The Role of Oxidative Stress on Neural TRPC3, TRPC5, TRPC6 Expression and/or Function and Relevance to
Bipolar Disorder
by
Steven Yi Sheng Tong
A thesis submitted in conformity with the requirements for the degree of Masters of Science
Department of Pharmacology and Toxicology University of Toronto
© Copyright by Steven Tong 2012
ii
The Role of Neural TRPC3, TRPC5, and TRPC6 Expression
and/or Function and Relevance to Bipolar Disorder
Steven Yi Sheng Tong
Masters of Science
Department of Pharmacology and Toxicology University of Toronto
2012
Abstract
The etiology of bipolar disorder (BD) is multidimensional and thought to involve several
factors that increase neuronal oxidative stress and disrupt intracellular calcium
homeostasis. As calcium-permeable canonical transient receptor potential channels
(TRPC) have been linked to bipolar pathophysiology, I sought to determine whether
oxidative stress affects TRPC3/TRPC5/TRPC6 expression and/or function. Chronic (4-
day) but not acute (24-hour) rotenone-induced oxidative stress dose-dependently
reduced TRPC5 and TRPC6 protein levels in primary rat cortical neurons. A decrease in
TRPC5 mRNA levels was only found following acute but not chronic rotenone whereas
TRPC6 mRNA levels did not change significantly with either treatment. Reduced
TRPC3 function was seen after chronic stress when stimulated by TRPC3/6 activator, 1-
oleoyl-2-acetyl-sn-glycerol. Lithium pre-treatment attenuated the rotenone-induced
reduction in TRPC3 but not TRPC6 protein levels. These results suggest TRPC
subtypes are differentially regulated by oxidative stress and support a potential
mechanistic link between oxidative stress and calcium dyshomeostasis in BD.
iii
Acknowledgments
The completion of this thesis and research project would not be possible without the
unwavering support of several people. First and foremost, I would like to express my
gratitude to my supervisor, Dr. Jerry Warsh, who provided me with this opportunity and
at every turn, provided sound advice. He not only taught and guided me but challenged
me to learn, to grow, and to think critically.
I would also like to acknowledge my fellow colleagues that have helped me
acquire the technical skills needed to collect the data presented here. Many thanks to
Angela Roedding for sharing your expertise in primary rat cortical neurons and
answering any and all questions I had about it; Marty Green for teaching me the
intricacies PCR; Wynne Au-Yeung for showing me the immunoblotting process; Dr.
Takuji Uemura for helping me perfect my techniques as well as some great guidance;
Dharshini Ganeshan for your expertise in handling cell cultures; and Dr. Michael Tseng
for sharing your knowledge of live cell calcium kinetics imaging assays.
Lastly, the morale support and love I received from my family, partner, and
friends were invaluable and have been a constant source of strength and
encouragement. Thank you everyone and I feel blessed to have each and every one of
you in my life.
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Table of Contents
Acknowledgments ........................................................................................................... iii
Table of Contents ............................................................................................................ iii
List of Tables................................................................................................................. viii
List of Figures ................................................................................................................. ix
1 Introduction .................................................................................................................. 1
1.1 Bipolar Disorder .................................................................................................... 2
1.1.1 Classification .............................................................................................. 2
1.1.2 Epidemiology .............................................................................................. 2
1.1.3 Burden of Disease ...................................................................................... 3
1.1.4 Treatment ................................................................................................... 4
1.1.5 Etiology ....................................................................................................... 5
1.2 Evidence of Neuropathology in BD ....................................................................... 7
1.2.1 Structural Abnormalities in the Brain .......................................................... 7
1.2.2 Cellular Disturbances in BD ........................................................................ 8
1.2.3 Genetic Associations with BD ................................................................... 10
1.2.4 Irregularities in Signal Transduction in BD ................................................ 12
1.2.5 Alterations in Intracellular Ca2+ ................................................................. 20
1.3 Oxidative Stress in BD ........................................................................................ 23
1.3.1 Overview of Oxidative Stress.................................................................... 23
1.3.2 Evidence of Oxidative Stress in BD .......................................................... 24
1.4 Lithium and BD .................................................................................................... 28
1.4.1 Overview of Lithium and Lithium Treatment in BD.................................... 28
1.4.2 Evidence of Neuroprotective and Anti-oxidative Effects of Lithium ........... 29
v
1.5 TRP Channels ..................................................................................................... 31
1.5.1 Overview of TRP Channels ...................................................................... 31
1.5.2 Molecular Structure of TRP Channels ...................................................... 32
1.5.3 Structural Aspects of TRP Channels ........................................................ 33
1.6 The Canonical TRP Subfamily of TRP Channels ................................................ 35
1.6.1 Overview and Distribution of TRPC Channels .......................................... 35
1.6.2 Biophysical Characteristics of TRPC Channels Pores .............................. 37
1.6.3 Activation Mechanisms of TRPC Channels .............................................. 38
1.6.4 Pharmacological Properties of TRPC Channels ....................................... 39
1.6.5 Physiological Role of TRPC Channels ..................................................... 40
1.7 Main Objectives and Hypothesis ......................................................................... 41
2 Materials and Methods .............................................................................................. 43
2.1 Materials .............................................................................................................. 44
2.1.1 Chemicals, Reagents, and Drugs ............................................................. 44
2.1.2 Animals ..................................................................................................... 44
2.1.3 Cell Culture Reagents .............................................................................. 45
2.1.4 Antibodies ................................................................................................. 45
2.1.5 Analytical Kits ........................................................................................... 46
2.1.6 Calcium Kinetic Assay Solutions .............................................................. 46
2.2 Primary Rat Cortical Neuronal Cell Culture and Drug Treatment ........................ 47
2.2.1 Preparation of Cell Culture Plastics .......................................................... 47
2.2.2 Fetal Rat Cortical Dissection .................................................................... 47
2.2.3 Establishment of Primary Rat Cortical Neuronal Cultures ........................ 48
2.2.4 Maintenance of Cell Cultures ................................................................... 48
2.2.5 Drug Treatment ........................................................................................ 49
2.3 Immunocytochemistry ......................................................................................... 52
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2.4 Measurement of Cell Viability .............................................................................. 53
2.5 Immunoblotting .................................................................................................... 54
2.5.1 Preparation of Cell Lysates for Immunoblotting ........................................ 54
2.5.2 SDS-PAGE and Transfer .......................................................................... 54
2.5.3 Probing and Development ........................................................................ 55
2.6 Quantitative Real-time PCR ................................................................................ 58
2.6.1 RNA Extraction ......................................................................................... 58
2.6.2 First Strand Synthesis .............................................................................. 58
2.6.3 Primer Design and Optimization ............................................................... 58
2.6.4 Polymerase Chain Reaction ..................................................................... 59
2.7 Live Cell Calcium Kinetics Imaging ..................................................................... 62
2.7.1 Preparation of cells ................................................................................... 62
2.7.2 Imaging ..................................................................................................... 62
2.8 Data Analysis ...................................................................................................... 64
3 Results ....................................................................................................................... 65
3.1 Optimal conditions for real-time RT-PCR ............................................................ 66
3.2 Detection of TRPC5 and TRPC6 protein levels in Primary Rat Cortical Neurons ............................................................................................................... 73
3.3 Effect of Oxidative Stress on Primary Rat Cortical Neurons ................................ 75
3.4 The Effect of Acute and Chronic Oxidative Stress on TRPC5 and TRPC6 ......... 79
3.5 OAG Induces a TRPC3-mediated Response in Primary Rat Cortical Neurons ... 84
3.6 The Effect of Chronic Rotenone Induced Oxidative Stress on the Mobilization of Calcium in Primary Rat Cortical Neurons ........................................................ 88
3.7 Effect of Chronic Lithium on the Levels of TRPC3, TRPC5, and TRPC6 Protein and mRNA under Chronic Rotenone-induced Oxidative Stress .............. 91
4 Discussion ................................................................................................................. 97
4.1 Acute and Chronic Oxidative Stress on TRPC5 and TRPC6 .............................. 99
vii
4.2 The Functional Significance of Chronic Oxidative Stress on TRPC3/6 ............. 104
4.3 The Effect of Chronic Lithium Treatment on Chronic Rotenone-induced Changes in TRPC3, TRPC5, and TRPC6 ......................................................... 107
4.4 Methodological Considerations and Limitations ................................................ 110
4.4.1 Cell Model and Stressor ......................................................................... 110
4.4.2 mRNA Quantification: Real-time RT-PCR .............................................. 111
4.4.3 Protein Quantification: Western Blot ....................................................... 111
4.4.4 Functional Assay: Calcium Kinetics ........................................................ 113
4.5 Future Studies ................................................................................................... 116
4.6 Conclusions ....................................................................................................... 120
References................................................................................................................... 122
viii
List of Tables
Table 1.1 Commonly prescribed treatments to BD episodes ………………………….6
Table 1.2 Disturbances in G-protein signaling in BD ……………………………. …...18
Table 1.3 Disturbances in cAMP-pathway in BD ………………………………… …...19
Table 2.1 Immunoblotting conditions for TRPC3, TRPC5, TRPC6, α-tubulin, and β-
actin ……………………………………………………………………….. …...56
Table 2.2 Accession number, sequence, and expected product length of primer pairs
used for TRPC3, TRPC5, TRPC6, HO-1, and GAPDH.…………………..59
Table 3.1 Optimal primer concentration and template concentration for the
amplification of TRPC3, TRPC5, TRPC6, HO-1 and GAPDH .…………..70
ix
List of Figures
Figure 1.1 The phosphoinositide (PI) signal cascade ………………………………….17
Figure 1.2 Detoxification of Reactive Oxygen Species in the body …………………..26
Figure 2.1 Schedule of oxidative stress and lithium of rat cortical neurons …………50
Figure 3.1 Gel electrophoresis of RNA extracted from RCN and BLCL ……………..66
Figure 3.2 Dissociation curves of TRPC3, TRPC5, TRPC6, HO-1 and GAPH primers
…………………………………………………………………………………...67
Figure 3.3 Agarose gel electrophoresis of TRPC5, TRPC6 and GAPDH amplicons 68
Figure 3.4 Relative efficiency curves of TRPC5 and TRPC6 primers ………………..69
Figure 3.5 Western blot assay and linearity of detection vs lysate protein
concentration of TRPC3, TRPC5, and TRPC6 in primary rat cortical
neurons …………………………………………………………………………72
Figure 3.6 Representative dual labelled immunocytochemistry images of primary rat
cortical neuron cultures ……………………………………………………….74
Figure 3.7 Representative phase contrast microscopy images of primary rat cortical
neuron cultures over 4 day rotenone-induced oxidative stress treatment 75
Figure 3.8 Effects of rotenone on HO-1 expression and cell viability in primary rat
cortical neurons ………………………………………………………………..76
Figure 3.9 The effect of acute and chronic rotenone treatment on TRPC3 protein and
mRNA levels in rat primary cortical neurons ……………………………….79
Figure 3.10 The effect of acute and chronic rotenone treatment on TRPC5 protein and
mRNA levels in rat primary cortical neurons ……………………………….80
x
Figure 3.11 The effect of acute and chronic rotenone treatment on TRPC6 protein and
mRNA levels …………………………………………………………………...81
Figure 3.12 Pry3 has a significant effect on mean normalized maximal intensity, rate of
rise, and area under the curve but not the percentage of OAG activated
responding ROIs in primary rat cortical neurons …………………………..84
Figure 3.13 OAG induced a Ca2+ response in rat primary cortical neurons is mediated
in part by TRPC3 ……………………………………………………………...85
Figure 3.14 Chronic rotenone treatment reduces OAG-induced Ca2+ response in
primary rat cortical neurons ………………………………………………….87
Figure 3.15 Rotenone treatment significantly attenuates calcium responses in rat
primary cortical neurones stimulated with OAG …………………………...88
Figure 3.16 The effect of lithium pretreatment on the downregulation of TRPC5 by
chronic rotenone treatment ………………………………………………….92
Figure 3.17 The effect of lithium pretreatment on the downregulation of TRPC6 by
chronic rotenone treatment ………………………………………………….93
Figure 3.18 The effect of lithium pretreatment on the downregulation of TRPC3 by
chronic rotenone treatment ………………………………………………….94
1
1 Introduction
2
1.1 Bipolar Disorder
1.1.1 Classification
Bipolar disorder (BD) is a chronic and recurrent mood disorder best characterized by
cycling episodes of manic and depression between periods of euthymia. The Diagnostic
and Statistical Manual IV – Text Revision (DSM IV-TR) defined symptoms and signs for
manic episodes include inflated self-esteem or grandiosity, decreased need for sleep,
over activity, decreased attention span, distractibility, and increased engagement in
pleasurable and risky activities. Symptoms of depression, in contrast, are described as
diminished interest in previously pleasurable activities, weight loss, insomnia, fatigue,
diminished ability to focus, feelings of worthiness and suicidal ideation. There are
currently four subtypes of BD recognized by the DSM IV-TR – BD type I, BD, type II,
cyclothymia and BD- Not Otherwise Specified (BD-NOS), which are distinguished based
on clinical symptom severity, duration of mania or hypomania, and symptom complexes.
