through an unknown
mechanism and copper is loaded by Cu chaperone for SOD1 (CCS) (Culotta et al., 1997).
After that, SOD1 is kept inactive until activation upon oxidation of its cysteine residues
forming intramolecular disulphide bond (Inarrea et al., 2005).
In order to investigate how destabilized SOD1 may cause mitochondrial dysfunction,
we analyzed mutant SOD1 association to mitochondria. Mutant SOD1 associated to spinal
cord mitochondria to a higher extent than to mitochondria in the cortex of G93A-SOD1 rats.
However, as the mutant SOD1 association to mitochondria may also be related to the over-
expression of the transgene (Bergemalm et al., 2006), we analyzed mutant SOD1 toxicity not
in mitochondria from transgenic rats, but from wt liver mitochondria by exposing mitoplasts
(mitochondria devoid of outer membrane) with cytosolic tissue homogenates of G93A-SOD1
82
rats from the spinal cord and cortex, with or without destabilized SOD1, respectively. This
model allowed us to re-create the situation where mutant SOD1 is acting in the
intermembrane space and we also were able to distinguish mutant SOD1 from endogenous rat
SOD1 residing in the mitochondrial fraction, as mitochondria and mitoplasts were isolated
from wt rats. The association of destabilized SOD1 from the spinal cord cytosol to wt liver
mitoplasts after exposure was increased and also ROS production was elevated by 28% at the
pre-symptomatic timepoint. The result was reproducible and in our opinion truly noteworthy,
as exposed mitoplasts were from the liver of healthy young wild type rats. Taken together,
exposing mitoplasts to destabilized SOD1 may damage mitochondria, as destabilized mutant
SOD1 bound to the inner membrane of mitochondria and increased ROS production.
Our results indicate that the destabilization with loss of quaternary structure involving
disulphide bonds of mutant SOD1 in the spinal cord makes SOD1 toxic to mitochondria, as
destabilized SOD1 associated to the inner mitochondrial membrane and increased ROS
production. The destabilization may help possibly the mutant SOD1 to enter mitochondria, as
the import of SOD1 requires the reduction of disulphide bonds and demetallination, and the
preferential import of mutant SOD1 may cause toxicity to mitochondria in the intermembrane
space. Still, the mechanism for this toxic function of mutant SOD1 in mitochondria has not
been identified - until now.
6.4 Elevated SOD1 activity in the intermembrane space leads to increased hydrogen
peroxide production resulting in cytochome c catalyzed oxidation (III)
Even though several theories on mechanisms explaining how mutant SOD1 may cause
mitochondrial dysfunction are proposed, including oxidative damage, excitotoxicity with
calcium buffering, and aggregation, there is a lack of conclusive proof of mutant SOD1
toxicity in mitochondria. In fact there is no conclusive proof for one general mechanism of
mutant SOD1 toxicity in ALS at all. What is generally accepted is that many harmful events
like protein aggregation, oxidative damage and excitotoxicity take place in ALS pathogenesis,
occur in concert, and together are combined to ALS disease. What comes to oxidative damage
and mutant SOD1, after the discovery of SOD1 mutations in 1993, mutant SOD1 was
believed to lack activity and the lack of dismutase activity with increased superoxide levels
was thought to be a key contributor for the disease mechanism - this was not the case, as
SOD1 knock out mice do not develop motor neuron disease (Reaume et al., 1996). Secondly,
mutant SOD1 was believed to produce ROSs from aberrant substrates (Beckman et al., 1994;
83
Estevez et al., 1999; Wiedau-Pazos et al., 1996) - which is most likely not the case as,
increasing the levels of endogenous wild type SOD1 activity did not have any improvement
on the disease pathogenesis in transgenic mouse models. Still, SOD1 activity must play a role,
as increasing the levels of human wt SOD1 expression in mutant SOD1 mice accelerates
disease progression in ALS mice (Deng et al., 2006). From this we come back to the SOD1
activity, not aberrant, but the known accepted SOD1 dismutation activity where superoxide is
converted to oxygen and hydrogen peroxide.
In the cytosol, SOD1 converts superoxide to oxygen and hydrogen peroxide and
hydrogen peroxide is further on converted to water by glutathione peroxidase or catalase as
hydrogen peroxide itself is a strong oxidant. So, in one way of saying, SOD1 takes care of the
job of detoxifying superoxide only halfway, as hydrogen peroxide needs to be cleared by
other enzymes. This is still fine as long as glutathione peroxidase or catalase is present.
However, glutathione peroxidase or catalase are only present in low levels in the
intermembrane space (Martin et al., 1998) and superoxide detoxification is thought to depend
on cytochrome c, which can efficiently oxidize superoxide to oxygen, acting as true
antioxidant (Pereverzev et al., 2003) as none of the products needs to be processed any
further.