1.1.2 Epidemiology
Epidemiological data show that BD can develop at any stage of life but most commonly
during late adolescence and early adulthood. One study found that most patients were
diagnosed with BD before the age of 25 [1]. The ratio of BD prevalence in men and
women is generally accepted as about equal [2]. However, women are more likely than
men to experience depressive episodes and rapid cycling BD, characterized by four or
more major mood shifts within one year [3, 4]. The overall lifetime prevalence of BD in
its broadest definition (includes bipolar spectrum disorders) is estimated to be between
3
3% and 10% in the U.S. [5]. A more recent report estimated an overall prevalence of 9%
in the European Union [6].
1.1.3 Burden of Disease
With regard to the burden of disease, BD was ranked sixth among the top ten most
disabling conditions in the world [7]. A significant contributor to this burden is the noted
increase in comorbidities associated with BD, with an estimated rate reported to range
from 30% to 75% [8]. In an adult population of BD type I and II patients, it was found
that over two thirds of the patients were diagnosed with substance use disorders,
anxiety disorders (panic disorder, obsessive compulsive disorder) and eating disorders
during their lifetime [9]. Axis I psychiatric disorders such as substance abuse and
anxiety disorders were equally prevalent and accounted for the majority of concurrent
diagnoses [8, 10-12]. Outside the realm of psychiatric disorders, BD patients are more
likely to develop cardiovascular disease, hypertension, respiratory diseases, illnesses
from musculoskeletal system, endocrine system, and nervous system, diabetes, and
obesity [13-18]. Lastly, the frequency of suicide is dramatically increased in BD patients
[19]. It has been estimated that mood disorders are responsible for 70% of all suicides
[20, 21]. While there is currently no consensus on whether comorbidity is a result of BD
or secondary to the adverse effects of medication and treatment, comorbid diseases
negatively impact functionality, quality of life, response to mood stabilizers and
prognosis [11, 16, 22].
In addition, patients suffering from BD have demonstrated impairment in mental
and social function. Multiple studies have established that BD patients performed worse
than demographically-matched healthy controls on neuropsychological tests in areas of
4
attention span and memory [23, 24]. Further studies showed that these functional
impairments persisted during periods of normal moods highlighting the fact that
deficiencies are independent of the episodic nature of BD [24-27]. Symptoms of BD
also significantly impact the social daily activities and productivity of BD patients.
The combined factors of comorbidity and functional decline result in a state of
significantly increased health care costs but reduced productivity which creates a heavy
burden on the patient, the caregivers and society at large. Indeed, in 2011 it was
estimated that the combined cost of BD type I and II was a staggering 151 billion dollars
in the United States in 2009 [28]. The author noted that this study only took into account
32.3% to 47.7% of the bipolar spectrum and the entire economic burden of BD would
greatly exceed that number.
1.1.4 Treatment
Although there are non-pharmaceutical treatments to BD such as electroconvulsive
therapy (ECT), currently, treatment with lithium and anticonvulsant mood stabilizers,
antipsychotics and antidepressants are the most common approaches [29]. Due to the
episodic nature of BD, the course and nature of treatment depends on the current state
(i.e. mania or depression) that the patient is experiencing. However, a common first-line
of treatment is mood stabilizers, most notably lithium [30]. Placebo-controlled trials have
shown that 50% of patients treated with lithium had a partial reduction of mania and
36% of patients treated with lithium have a strong response in treatment for depression
[31]. In addition, anticonvulsants, lamotrigine, carbamazepine, and valproate, are often
used as alternatives or adjunctive to lithium treatment and can act as mood stabilizers
[32-35]. Table 1.1 provides a summary on the efficacy of commonly prescribed
5
psychotropic medications for mood episodes in BD. While antipsychotics have been
reported to be less efficacious than lithium and anticonvulsants, these interventions still
demonstrate effectiveness in the treatment of BD symptoms [36-38]. Patients with acute
depression are also prescribed antidepressants [39] but most often under the cover of a
mood stabilizer. Unfortunately, although there are a number of pharmacological agents
for treatment of BD, there is still a dire need to develop more efficacious and tolerable
interventions as the degree of improvement in BD symptoms have not correlated to a
similar improvement in functionality [40].
1.1.5 Etiology
The ethiopathogenesis of BD has been recognized to be complex involving
disturbances in neurobiological function as a result of both environmental and genetic
factors. Multiple studies have reported traumatic life events and lack of social support to
adversely affect mental health and play a role in the development and progression of
BD [41, 42]. On the other hand, BD is considered a heritable disease and it has been
found that children with parents or siblings suffering from BD are 4 to 6 times more likely
to develop the illness [43]. Interestingly, anomalies associated with BD have been well-
documented at all levels: tissue, cellular, molecular and genetic, of physiology. While it
remains unclear whether these abnormalities are causal or consequential, they provide
valuable insight into the physiopathology of BD and will be reviewed in the next section.
6
Table 1.1 Commonly prescribed treatments to BD mood episodes [44].
Episode Available Treatments
Mania or Hypomania Lithium
Carbamazepine
Valproate
Atypical antipsychotic
Benzodiazepines
Depression Lithium
Lamotrigine
Antidepressants
Atypical antipsychotics
Euthymic Maintenance treatments –Mood Stabilizers with or without atypicals
7
1.2 Evidence of Neuropathology in BD
1.2.1 Structural Abnormalities in the Brain
Irregularities in brain structure are often studied by means of powerful imaging
techniques such as magnetic resonance imaging (MRI) and computed tomography (CT)
which allow the measurement of brain volume and morphometry. Currently, MRI is the
preferred method as it exposes patients to less radiation and produces images at higher
resolutions [45]. Overall, numerous studies have linked reductions in grey matter
volume and density with BD in the prefrontal cortex, hippocampus, parietal lobe,
cingulate, and temporal lobe [46-50]. Two longitudinal studies reported greater
volumetric decline in the subgenual cingulate gyrus and hippocampus in BD patients
relative to healthy controls [47, 51]. Similar to grey matter, imaging studies have
revealed significant decreases in white matter volume in the inferior cingulate cortex,
frontal, parietal, and temporal lobes of BD patients relative to healthy controls [52-54].
Interestingly, a reduction in white matter volume, albeit modest, was seen in patients
experiencing their first manic episode compared to healthy controls [55]. In addition,
total cerebral white matter was found to be reduced in euthymic BD males compared to
healthy controls [56].
Another aspect of white matter of interest is the presence of white matter
hyperintensities (WMH), seen as intense bright spots in T2-weighted MRI images [57].
While no consensus was reached in the association of white matter with BD in the early
2000s, a recent meta-analysis of 98 independent neuroimaging studies has reported a
significant increase in WMH in BD patients compared to controls [58]. Furthermore,
escalation in WMH has been demonstrated to be positively correlated to the frequency
8
of manic episodes and number of relapses requiring hospitalization and recovery from
mania or depression and length of euthymia are negatively correlated to WMH
frequency [59-61]. In summary, volumetric and densometric decreases in brain regions
and increases in WMH are correlated with BD and most likely represents physiological
changes at the cellular level.
1.2.2 Cellular Disturbances in BD
Evidence of structural abnormalities also directly implicates changes at the neurocellular
level. Indeed, studies using post-mortem brain samples found lower glial density in the
subgenual prefrontal cortex of patients with a familial history of BD relative to healthy
controls [62]. Glial density was also observed to be reduced in the dorsolateral
prefrontal cortex in BD patients [63]. Other studies using magnetic resonance
spectroscopy (MRS) and microscopic morphometry have corroborated these results
[64-66]. In addition, observations of reductions in neuronal soma size were also found
in the lateral amygdaloid nucleus and the accessory basal parvocellular nucleus within
the specific amygdaloid nuclei in BD patients [67]. Patients with BD also demonstrated a
significant decrease in neuronal cell density in the anterior cingulate cortex compared to
healthy controls [68]. Similar observations were also reported in the dorsolateral
prefrontal cortex and the anterior cingulate [63, 66]. Therefore, based on evidence from
these post-mortem studies, it has been hypothesized that loss of neuronal integrity and
atrophy are involved in the pathogenesis of BD.
Interestingly, a reduction in the concentration of amino acid derivative N-
acetylaspartate (NAA), found at high levels in neurons, can be used as a marker of loss
of neuronal function [69, 70]. In a proton MRS study, male patients suffering from
9
chronic BD showed lower NAA to creatine levels within the prefrontal cortex compared
to healthy controls [71]. These results supported findings from an older 1H-MRS study
also reporting a decrease in the ratio of NAA to creatine levels in the dorsolateral
prefrontal cortex [72]. In addition, lower NAA ratios were seen in the hippocampus of BD
type I patients compared to age, age of onset, and duration of illness matched healthy
controls [73]. A more recent study found that NAA/creatine ratios were lower in
euthymic BD type I patients compared to healthy controls implying that this change is
state-independent [74]. Furthermore, lower NAA/creatine ratios were detected in the left
dorsolateral prefrontal cortex of a mixed sample of young patients but in the right
dorsolateral prefrontal cortex of a sample of paediatric patients with a family history of
BD, acting as evidence of a difference in the pathobiology of different subtypes of BD
[75, 76]. In summary, reports of lower NAA levels combined with evidence of changes in
neuromorphology strongly suggest the loss of neuronal function and cell loss as a major
component of BD.
These changes in neuronal densities and function are postulated to be
associated with a decrease in cellular resilience [77]. In one study, changes in gene
expression in response to glucose deprivation induced stress were compared between
B lymphocyte cell lines (BLCLs) from BD patients and healthy controls. It was found that
at normal glucose concentrations, no differences between the two groups were
detected. However, an upregulation of mitochondrion and electron transport chain
related genes were found in lymphocytes from healthy controls while lymphocytes from
BD patients demonstrated a downregulation in electron transport chain related genes
[78]. This differential response supports anomalies in cellular response to stress which
may contribute to BD. Additionally, significant increases in cell death were observed in
10
differentiated neuronal cultures of biopsied olfactory mucosa from BD type I patients
relative to health controls [79]. The same study also discovered lower levels of
enzymatic gene expression, including inositol-1,4,5-triphosphate kinase A (IP3KA) in
inositol phosphate metabolism and phosphatidyl inositol signalling pathways. Such a
decrease in IP3KA levels could adversely affect neuronal adaptation as this enzyme has
been demonstrated to be involved in intracellular calcium homeostasis and regulation
by terminating IP3 signalling and neuronal structural plasticity by bundling actin [80, 81].
Further consideration of the irregularities in the cellular signalling pathways will be
discussed in Section 1.2.4. Nonetheless, a growing body of evidence strongly
implicates the involvement of neural impairment in function, size and response to
cellular stress to BD pathogenesis.
1.2.3 Genetic Associations with BD
Accumulated evidence from the past 4 decades, demonstrating a correlation between
risk of BD and relatedness to BD patients within families, strongly supports a genetic
predisposition to BD through numerous family, twin and adoption studies [82, 83]. Using
linkage analysis, a locus on chromosome 21q22.3 demonstrated significant association
with BD [84]. Interestingly, this region contained the C21ORF29/TSPEAR and transient
receptor potential melastatin subtype 2 (TRPM2) genes. TRPM2 is a member of larger
family of TRP channels which are Ca2+ permeable ion channels and will be discussed
more in depth in section 1.5. TRPM2 is highly expressed in the brain and several
studies have implicated an association between the genetic variants of TRPM2 and BD
[85, 86]. In addition, two regions, 6q21-q25 and 8q24, were reported to show
significance in a genome wide scan analysis of 11 bipolar linkage [87]. An earlier meta-
11
analysis of 7 bipolar linkage studies found evidence for BD susceptibility on
chromosomes 13q and 22q [88]. While linkage studies have provided some insight into
the genetics of BD, the findings have not revealed any particular gene(s) to explain
most cases of BD.
Candidate gene association studies have classically investigated polymorphisms
focusing on neurotransmitter systems such as dopamine, serotonin and noradrenaline.
More recently, more interest has been placed in candidate genes that are associated
with schizophrenia, implicated from linkage studies or predicted from models of
pathogenesis [82]. Currently, two schizophrenia genes, D-amino acid oxidase activator
(DAOA) and neuregulin1 (NRG1), have also been associated with BD [89-93]. While
evidence for the DAOA association with BD has been reaffirmed by multiple studies,
these studies have identified different single nucleotide polymorphisms (SNPs) and
haplotypes showing this association. Of note, the DAOA gene is located on
chromosome 13q33 which was implicated in earlier linkage studies in BD [89, 90]. The
gene product of NRG1 has broad biological functions and thus, it is not clear which one
is of major importance to pathogenesis [94]. Lastly, brain-derived neurotrophoic factor
(BDNF) has also been reported to be associated with BD in candidate gene screens
[95].
With coming of the genomic age, high-throughput genotyping allows countless
DNA polymorphisms over the entire genome can be assayed in genome-wide
association studies (GWAS) [96]. This powerful method is able to examine prospective
disease-related gene variations over the genome. GWAS is appealing for studying
complex diseases because it is able to investigate multiple loci and provide candidate
genes for further study [97]. In four published studies of GWAS in bipolar disorder,
12
associations with BD have been found in SNPs in the diacylglycerol kinase eta gene
(DGKH), a gene-rich locus including BRCA2, NADH dehydrogenase 1 (NDU-FAB1),
and dynactin 5 (DCTN5), a single SNP in myosin 5B (MYO5B), and Src kinase-
associated phosphoprotein 1 (SKAP1) [98-101]. A meta-analysis of 3 of the 4 GWAS
studies above identified two very strong SNPs associated with BD – one in the Ankyrin-
G gene (ANK3) and another within the gene encoding the α-1C subunit of the L-type
voltage-gated Ca2+ channel (CACNA1C) [102]. Ankyrin-G links integral membrane
proteins to the cytoskeleton and affects proliferation, activation, maintenance of
membrane domains and cell motility [103]. Therefore, both genes identified by the meta-
analysis are likely to be involved in neuronal excitability which implicates the importance
of ion channels in bipolar disorder.
1.2.4 Irregularities in Signal Transduction in BD
Given the amount of evidence connecting cellular function and response to BD, it is not
surprising that irregularities in multiple signalling pathways have been reported in
association with BD. As signalling systems are responsible for the conversion of
extracellular stimuli into intracellular response, an appropriate cellular response to the
environment is heavily dependent on the integrity of these mechanisms.
1.2.4.1 B Cell Lymphoma 2
The Bcl-2 (B cell lymphoma 2) protein family, identified by the presence of Bcl-2-
homology (BH) domains, is known for its role in cell death and regulation [104]. It is
divided into three subfamilies: the anti-apoptotic Bcl-2 subfamily, the pro-apoptotic Bax
subfamily, and the Bax-activating subfamily [105]. In the cell, anti-apoptotic Bcl-2 family
13
members promote cell survival by binding to and inhibiting the activity of pro-apoptotic
Bcl-2 family members and reducing ER-to-mitochondria Ca2+ flux [105]. Within the
context of BD, it is suspected that Bcl-2, by interacting with IP3R, plays an important role
in modulating IP3-evoked Ca2+ elevation [106]. Interestingly, mood stabilizers such as
lithium have been shown to attenuate Ca2+ abnormalities and increase Bcl-2 levels in
the brain in animal models [107]. Also, a post-mortem study found reduced levels of bcl-
2 mRNA and protein levels in the frontal cortex of BD patients [108]. A more recent
study has found that a genetic polymorphism in the Bcl-2 gene increased both basal
cytosolic Ca2+ and IP3R-mediated Ca2+ elevations in lymphocytes from BD patients
[109].
1.2.4.2 Serine/Threonine Kinases – GSK3 and MAPK
Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that is widely
expressed in the brain and appears in 2 isoforms, α and β, in humans. In the cell, GSK3
modulates important cellular signalling pathways including insulin, neurotrophic factors,
and the Wnt pathway and is in term regulated by signals from the Wnt pathway,
phosphoinositide 3-kinase (PI3K) pathway, PKA, and PKC among others [110]. While it
is considered to be a major regulator of inflammation, dysfunctions of this enzyme have
also been implicated in diabetes, cancer, Alzheimer’s disease, and psychiatric mood
disorders [111]. Indeed, lithium and other mood stabilizers such as valproate have
been shown to reduce GSK-3 activity either through direct inhibition via competitive
inhibition for Mg2+ ions or indirectly by reducing GSK3 expression and increasing GSK3
protein phosphorylation [112-114]. Therefore, it is suspected that mood stabilizers such
14
as lithium, by inhibiting GSK3, result in changed cellular processes which promote
neuroplasticity, neurogenesis, and survival [115].
Similar to GSK3, mitogen-activated protein kinases (MAPKs) are
serine/threonine-specific protein kinases with the most well known being the classical
MAPKs, extracellular signal-regulation kinases (ERK) 1 and 2. Among others, the
MAPK pathways have been shown to modulate neuronal survival and long-term
neuroplasticity [116]. As treatment with mood stabilizers such as lithium and valproate
reverse or attenuate structural changes in the brain, it is thought that the efficacy of
these therapeutics may lie, at least partially, in their ability to activate the MAPK
pathway through ERK2 [117, 118]. In support of this hypothesis, it has been found that
treatment of rodent neuronal cultures with mood stabilizers resulted in increased
phosphorylation of ERK2 [119-121].