Previous studies have demonstrated that the reaction of cytochrome c with hydrogen
peroxide results in the formation of oxoferryl cytochrome c (peroxidase compound I-type
intermediate), which is highly reactive and has a potential to oxidize proteins, DNA and
lipids, as well as endogenous antioxidants such as glutathione, NADH and ascorbate
(Lawrence et al., 2003). In particular, oxidation of cardiolipin, a phospholipid which is in
complex with cytochrome c on the surface of the inner mitochondrial membrane, leads to the
release of proapoptotic factors from mitochondria (Belikova et al., 2006; Kagan et al., 2005).
This leads to a scenario where upon mitochondrial stress, SOD1 might compete with
cytochrome c for superoxide in the intermembrane space and generate hydrogen peroxide,
which then could react with cytochrome c and oxidize the cytochrome c molecule to oxoferryl
heme, a highly reactive oxidant that is able to react with a number of intracellular targets
including proteins, nucleic acids and lipids (Lawrence et al., 2003), eventually leading to a
paradoxical increase in ROS production and cellular injury. The hydrogen peroxide produced
by increased SOD1 activity in the intermembrane space would thus also serve as a substrate
for cardiolipin-bound cytochrome c, and consequently switch on a very early proapoptotic
processes, leading to consecutive programmed cell death.
Our results indicated that upon inhibition of mitochondrial respiration, the elevated
SOD1 activity is responsible for the increased hydrogen peroxide production in the
84
intermembrane space, resulting in cytochrome c-catalysed oxidation not seen in the
mitochondria isolated from SOD-/- mice. This could trigger a vicious circle where oxidative
damage to mitochondrial respiratory components leads to further ROS production and
peroxidation. Indeed, inhibition of mitochondrial respiration at the level of complex III causes
SOD1-dependent ROS production and apoptotic death of isolated blood lymphocytes.
Moreover, accumulation of mutant human G93A-SOD1 in the intermembrane space that is
observed in tg animal models of ALS, leads to elevated SOD1 activity and increased
cytochrome c-catalyzed oxidation in the intermembrane space.
SOD is generally thought to protect cells from oxidative damage. Accordingly, as a
cytosolic antioxidant SOD1 provides protection in models of transient myocardial (Chen et
al., 2000), brain ischemia (Chan, 2005) and Parkinson's disease (Barkats et al., 2002). Some
other studies, however, suggest that the increased SOD1 activity promotes injury. For
instance, immature mouse brain overexpressing SOD1 shows an increased propensity for
injury and accumulates more hydrogen peroxide after hypoxia-ischemia than wt mouse brain
(Fullerton et al., 1998). Also, elevation of SOD1 increases acoustic trauma from noise
exposure (Endo et al., 2005), and mice deficient in SOD1 are resistant to acetaminophen
toxicity (Lei et al., 2006). Moreover, a superoxide generator, menadione, produces
significantly increased DCF fluorescence and greater death in neurons with mutant SOD1
than in wt neurons, suggesting increased hydrogen peroxide formation in the mutant SOD1
expressing cells (Ying et al., 2000). This apparent discrepancy concerning the role of SOD1 in
cellular injury is explained by the results showing that increased SOD1 activity in the
intermembrane space paradoxically produces peroxides that are converted to highly toxic
ROS.
Mitochondrial dysfunction, including altered function of respiratory complexes, has
been described in arteriosclerosis, diabetes mellitus, and a number of acute and degenerative
brain diseases such as stroke, Parkinson's disease and ALS. Increased ROS production is also
a characteristic of these diseases (Barnham et al., 2004). The increased SOD1 activity
accompanied with high hydrogen peroxide production in the intermembrane space may be
one mechanism of neurodegeneration. Importantly, the toxicity of ALS-linked SOD1 mutants
originates from their selective recruitment to the spinal cord mitochondria (Bergemalm et al.,
2006; Deng et al., 2006; Liu et al., 2004; Vijayvergiya et al., 2005). SOD1 activity in the
intermembrane space may be a relevant therapeutic target for ALS and other degenerative
diseases involving mitochondrial pathogenesis.
85
6.5 Hypothesized role of destabilized mutant SOD1 toxicity in mitochondria
The reports in the present thesis work provide important information regarding the
possible toxic role of mutant SOD1 in mitochondria in ALS pathogenesis (Figure 5), where
destabilized but active mutant SOD1 is preferentially imported to mitochondria in the spinal
cord. PDI upregulation and conversely correlating levels of reduced cysteine residues of
SOD1 implicate SOD1 inter- and intramolecular disulphide bridges to have a key role in
stability and activity of SOD1. Moreover, destabilized SOD1 may well have implications also
on aggregation, which is a dominant feature of ALS pathogenesis. One striking feature of the
immunoproteasome inhibition by PDTC is that it only occurs in glial cells, namely microglia
and astrocytes, but the damaging effect is seen as reduced survival in rat a model of ALS
despite upregulated GLT-1 levels and anti-inflammatory/oxidant properties of the drug used,
indicating the importance of glial immunoproteasome in ALS pathogenesis. As the glial
immunoproteasome machinery was subdued, the toxicity was focused against motor neurons.