1.2.4.3 G-protein Coupled Signaling Pathways
A classical model of the phosphoinositide (PI) pathway is activated by the binding of
specific ligands to the extracellular ligand-binding domains of heteromeric G-protein
coupled receptors (GPCRs) (Figure 1.1). GPCRs are so named for the tight association
of the receptor to a G protein complex consisting of Gα bound to guanine diphosphate
(GDP), Gβ and Gγ, in the resting state. Ligand binding induces a conformation change
in the protein complex resulting in an exchange of GDP for guanine triphosphate (GTP)
and the dissociation of Gα-GTP and Gβγ subunits. These subunits then interact with
further downstream effectors. The PI signalling cascade is initiated by the activation of
phospholipase C-β (PLC-β) by Gq/11, resulting in enzymatic hydrolysis of
phosphatidylinositol-4,5-biphosphate (PIP2) into inositol-1,4,5-triphosphate (IP3) and
15
diacylglycerol (DAG) [122] . The formation of IP3 and DAG is also possible through
tyrosine kinase activation of PLC-γ and GPCR induction of PLC-ε [123, 124]. IP3 is
involved with the release of Ca2+ from the ER by binding to IP3 receptors (IP3R) on the
ER membrane [125]. DAG activates protein kinase C as well as some subtypes of
canonical transient receptor potential (TRPCs) channels (to be discussed further in
section 1.6) [126]. It has been reported that increased concentration of PIP2 in platelets
was found to be associated with BD patients in both the manic and depressed state
relative to controls. This suggests higher levels of PIP2 may be a state or active illness-
dependent characteristic of BD [127, 128]. In addition, increased PKC activity has been
found in platelets and post-mortem brain studies comparing BD patients to
demographically-matched control subjects [129, 130]. Furthermore, chronic lithium
therapy has been demonstrated to reduced PIP2 levels in euthymic BD patients relative
to controls [131]. Thus, irregularities in the PI signal transduction mechanism such as
increased PIP2 and PKC activity levels are associated with BD.
In addition, studies of peripheral blood cells and post-mortem brain tissue have
suggested irregularities in G protein signalling, which may affect downstream signal
transduction cascade and have further implications on cellular function and response.
These studies are summarized in Table 1.2. Another major intracellular signalling
pathway implicated in BD is the cyclic adenosine monophosphate (cAMP) pathway.
Briefly, the cAMP – generating pathway, as the name implies, is characterized by the
conversion of adenosine triphosphate (ATP) into cAMP by the action of adenyl cyclase
[132]. Adenyl cyclase is regulated by α-subunits of G-protein coupled receptors
(GPCRs). Within the cell, cAMP acts as a second messenger and plays an important
role in regulation of cellular functions though the activation of protein kinase A (PKA).
16
PKA is responsible for the phosphorylation of other cellular proteins including
transcription factors such as cAMP-response element-binding protein (CREB). The
activation of transcription factors by PKA represents the essential link between
signalling pathways and a cellular response to external stimuli through the expression of
relevant genes, including the neuroprotective growth factor, BDNF[133]. A summary of
disturbances within this pathway linked to BD is summarized in Table 1.3.
In general, alterations in signal transduction cascades such as the PI and cAMP
pathways may have strong implications for downstream signalling and subsequent
cellular response to external stimulus. These consequences then could be critical in the
pathogenesis of disease and are of significant interest.
17
Figure 1.1 The phosphoinositide (PI) signal transduction cascade. Similar to The cAMP signalling
system, the PI pathway is activated by the ligand-binding of G protein coupled receptor (GPCR) resulting
in unbound G protein alpha subunit (Gα). Gαq/11 activates phospholipase C-β (PLC-β) hydrolyzes
phosphatidylinositol-4,5-biphosphate (PIP2) into intoinositol-1,4,5-triphosphate (IP3) and diacylglycerol
(DAG). IP3 is involved with the release of Ca2+
from the ER by binding to IP3 receptors on the ER
membrane. DAG activates protein kinase C (PKC) as well as some subtypes of canonical transient
receptors.
18
Table 1.2 Disturbances in G-protein signaling in BD
Model Observation Study
Post-mortem cerebral
cortex
↑Gαs levels, ≈ Gβ levels [134, 135]
≈ Gαs mRNA levels [136]
↓Gαs in Li-treated patients [137]
Leukocytes and Platelets ↑Gαs and Gαi levels in depressed patients [138]
↑Gαs levels in Li-treated depressed
patients
[139]
↑Gαs levels in BD patients [140]
↑Gαs levels in BD type I and II patients,
regardless of treatment
[141]
↓Gαs levels in Li-treated patients [142]
≈ Gαs levels in Li-treated BD patients [143]
19
Table 1.3 Disturbances in the cAMP-pathway in BD
Model Observation Study
Post-mortem cerebral cortex ↑Forskolin-stimulated cAMP production [134, 135]
↓ cAMP binding [144]
≈ AC levels [145]
↑ Maximal and basal cAMP-dependent PKA
activity
[146]
↓Forskolin-stimulated AC [137]
Leukocytes and Platelets ↑cAMP-stimulated PKA activity, ↓ cAMP binding
in euthymic patients
[147, 148]
↑PKA activity, PKA catalytic subunit, pCREB [149]
↓PGE1-stimulated cAMP in depressed BD
patients
[150]
↓Isoproterenol-stimulated cAMP production in
depressed BD patients
[151, 152]
↓Forskolin-stimulated AC activity after Li
treatment
[153]
↓Basal and stimulated AC activity in Li-treated
patients
[154]
↑cAMP dependent protein phosphorylation after
Li treatment
[155]
↑Basal and cAMP-stimulated protein
phosphorylation after Li treatment
[156]
↑PKA catalytic subunit levels compared to
untreated BD and control subjects
[157]
20
1.2.5 Alterations in Intracellular Ca2+
Based on the disturbances seen in intracellular signalling cascades, it should not be
surprising that changes in Ca2+, an important second messenger, are also linked with
BD. Aside from synaptic transmission and neurotransmitter release, Ca2+ are critical in
the cellular regulation of a diverse range of functions including neuronal survival, death,
exocytosis, axon outgrowth, development, and synaptic plasticity [158]. Thus,
intracellular Ca2+ concentration ([Ca2+]i) is tightly controlled. It has been found that
alterations in [Ca2+]i can lead to physiological changes that affect neuronal functions
over time periods from milliseconds to days or longer [159]. Interestingly, the amplitude,
frequency and duration of the change in [Ca2+]i are important in the physiological
function of the signal, providing this cascade with enormous diversity and flexibility. For
instance, resting [Ca2+]i is on the order of 100 nM but increased sustained
concentrations of 200-300 nM can lead to apoptosis. [160, 161]. Inside the cell, Ca2+
also binds to effector proteins, including calmodulin, resulting in activation of
downstream kinases, phosphorylation of transcriptional factors such as CREB, and
subsequent regulation of gene expression [162]. Although the role of calmodulin has
been considerably studied, the effects of other proteins known to bind Ca2+ such as
neuronal Ca2+ sensory (NCS) family of proteins and Ca2+ -binding protein
(CaBP)/calneuron are not so well elucidated [162]. Despite this, disturbances in the
regulation of intracellular Ca2+ and Ca2+-dependent signalling could result in a
phenotype characterised by aberrant response and function such as that seen in BD.
Indeed, growing evidence from studies investigating signal transduction systems
in peripheral blood cells support the idea of Ca2+ aberrations in association with BD.
21
The link between Ca2+ and BD first germinated in the early 1920s when it was found
that manic patients had significantly decreased levels of Ca2+ in their cerebral spinal
fluid compared to depressed patients [163]. It was not until the discovery that red blood
cells (RBC) from bipolar patients demonstrated more variable Ca2+ ATPase activity than
matched controls from multiple investigations that the field expanded [164-167]. The
notion of disrupted intracellular Ca2+ homeostasis in BD began to blossom when
evidence of elevated basal [Ca2+]i was found in platelets of BD patients relative to
controls, although only significantly different between manic patients and healthy
controls [168]. In addition, it was reported that higher levels of basal and agonist-
stimulated (platelet activating factor, thrombin and 5-HT) [Ca2+]i in platelets and
leukocytes of BD patients compared to such blood cells from healthy volunteers [169-
171]. Interestingly, when ultrafiltrates of plasma from BD patients were incubated with
platelets from health patients , the [Ca2+]i of the platelets did not change [172]. These
results hint that the change in [Ca2+]i is a result of intrinsic and not circulating factors.
Subsequent studies further substantiated this hypothesis by detecting higher levels of
basal Ca2+ in cellular models, such as white blood cells, and elevated [Ca2+]i in
response to thapsigargin in BD patients relative to controls [173-175]. In euthymic
patients undergoing lithium therapy, serum Ca2+ levels were reported to be higher than
healthy controls [176]. Although there are conflicting reports with regards to the direction
of change of [Ca2+]i, mounting evidence supports that disruptions of intracellular Ca2+
homeostasis is linked with BD.
Another line of evidence associating BD with Ca2+ disturbances stems from
observations of the effect of mood stabilizing agents on signal transduction cascades
such as the G proteins, PI and importantly, Ca2+. Indeed, studies in various rat neuronal
22
subtypes, including astrocytes and pituitary GH3 cells, have demonstrated a
suppression of [Ca2+]i by long-term treatment of lithium [177, 178]. Chronic lithium
therapy also attenuated norepinephrine, thyrotropin-releasing hormone, 5-HT, thrombin
and NMDA receptor stimulated increases in [Ca2+]i [177-181]. Clinical studies have
found that chronic lithium treatment is able to reduce the higher levels of [Ca2+]i in
untreated depressed BD patients to levels of treated euthymic controls [182].
In BLCLs, it was found that BD patients exhibited greater Ca2+ responses to the
bioactive lipid, 1-oleoyl-lysophosphatidic acid (LPA), relative to cell lines from healthy
controls [183]. BD patients had higher levels of basal [Ca2+]i and more rapid LPA and
thapsigargin-induced increase in [Ca2+]i [184]. The implication of an irregular
thapisgargin response is the disturbance of store-operated Ca2+ (SOC) flux from ER
stores. As mentioned earlier, B lymphoblasts from BD patients demonstrated greater
Ca2+ response to thapsigargin mediated store depletion [171, 175]. Thapsigargin was
also used to experimentally approximate the size of ER stores and the magnitude of
SOC entry. Data suggests that BD patients have elevated ER Ca2+ stores and [Ca2+]i
following SOC entry [185]. The increased LPA kinetics may be in part mediated by
canonical TRP channels such as TRPC3 which have been linked to BD. It was reported
that TRPC3 levels were reduced after chronic, but not acute lithium treatment in
lymphoblast cell lines from BD patients [186]. As the TRPC subfamily will be a major
point of focus in this dissertation, TRPC channels will be discussed more in dept in
section 1.6. In summary, the substantial body of evidence of abnormalities in
intracellular Ca2+ homeostasis strongly suggest that the cellular regulation of Ca2+ is an
important component of the physiopathology of BD.
23
1.3 Oxidative Stress in BD
1.3.1 Overview of Oxidative Stress
Aerobic organisms produce reactive oxygen species (ROS), such as hydrogen peroxide
(H2O2), superoxide (O2-) and hydroxyl radicals (OH-), under physiological conditions as
part of respiration within the mitochondria [187-189]. While low levels of these partially
reduced oxygen species are required for cellular activities such as protein folding,
activation of kinases and phosphatases, gene expression and general maintenance of
intracellular redox environments, situations where the generation of ROS exceeds the
capacity of antioxidant mechanisms, damage to cellular macromolecules including
lipids, nucleic acid and proteins can result [190-192]. Within the body, O2- radicals are
generated at the complexes I and III of the mitochondria by the donation of an electron
to O2 [193]. Being highly reactive, it is inactivated through the activity of the enzyme
superoxide dismutase (SOD) into H2O2 and then subsequently into water through the
glutathione detoxification system (Figure 1.2) [194]. The presence of ferrous iron (Fe2+)
can also catalyze the Fenton reaction resulting in the production of OH- radicals from
H2O2 [195]. Cellular mechanisms to counteract reactive radicals include ROS
neutralizing compounds, like thiols, ascorbic acid, and α-tocopherol, and
aforementioned enzymes, such as SOD, catalase (CAT), glutathione enzymes,
peroxidases, and reductases [191, 196]. With the brain metabolizing 20% of total
oxygen consumed in the body and having limited antioxidant capacity, it is not
surprising that oxidative stress has come to be implicated in the physiopathology
neurodegenerative diseases and of neuropsychiatric disorders [197, 198].
24
In addition to ROS, nitric oxide (NO-), a reactive nitrogen species (RNS), is
generated by the nitric oxide synthase (NOS) enzyme family in neurons [199]. NO- is an
important signaling molecule and can activate guanylate cyclase and transduce
signaling cascades through cyclic guanine monophosphate (cGMP) [200]. Thus,
excessive NO- concentrations and associated glutamate-receptor activation is
correlated to several brain pathologies [201]. Further, NO- is able to undergo a chemical
reaction with O2- to form the peroxynitrite anion (ONOO-). While ONOO- readily breaks
down into OH- and nitrogen dioxide, it can create function-altering abducts with tyrosine
residues in proteins through nitration [202]. Indeed, the role of excess ONOO- in the
pathogenesis of amyotrophic lateral sclerosis (ALS) is well documented [203]. Aside
from proteins, ROS and RNS are able to interact with other cellular constituents such as
nucleic acids, lipids, and thiols, leading to neuronal damage and dysfunction [204-206].
1.3.2 Evidence of Oxidative Stress in BD
At present, there is a strong body of evidence that oxidative stress contributes to the
pathogenesis of BD [207-209]. In comparing BD and control post-mortem anterior
cingulate cortex, it was found that there was a 59% increase in 4-hydroxynonenal
levels, a marker of lipid peroxidation [210]. In studies of peripheral blood, several groups
have reported increased levels of thiobarbituric acid reactive substances (TBARS),
another marker of lipid peroxidation, [207, 211-214]. Finally, a meta-analysis conducted
in 2008 demonstrated that TBARS levels are increased in patients suffering from BD,
providing strong evidence of oxidative damage in BD [215].
At the molecular level, another marker for oxidative stress is DNA damage in the
form of chemical and structural modifications to nitrogenous bases or DNA strand
25
breaks [216, 217]. Interestingly, it has been reported that the frequency of DNA damage
was not only increased in BD compared to controls but also that the amount of DNA
damage correlated with severity of mania or depression episodes [212]. In a
monozygotic twin study, bipolar twins were found to have a greater degree of DNA
damage compared to the healthy control. Moreover, even after 6 weeks of mood
stabilization, DNA damage still remained altered [218]. In a study investigating post-
mortem anterior cingulate cortex, non-GABAergic neurons from bipolar patients
displayed higher levels of DNA fragmentation compared to healthy controls [219].
Another post-mortem study found significant increases in protein carbonylation and
reduced mitochondrial complex I activity in the bipolar group but not depressed or
schizophrenic groups compared to healthy controls [220].
Not surprisingly, innate antioxidant defence systems can be a target for ROS.
While it has been thought that BD is associated with increased levels of SOD, a recent
study demonstrated that SOD levels were increased in the peripheral blood of patients
only in the acute phases of BD and not in the euthymic phase or in healthy controls
[207, 213, 221]. A separate study, however, reported reduced CAT and SOD levels in
BD patients compared to healthy controls [208]. With regards to the glutathione system,
it has been found that there are increased levels of glutathione enzymes, including
glutathione S-transferase (GST) and glutathione reductase in BD patients [222]. Multiple
studies have also demonstrated that GST may mediate the neuroprotective effects of
lithium, valproate, lamotrigine or olanzapine, against oxidative stress [222-225]. These
findings suggest that the anti-oxidant glutathione system could contribute significantly to
the therapeutic effects of mood stabilizing drugs in BD [226] and support the
pathophysiological relevance of oxidative stress and antioxidant defences in BD.
26
27
Figure 1.2 Detoxification of reactive oxygen species in the body. Highly reactive superoxide ions (O2-
) are reduced into less reactive hydrogen peroxide (H2O2) and oxygen by superoxide dismutase (SOD).
Catalase and glutathione peroxidase (GPx) are responsible for the conversion of hydrogen peroxide into
water. Glutathione (GSH) is an important co-factor for the activity of GPx and is oxidized into glutathione
disulfide (GSSG).
28
1.4 Lithium and BD
1.4.1 Overview of Lithium and Lithium Treatment in BD
The first reported use of lithium in the treatment of mania was by Cade in 1949 and then
established as evidence-based by work done by Schou and Braastrup in the 1960s
[227, 228]. Shortly afterwards, it was approved by the FDA for treatment of mania in
1970 and for BD in 1974 [229]. Since then, it has remained as a primary agent in the
treatment of BD [230]. The typical maintenance dose of lithium is 900 mg/day to 1200
mg/day, usually starting at 300 mg [231, 232]. The target drug blood level is 0.8 meq/L
to 1.2 meq/L for management of acute phase symptoms and 0.6 meq/L to 0.8 meq/L for
maintenance therapy [233]. Unfortunately, lithium has a low therapeutic index and
patients undergoing lithium treatment must be monitored closely to minimize toxicity
[234]. Cognitive side effects, tremors, weight gain, polyuria, and loose stool are
common side effects of lithium therapy while long-term complications include
hypothyroidism, disturbances in cardiac rhythm, and renal impairment [30].
Although the exact mechanism through which lithium operates is unclear, there is
accumulating evidence of enhanced neuroresilient effects of lithium, mediated though
altering signalling pathways in the CNS. As alluded to in earlier sections, lithium is also
able to ameliorate alterations in Ca2+ homeostasis in BD. Lithium has also been noted
to inhibit GSK-3, a major pro-apoptotic enzyme involved in cell death due to neuronal
insults including glutamate-induced excitotoxicity [113, 235, 236]. Finally, lithium is
reported to upregulate the expression of cytoprotective or survival molecules such as
Bcl-2, BDNF, and VEGF, induce autophagy by reducing IP3 levels, and induce
neurogenesis in cultured neurons or brain [118, 237-240].
29
1.4.2 Evidence of Neuroprotective and Anti-oxidative Effects of Lithium
Due to the increasing body of evidence of disturbances at the neurocellular level and
the altered response profiles of neurons and glial cells to stress, it has been postulated
that neuroprotection mediated by mood stabilizers are a major component of the
therapeutic efficacy of these drugs. The in vitro neurotoxicity of inhibitors of respiration,
ROS or ROS-generating compounds, deprivation of cell culture media components,
NMDA receptor agonists, ethanol and even β-amyloid have been reduced by treatment
with lithium [240-244]. In addition, lithium attenuated levels of neuronal damage by
kainate, and ischemia has been reported in vivo [245]. Moreover, lithium has also been
implicated to be involved with several signalling pathways including stress-induced
kinases, transcription factors for stress responses and inhibition of apoptosis [246-251].
Interestingly, the neuronal death induced by stressors used in several of the
above studies implicates oxidative stress as a major insult. For example, decreased
levels of oxidatiave stress markers, lipid peroxidation, protein carbonyl formation, and
malondialdehyde, were found in cultured primary rat cortical neurons [252, 253]. The
lack of oxidant specificity in the attenuation of oxidative stress-induced cell death further
implies an anti-oxidant relevant role of lithium. In in vivo models, pre-treatment with
lithium in amphetamine-induced hyperactivity in rodents, an animal model of manic
behaviour, resulted in reduced levels of amphetamine-induced TBARS formation and
DNA damage [254-256]. With regards to BD patients, higher TBARS levels were found
in unmedicated patients compared to medicated patients in a cross-sectional study
[257]. In a longitudinal study, patients were treated with lithium and olanzapine. At the
end of the study, patients had lower levels of TBARS and increased antioxidant enzyme
activities compared to baseline [258]. Evidence collected from in vitro, in vivo and
30
clinical studies strongly support a link between lithium’s neuroprotection and anti-
oxidant properties.
31
1.5 TRP Channels
Increased neuronal oxidative stress and alterations in intracellular Ca2+ homeostasis are
implicated in the pathology of BD. As these two processes are interwoven at the
molecular level, Ca2+ channels which regulate intracellular Ca2+ and are modulated by
oxidative stress are an important interface for investigation. In this regard, the transient
receptor potential family represents one such candidate.