Destabilization of SOD1 may also make it more preferable for mitochondrial import,
as SOD1 needs to be demetallinated and intaramolecular disulphide bond reduced in order to
enter mitochondria. In the mitochondrial intermembrane space, destabilization may also have
a role in SOD1 activation via PDI and production of hydrogen peroxide by activated mutant
SOD1 in the early stages of the disease. In addition to immunoproteasome inhibition, PDTC
also increased intracellular copper concentrations in the CNS, which may lead to higher
SOD1 activity as copper is required in the dismutation reaction, therefore possibly resulting in
increased hydrogen peroxide production. Hydrogen peroxide can react with cytochrome c,
producing highly reactive oxoferryl heme, resulting in oxidation far more damaging than that
of superoxide or hydrogen peroxide, initiating a process of cellular death.
The mechanism presented here for the toxicity of SOD1 in mitochondria provides new
and important information about SOD1 toxicity and enables us to understand better the
underlying factors of motor neuron cell death in ALS, where, SOD1 activation upon stress in
mitochondria and production hydrogen peroxide are the key initiators of oxidative damage
and the process of cellular damage and death. In the future, controlling SOD1 activity in the
intermembrane space or targeting anti-oxidants to the intermembrane space of mitochondria
may be relevant therapeutic approaches for many degenerative diseases involving
mitochondrial pathogenesis, especially ALS.
86
Figure 5. Hypothesized role of mutant SOD1 in mitochondria and in ALS pathogenesis.Misfolded but active mutant SOD1 localizes to mitochondria, where hydrogen peroxideproduced by SOD1 dismutase activity leads to the formation of oxoferryl heme of cytochromec and oxidation of biological targets. Misfolded SOD1 may well have implications onaggregation as PDI is upregulated in ALS pathogenesis and inhibition of immunoproteasomewith PDTC had adverse effects. PDTC increased copper concentrations and therefore possiblyalso increased SOD1 activity and ROS production. Activation of caspases is the final eventleading to programmed cell death.
SOD1•O2
–
+ H2O2
cyt coxoferryl-cyt c
H2OO2
cyt c
cyt c
ActiveSOD1
Mitochondria
Proteasome Chaperones
Caspase
Aggregatescontaining SOD1
Immuno-proteasome
PDTC
SOD2•O2–
MisfoldedSOD1
PDI
Cu2+
87
7. SUMMARY AND CONCLUSIONS
The present study was carried out to study the stability and oxidation of mutant SOD1 and the
toxic role of mutant SOD1 in mitochondria in ALS pathogenesis. In addition, we tested an
anti-inflammatory/oxidant drug with PDTC treatment in an ALS rat model.
PDTC inhibited immunoproteasome induction in glial cells and resulted in decreased
survival despite upregulated GLT-1 and other possible beneficial effects including anti-
inflammatory and anti-oxidative mechanisms by the drug. These results indicate that
immunoproteasome induction in the disease pathogenesis may be an important mechanism for
coping with the toxic consequences of mutant SOD1 in tissues affected in ALS, and also in
the surrounding glial cells. PDTC also increased intracellular copper concentration.
Oxidation of SOD1, destabilization of the quaternary structure with reduction of the
cysteine residues of mutant SOD1 combined with upregulation of PDI, a disulphide bond
rearranging chaperone, were specific for the affected spinal cord tissue in rat and mouse
models of ALS, indicating a structural modification that may have an impact on cellular
localization, activation and aggregation of the protein.
Although SOD1 is classically considered to be a cytosolic enzyme, mutant SOD1
associates to mitochondria in affected spinal cord tissue to a higher extent than to
mitochondria in other regions of the CNS. In addition, destabilized SOD1 associated to the
inner mitochondrial membrane and increased ROS production. The destabilization may
possibly help the mutant SOD1 to enter mitochondria as the import of SOD1 requires
reduction of the disulphide bond and demetallination. Because of the destabilization, mutant
SOD1 is imported preferentially to mitochondria, and may cause damage in the mitochondrial
intermembrane space by interacting with the machinery of the oxidative phosphorylation.
The mechanism of this interaction was shown to be a situation where upon
mitochondrial stress SOD1 might compete with cytochrome c for superoxide in the
mitochondrial intermembrane space and generate hydrogen peroxide, which then could react
with cytochrome c and oxidize the cytochrome c molecule to oxoferryl heme. Cytochrome c
oxidized to oxoferryl heme is a highly reactive oxidant, far more reactive than superoxide or
hydrogen peroxide, capable of rapidly reacting with a number of intracellular targets
including proteins, nucleic acids and lipids, eventually leading to a paradoxical increase in
ROS production and cellular injury leading to consecutive programmed cell death.
The mechanism presented here for the toxicity of SOD1 in mitochondria provides new
and important information about SOD1 toxicity and enables us to understand better the
underlying factors of motor neuron cell death in ALS, where SOD1 activation upon stress in
88
mitochondria and production of hydrogen peroxide are the key initiators of oxidative damage
and the process of cellular damage and death. In the future, controlling SOD1 activity in the
intermembrane space or targeting anti-oxidants to the intermembrane space of mitochondria
may be relevant therapeutic approaches for many degenerative diseases involving
mitochondrial pathogenesis, especially ALS.
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