1.5.1 Overview of TRP Channels
The Transient Receptor Potential (TRP) superfamily consists of channel proteins that
share a common six transmembrane (TM) motif, sequence homology and permeability
to cations such as Ca2+, Mg2+, and Na+. TRP channels also possess a remarkably wide
range of activation mechanisms and cation selectivity, an unusual trait among known
families of ion channels. Most interestingly, many TRPs function as signal integrators
single TRP channels can be activated by seemingly distinct mechanisms and the
response to one input can be modulated by another [259-261]. It should not be
surprising that mutations in and dysregulation of channel function or expression can
lead to altered physiological states and cellular responses culminating in pathologies
[262-264]. Indeed, there are currently twelve recognized channelopathies, diseases
arising due to mutations in TRP channel-encoding genes [265]. Further, as TRPs are
critical in various sensory responses, hormone secretion, organ and bone function, and
neuronal cell death, they are implicated in a wide variety of diseases and disorders.
The first TRP channel was identified in Drosophila as a spontaneous mutation
which resulted in a specific phenotype of the visual system where prolonged exposure
32
to light caused visual excitation currents to drop to baseline levels [266, 267]. It was
then found that TRPs are widely expressed amongst multicellular organisms including
worms, fruit flies, zebrafish, mice and humans due to the essential role of ion channels
in cellular function [3]. In mammals, the TRP superfamily encompasses seven
subfamilies which are divided into two groups, group 1 and group 2 [268]. Group 1
consists of the TRPC (canonical), TRPV (vallinoid), TRPM (melastatin), TRPA (ankyrin),
and TRPN (no mechanopotential) subfamilies and group 2 is comprised of TRPP
(polycystin), and TRPML (mucolipin). The separation of the two groups of TRPs is
largely based on sequence homology and topological differences [269]. In this sense,
group 1 TRPs display the greatest sequence homology to the founding member of the
TRP superfamily, Drosophila Trp [270]. The subfamily most related to the Drosophila
Trp is referred to as the canonical or classical TRPs (TRPC). All other group 1 TRPs are
named based on the first described member of each subfamily [271]. The TRPP and
TRPML subfamilies are more distantly related to the group 1 TRPs. Similar to group 1
TRPs, group 2 TRPs are also named after their founding member [8].
1.5.2 Molecular Structure of TRP Channels
Being of significant physiological importance, it is not surprising that the TRP membrane
topology bears strong resemblance to voltage-gated K+, Na+, and Ca2+ channels. TRP
channel subunits contain six TM domains and a pore forming region between the last
two membrane spanning segments. Functional TRP channels are composed of the
polymerization of four subunits in which residues between the fifth and sixth TM
domains line and create the pore[272]. The tetramerization, like other ion channels,
provides an additional level of regulation, diversity and specificity in function among cell
33
types and across species. Unlike voltage-dependent ion channels, TRPs do not have a
classic voltage sensor present in the S4 domain as described by the pioneering work of
MacKinnon et al. in voltage-gated potassium channels [273, 274]. However, this does
not mean that TRP channels have no voltage sensing capability. TRPM8, for example,
does have voltage gating potential possibly due to positive residues at the end of the
fourth TM domain and in the putative loop between the fourth and fifth TM domains
[275].
As the molecular mechanisms of TRP channels have not been fully elucidated,
the current model is heavily based on the voltage-gated K+ channels under the
assumption that the structure-based functional arrangement is conserved [276]. In the
voltage-gated K+ channels, there are two main domains – the voltage sensor on S4 and
the pore-forming loop comprised by S5 and S6 domains. Under this hypothesis, the
TM1-4 domains of TRPs would function as sensors and regulators of the gating ability
of the TM5-TM6 pore. Since TRPs can be activated by many agonists, it would mean
that the TM1-4 region could function as chemical receptors and the site of action of
agonists, such as diacylglycerol (DAG) in the case of TRPC3/6/7. In addition, the
mechanism behind temperature sensitivity of TRP channels may follow a similar
paradigm or a completely different one. In either case, extensive experimentation is
required to understand the sensory capabilities of the TRP channels.
1.5.3 Structural Aspects of TRP Channels
In contrast to classical ion channel types, many of the TRP proteins were discovered
only after their encoding genes were overexpressed in heterologous cellular systems.
As a result, this provided the field with a better understanding of the structural aspects
34
of these channels. In the group 1 TRPs, the six transmembrane domains represent the
region of greatest sequence homology, including the pore-forming loop between TM5
and TM6 [269, 277]. Following the sixth TM domain is a TRP domain shared by TRPC,
TRPM and TRPN channels. The TRP domain is a highly conserved region of
approximately 23-25 amino acids with the greatest homology in the two regions known
as TRP box 1 and TRP box 2. The TRPs in group 1, with the exception of TRPM, have
multiple ankyrin repeats at the N-terminus. Furthermore, three TRPM proteins possess
C-terminal enzyme domains and are known as chanzymes [278]. The group 2
subfamilies, TRPP and TRPML, are more distantly related to the group 1 TRPs. The
most striking aspect is that the two subfamilies contain an extracellular loop between
TM1 and TM2. Similarly to the group 1 TRPs, the group 2 TRPs demonstrate a high
degree of sequence homology over the transmembrane segments [269, 277].
35
1.6 The Canonical TRP Subfamily of TRP Channels
1.6.1 Overview and Distribution of TRPC Channels
Members of the TRPC subfamily were the first described mammalian homologs of
Drosophila Trp [279]. Although initially only three homologs were identified, TRPC1,
TRPC2 and TRPC3, there are seven recognized mammalian TRPC proteins today
[280-282]. However, only six are expressed in humans as human TRPC2 is a
pseudogene [281]. In terms of relatedness, the seven TRPCs share with each other and
Drosophila Trp over 70% amino acid identity over the 750-900 N-terminal residues [280,
281]. The members of the TRPC subfamily can be further divided into four phylogenetic
groups or subsets: TRPC1, TRPC2, TRPC3/6/7, and TRPC4/5 [271, 283, 284].
The functional nature of TRPC1, while being the first described member of the
TRPC subfamily, is still widely debated in the field. There have been reports of
homomeric TRPC1 acting as store-operated, receptor-operated, IP3R-gated cation
channels, or even a non-functional subunit [285]. However, within heteromeric systems,
TRPC1 complexes with TRPC3, TRPC4, or TRPC5 as part of G-protein mediated
receptor-operated cation channels [286]. This heterogeneity in tetramerization highlights
a major hurdle in discerning individual TRPC protein function and the role of TRPCs as
subunits within a heterotetramerized unit. hTRPC1 has been found to be widely
distributed throughout the body but with expression higher within the central nervous
system (CNS) [287]. In the CNS, the cerebellum represents the region with greatest
TRPC1 expression, by a factor of almost 2 compared to other regions. Outside the
CNS, TRPC1 is seen in many tissues including the muscle, pancreas, prostate, kidney,
and lung but is most common in the heart and pituitary gland.
36
In humans, the gene encoding TRPC2 contains a premature stop codon resulting in a
non-functional truncated protein [288]. However, rodent clones of TRPC2 have been
investigated and may be activated by DAG [289]. Furthermore, TRPC2 appears to be
required for neuronal excitability in pheromone signal transduction [290].
The TRPC4 and TRPC5 share 64% homology and are the group most closely
related with TRPC1 [285]. These channels act as non-selective cation channels that
function as downstream effectors of GPCRs [291]. Under homomeric expression,
TRPC4 and TRPC5 both exhibit currents with an unusual double rectifying current-
voltage (I-V) relationship [286, 292]. There is currently some speculation that the
complexity of the outward TRPC5 current could be due in part to voltage-dependent
Mg2+ block at an intracellular site [293]. However, heteromeric TRPC1+C4 and
TRPC1+5 channels generate currents with a gentle negative slope at negative
potentials and smooth outward rectification [286]. Single channel conductance for
homomeric TRPC5 channels is significantly greater than TRPC1+C5 heteromers [286].
The hTRPC4 is distributed ubiquitously throughout the CNS and largely in the bone and
less so in the heart, prostate, placenta, and pancreas [287]. On the other hand,
hTRPC5 demonstrates the greatest CNS specificity of all the TRPCs with expression in
the CNS over ten-times greater than the peripheral tissues [287]. Despite this, hTRPC5
has been detected in other tissues such as the heart, muscle, liver and pituitary gland.
The final subset of TRPC channels, the TRPC3, TRPC6, and TRPC7 group,
exhibit between 65% and 78% homology [285]. These TRPCs are non-selective and
generate similar double-rectifying I-V curves when activated by GPCRs or DAG either
as homomeric or heteromeric units [126]. The mechanism of receptor-mediated channel
activity for this group of TRPCs is still mostly unclear. However, TRPC6 has been found
37
to be highly cooperative with muscarinic receptors and synergize with DAG [294]. In
terms of distribution, hTRPC3 is found in all regions of the CNS with the highest levels
concentrated in the cerebellum, caudate nucleus, putamen, and striatum [287]. In
peripheral tissues, there is generally low expression of TRPC3 with the exception of the
vascular epithelium [295]. In contrast, hTRPC6 had the highest expression in the CNS,
specifically the caudate nucleus, nucleus accumbens, striatum, cingulate gyrus, and
superior frontal lobe but is also highly expressed in the placenta and lung [287].
hTRPC7 showed broad expression within the CNS, with highest levels in the nucleus
accumbens, hypothalamus, putamen, striatum, caudate nucleus and locus coereleus
[287].
1.6.2 Biophysical Characteristics of TRPC Channels Pores
The physiological functions of an ion channel are defined by the characteristics of its
opening and closing (gating) and its ability to passage specific ion species (selectivity).
Unfortunately, insight into the permeation and pore structure of the TRP superfamily is
exceptionally limited, especially in comparison to the well studied voltage-gated ion
channel families. While being one of first TRP subfamilies to be identified, few studies
have described the pore properties and the pore region of TRPCs. Functional data is
often difficult to interpret due to a background of other endogenous store-operated or G-
protein activated channels. Moreover, as the TRPCs do not share significant sequence
homology to currently well described ion channel pore regions such as the bacterial K+
channels, it is difficult to make theoretical predictions about the pore elements [296].
Jung et al. were able to shed light onto TRPC pore characteristics through
studies of La3+ potentiation of TRPC5 [297]. Systematic mutagenesis was performed on
38
all negatively charged amino acids in the extracellular loops of TRPC5. It was found that
neutralization of amino acids in the loop between TM5 and TM6 resulted in a loss of
potentiation whereas no obvious effects were detected in the other regions. Mutations
localized to the distal parts of the loop had the most profound effect in contrast to
mutations localized in the central part of the loop which did not have any effect on
channel properties. A later study on TRPC1 revealed similar results, pointing to the fact
that the pore properties of TRPC5 and TRPC1 seem to be located in the distal parts of
the putative pore entrance [298].
1.6.3 Activation Mechanisms of TRPC Channels
In general, subfamily assignment is not a reliable way to predict the activation
mechanisms of a given TRP channel. Fortunately, in the case of TRPCs, all channels
are activated by the stimulation of phospholipase C (PLC) and pathways coupled to it.
Receptor stimulation that activates PLC, most commonly through the GPCR/Gq/11
pathway leads to activation of the IP3 receptor (IP3R) and the TRPC channels [277].
This mechanism has been well documented in the field by monitoring intracellular
calcium concentrations ([Ca2+]i) through the use of Ca2+ indicator dyes. The observation
that treatment with U73122, an inhibitor of PLC, eliminates ROCE provides strong
experimental data to the importance of PLC in the activation of TRPCs [276].
It is also thought that activation of the IP3R may result in a conformational
coupling between the IP3R and TRPC leading to TRPC activation. Evidence from co-
immunopreciptiation experiments showing TRPCs and IP3R interaction both in vitro and
in intact cells strongly support this hypothesis [299]. Furthermore, it was noted that IP3R
peptides contained sequences that interact with the TRPC proteins which affected
39
SOCE development and duration. In contrast, the non-interacting regions had no effect
on SOCE [300].
In addition to PLC and IP3R, DAG and its derivatives, OAG (oleyl-acetyl-glycerol)
and SAG (stearoyl-arachidonoyl-glycerol), are known to experimentally induce TRPC3,
TRPC6, and TRPC7 activation [126, 294]. However, the physiologic importance of this
mechanism is uncertain. Manoeuvres that stimulate endogenous DAG formation in
response to agonist activation of the GPCR-Gq-PLC pathway fail to activate TRPC3
whereas under the same conditions, exogenous OAG creates a TRPC3 response [301].
In contrast, provided that protein kinase is inhibited, TRPC4 and TRPC5 activation by
DAG produced from the GPCR-Gq-PLC pathway is observed [302]. Finally, a second
lipid, lysophosphatidylcholine (LysoPC), is known to activate TRPC5. The effects of
LysoPC on other TRPCs have not yet been reported [303].
1.6.4 Pharmacological Properties of TRPC Channels
Elucidation of the mechanism of pharmacological action on TRPC channels is heavily
dependent on their structural makeup. As discussed earlier, the heteromerization of
TRPs veil the specific effects of each subunit and thus, insight into which TRPCs
operate as homomers and heteromers and the impact of tissue-dependent
polymerization is still limited [304]. Drug development targeting specific TRP tetramers
(including TRPCs) could prove to be both challenging and rewarding.
Despite this, published data reports the use of a few compounds which target
TRPCs presumably by direct interaction. As none of these compounds are specific,
there is still debate and conflicting reports on the effects of pharmacological agents on
TRPCs [276].
40
1.6.5 Physiological Role of TRPC Channels
At the molecular level, TRPC channels are well documented to play important roles in
both receptor-operated Ca2+ entry as well as store-operated Ca2+ entry. As a result of
their importance in maintaining calcium concentration within the cell, they are also
implicated in a wide range of physiological processes. Within a neuroscience context,
there is a body of evidence implicating the TRPCs in cell death, regulation of
neurotransmitter release, plasticity, axonal regeneration, growth cone development,
dendritic morphology and even responses to fear [305].
41
1.7 Main Objectives and Hypothesis
Several disturbances have been characterized in the pathophysiology of BD, notably
altered intracellular Ca2+ homeostasis and increased markers of oxidative stress.
Investigations into the disrupted intracellular Ca2+ signaling in BD have suggested a
potential role of some TRP channels, such as TRPC3, in these disturbances which have
been separately noted to be potentially modulated by oxidative stress. As such, certain
TRP channels represent a potential cellular interface between Ca2+ signaling dynamics
and ROS signaling. Given this, I sought to examine the properties of several other
canonical TRP channels with regards to oxidative stress to expand the current
understanding of the mechanistic pathways behind TRP signaling and how this process
may contribute to BD pathology.
Previous work by Roedding et al. (2011, under review and in press), reported
that chronic treatment of primary rat cortical neurons (RCN) by oxidative stressor,
rotenone, resulted in dose-dependent reductions in TRPC3 protein and mRNA levels.
Following on these interesting findings, I sought to evaluate if two other canonical TRP
channels possessed similar rotenone-regulated properties, specifically TRPC6, a
subtype highly related to TRPC3, and TRPC5, a subtype reported to be modulated by
ROS [306]. Based on the results of the aforementioned TRPC3 investigations, I
hypothesized that:
Chronic (4 day) but not acute (24 hour) rotenone treatment of primary rat cortical
neuron cultures will result in a significant dose-dependent reduction in TRPC5
and TRPC6 protein and mRNA levels.
42
After examining expression, another objective of this work would be to investigate
whether there is also functional impairment in TRPC3 and TRPC6 as a result of chronic
oxidative stress. Unfortunately, due to a lack of specific pharmacological modulators, I
was only able to evaluate TRPC3 function. Based on the previously observed reduction
of TRPC3 mRNA and proteins levels after chronic rotenone treatment I hypothesized
that:
Chronic rotenone exposure will result in a dose-dependent reduction in TRPC3
function.
Finally, lithium, the standard pharmaceutical intervention for BD, has been shown to
possess neuroprotective properties which may be linked to ameliorating the effects of
oxidative stress through alternate cellular signalling mechanisms. As such, I
hypothesized that:
Pre-treatment with therapeutically relevant concentrations of lithium would result
in a rescue of the effects caused by chronic rotenone exposure in TRPC3,
TRPC5, and TRPC6 expression.
43
2 Materials and Methods
44
2.1 Materials
2.1.1 Chemicals, Reagents, and Drugs
Rotenone, dimethylsulfoxide (DMSO), lithium chloride, magnesium chloride,
probenecid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium
dodecyl sulphate (SDS), Ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-
(trifluoromethyl)-1H-pyrazole-4-carboxylate (Pyr3), β-mercaptoethanol, egg white
albumin, formaldehyde and DNase, RNase-free water were purchased from Sigma-
Aldrich (Oakville, ON). Invitrogen (Burlington, ON) supplied ImageEnchancer, ProLong
Gold antifade reagent, fura-2 acetoxymethyl ester (fura-2 AM) and goat serum.
Tris(hydroxymethyl)aminomethane (Tris), Tween 20, skimmed milk powder, glyercol,
agarose, and sucrose were obtained from BioShop (Burlington, ON). LI-COR blocking
buffer was obtained from LI-COR Biosciences (Lincoln, NE). Benson Life Technologies
(Markham, ON) provided the Isoflurane. Bromophenol blue was purchased from Bio-
Rad (Mississauga, ON). Triton X-100 was obtained from J.T. Baker (Mississauga, ON).
Calbiochem (La Jolla, CA) supplied 1-Oleoyl-2-acetyl-sn-glycerol (OAG).
2.1.2 Animals
Timed pregnant Sprague-Dawley rats were obtained from Charles River Laboratories.
The animals arrived at the CAMH animal facilities at gestation day 12 and were
maintained on a 12-hour light-dark cycle at 23C with chow and water provided ad
libitum.
45
2.1.3 Cell Culture Reagents
Horse serum, Hank’s balanced salt solution (HBSS), Dulbecco’s phosphate buffered
saline (DPBS), trypan blue dye, B27 supplement with and without antioxidants, and
neurobasal media were obtained from Gibco/Invitrogen (Burlington, ON).
Polyethyleneimine (PEI), penicillin/streptomycin, L-glutamine, and cytosine arabinoside
(ara-c) were purchased from Sigma-Aldrich (Oakville, ON). Serum-based media was
composed of 10% horse serum, 1% penicillin/streptomycin, and 1% L-glutamine and
88% neurobasal media. Serum-free media consisted of 2% B27 supplement with or
without antioxidants, 1% penicillin/streptomycin, 1% L-glutamine, and 96% neurobasal
media. All media solutions were warmed to 37C prior to feeding.
2.1.4 Antibodies
The antibodies raised in rabbit against TRPC3, TRPC5, and TRPC6 were obtained from
Alomone Labs Ltd (Jerusalem, Israel). Rabbit polyclonal anti-β-actin antibodies and
Mouse anti-α-tubulin were purchased from Cell Signalling Technologies and Sigma-
Aldrich (Oakville, ON), respectively. Horseradish peroxidise conjugated to protein A
antibodies were obtained from Bio-Rad (Mississauga, ON). LI-COR Biosciences
supplied the goat anti-rabbit IRDye 800CW and goat anti-mouse IRDye 680CW
polyclonal antibodies. Polyclonal antibodies against microtubule-associated protein 2
(MAP2), glial fibrillary acidic protein (GFAP), and 2’3’-cyclic nucleotide 3’-
phosphodiesterase (CNPase) were obtained from Abcam (Cambridge, MA).
Flourescent Alexa-conjugated antibodies were purchased from Invitrogen (Burlington,
ON).
46
2.1.5 Analytical Kits
ThermoFisher (Nepean, ON) supplied the bicinchoninic acid (BCA) protein assay kits
which were used to determine the protein concentrations in homogenized cell lysates.
RNA was extracted from cultured neurons using the RNeasy Plus Mini kits obtained
from Qiagen (Mississauga, ON). The SuperScript III RT kit and the Quant-iT High-
Sensitivity DNA assay kits purchased from Invitrogen (Burlington, ON) were used for
first strand synthesis and to determine DNA concentration, respectively. The ECLPlus
kit for detecting secondary antibodies crosslinked with horseradish peroxide (HRP)
during immunoblotting was supplied by GE Life Sciences (England).
2.1.6 Calcium Kinetic Assay Solutions
A fura-2 AM 1 mM stock solution was prepared by adding 1 ml of DMSO to 1 mg of
Fura-2 AM ester which was then aliquoted in volumes of 100 ul. The fura-2 AM stock
solutions were stored in an opaque-container at -30C. The fura-2 AM assay buffer was
composed of 1.25 mM probenecid and 5 mM HEPES in HBSS.
47
2.2 Primary Rat Cortical Neuronal Culture and Drug Treatment
2.2.1 Preparation of Cell Culture Plastics
Prior to animal dissection, cell culture plastics were coated with polyethyleneimine
(PEI). A working 0.25 mg/ml PEI solution was freshly prepared from a 25 mg/ml stock
PEI solution. Both solutions were prepared in DPBS with 0.901 mM CaCl2 and 0.493
mM MgCl2. The stock solution was stored at 4C for 1 month before it was discarded
and a new stock solution was prepared. Untreated 96-well plates, 6 cm Petri dishes and
coverslips were incubated for 1 hour at 37C in a 5% CO2/95% air atmosphere with the
0.25 mg/ml PEI solution. Following incubation, the PEI solution was removed and
replaced with serum-based media. The cell culture plastics were then incubated at 37C
with 5% CO2/95% air until seeding.
2.2.2 Fetal Rat Cortical Dissection
Pregnant Sprague-Dawley rats at gestation day 18 were anaesthetized using isoflurane
in an airtight chamber and sacrificed by cervical dislocation. An incision was made in
the lower abdominal region and the uterine horns and gestational sacs containing the
embryos were collected and placed on a Petri dish containing cold sterile HBSS with
1.26 mM CaCl2 and 1mM MgCl2. The embryos were removed from the sacs and
decapitated. Fetal brains were dissected from the skull and placed in a separate Petri
dish containing cold HBSS. Next, the cortical regions were isolated by excising the
hippocampi and striata, and removing the meninges under a stereomicroscope in cold
48
HBSS. The dissected cortices were placed in a 15 ml conical tube containing cold
HBSS.
2.2.3 Establishment of Primary Rat Cortical Neuronal Cultures
The resected cortices were then triturated 30 times using a fire-polished Pasteur pipette
to create a single suspension. The solution was then allowed to settle for 10 minutes
and the supernatant was collected in a separate 50 ml conical tube. The final cell
suspension was centrifuged at 800 x g for 10 minutes. The pellet was then resuspended
in HBSS at room temperature and the supernatant was discarded. An estimate of viable
cell number was determined using trypan blue staining and a hemocytometer. Finally,
the cells were plated at densities of 6 x 104 cells per well of 96-well plates, 6 x 105 cells
per cover slip and 3.5 x 106 cells per 6 cm Petri dish or at a density of approximately 1.5
x 105 cells/cm2.
2.2.4 Maintenance of Cell Cultures
The seeded cells were incubated at 37C with 5% CO2/95% air. On the day following
dissection and seeding or the first day in vitro (1st DIV), half of the media was replaced
with serum-free media supplemented with B27 with antioxidants (cocktail comprising of
vitamin E, vitamin E acetate, superoxide dismutase, catalase, and glutathione). An
additional component of 5 uM of cytosine arabinoside (ara-c), a mitotic inhibitor, was
included with the media change on the 3rd DIV in order to inhibit the proliferation of non-
neuronal cells. After 24 hours of ara-c treatment (4th DIV), media with ara-c was
replaced with fresh serum-free media supplemented with B27 with antioxidants. From
the 7th DIV and onwards, serum-free media supplemented with B27 without antioxidants
49
(less previous supplements listed) was used for feeding. Media was then replenished
every other day. The feed and treatment schedule is illustrated in Figure 2.1. In
addition, immunocytochemistry was performed on the 10th DIV to assess the cell
phenotype composition of the cultures.
2.2.5 Drug Treatment
2.2.5.1 Rotenone
Rat cortical neurons were treated with 15 nM (low dose) or 30 nM (high dose) rotenone
or vehicle for 24 hours (acute) and 4 days (chronic) starting on the 10th and 7th DIV,
respectively. For 'chronic' samples, media was refreshed and stressor re-added on the
9th DIV to maintain levels of oxidative stress throughout the 4-day period. On the 11th
DIV, the neurons were harvested for PCR or immunoblotting or imaged for functional
assays. Cell viability and morphology were monitored through phase contrast images
and viability assays.
2.2.5.2 Rotenone with Lithium
Lithium drug treatment was initiated on the 7th DIV. Cultures were fed with serum-free
media supplemented with B27 without antioxidants containing lithium chloride on the 7th,
9th, 11th and 13th DIV. Over the course of treatment, a therapeutically relevant
concentration of 1 mM lithium was maintained in the culture media [307].
Lithium treated rat cortical neurons were incubated with rotenone (15 nM or 30
nM) or vehicle for 4 days (chronic) starting on the 11th DIV. Media was refreshed and
stressor re-added on the 13th DIV to maintain levels of oxidative stress throughout the 4-
50
day period. On the 15th DIV, the neurons were harvested for PCR or immunoblotting.
Due to the additional numbers of neurons required, cell viability was not monitored for
lithium treated rat cortical neurons.
51
Figure 2.1 Schedule of oxidative stress and lithium treatment of rat cortical neurons. Primary rat
cortical neurons were cultured until the 7th DIV then subjected to oxidative stress treatment (0nM, 15nM or
30nM rotenone for 24 hours or 4 days) or lithium and oxidative stress treatment (1mM lithium for 8 days
and 0nM, 15nM or 30nM rotenone for 4 days). Solid triangles (▲) denote addition of rotenone and solid
circles (●) denote addition of lithium. On the 15th DIV, the cells were harvested for protein and RNA
samples or used in live cell calcium kinetics experiments.
52
2.3 Immunocytochemistry
The following protocol was adapted from that previously described by Marchenko and
Flanagan [308]. Rat cortical neuron (RCN) cultures grown on coverslips in 6-well plates
were incubated in a 4% paraformaldehyde fixative solution (4% paraformaldehyde, 4%
sucrose, 2% Triton X-100, 5 mM magnesium chloride, and 10 mM EGTA in PBS) pre-
warmed to 37C for 15 minutes. The coverslips were then washed 3 times with PBS for
5 minutes each. Following washing, the coverslips were coated with ImageEnchancer
and allowed to stand for 30 minutes at room temperature. Afterwards, the coverslips
were immersed in blocking solution (5% w/v skimmed milk, 2% goat serum in PBS) for 1
hour at room temperature. Primary antibodies to neural marker MAP2 (1:300 in blocking
solution), astrocytic marker, GFAP (1:100 in blocking solution), and oligodendrocyte
marker, CNPase (1:200 in blocking solution) were then added to the coverslips. The
coverslips were then sealed in parafilm overnight at 4C. After 3 washes at 5 minutes
each with PBS, the coverslips were incubated with secondary antibodies conjugated to
Alexa-488 (excitation 488nm, emission 519nm) and Alexa-594 (excitation 594nm,
emission 617nm) (both antibodies at 1:400 in PBS) for 2 hours at room temperature in
the dark. Another series of 3 washes at 5 minutes each with PBS was performed and
then the coverslips were mounted onto glass slides with ProLong Gold antifade reagent
and allowed to cure overnight at room temperature in the dark. The fixed cells were then
visualized using a LSM510 Zeiss confocal microscope with a 20x objective (Zeiss,
Munich, Germany).
53
2.4 Measurement of Cell Viability
Propidium iodide (PI) is a DNA binding dye that fluoresces (excitation maximum 535
nm, emission maximum 617 nm) upon intercalating with DNA. As the plasma
membrane of viable cells is impermeable to the dye, an increase in PI fluorescence is
correlated to an increase of dead or damaged cells. Hence, PI fluorescence was used
as a measurement and indicator of cell death [309]. Cell viability and death was
monitored on the 8th, 9th, 10th and 11th DIV, or every 24 hours after exposure to
rotenone-induced oxidative stress. Half the media was replaced with a 50 uM PI
solution in HBSS and the cells were incubated with the dye for 15 minutes at 37C with
5% CO2/95% air. Following incubation, the fluorescence was detected (excitation
485nm, emission 519nM) using the Fluoroskan Ascent microplate reader
(ThermoScientific, Fremont, CA). This protocol was performed according to the
manufacturer’s recommendations and optimized for the microplate reader by Angela
Roedding.
54
2.5 Immunoblotting
2.5.1 Preparation of Cell Lysates for Immunoblotting
A modified standard immunoblotting protocol using commercially available antibodies
was used to quantify the amounts of TRPC’s of interest in RCN. Protein lysates were
prepared using the hot SDS method [310]. Media was aspirated from 60 mm Petri
dishes and replaced with SDS pre-warmed to 100C and a cell scraper was employed
to collect the sample. The solution was then transferred to a 1.5 ml microcentrifuge tube
and sonicated (Vibra Cell Sonicator) 3 times for 10 seconds with 2 second pulses at
30% intensity. Following sonication, the suspension was placed in a heating block at
100C for 3 min. The sonication and heating steps were then repeated. The lysate was
aliquoted and stored at -80C. Quantification of the protein lysate concentration was
determined using the BCA (bicinchoninic acid) protein assay kit with bovine serum
albumin (BSA) as the standard. Absorbance at 562 nm was measured and subsequent
analyses were carried out using the UVMax microplate reader and SOFTmax PRO
analysis software (Molecular Devices, Sunnyvale, CA).
2.5.2 SDS-PAGE and Transfer
Prior to electrophoresis, protein lysates were diluted in sample buffer (62.5mM Tris, 3%
SDS, 10% glycerol, 5% mercaptoethanol, 0.01% bromophenol blue, pH 6.8) and placed
in a 100C heating block for 3 minutes. The samples, (10-40 μg) were loaded into 7.5%
resolving and 4% stacking polyacrylamide gels. Separation was carried out at 100V for
2 hours or until the dye front reached the end of the gel. Following electrophoresis,
55
proteins were then transferred electrophoretically onto a 0.45 µM nitrocellulose
membrane submerged in transfer buffer overnight at 150 mA.
2.5.3 Probing and Development
Membranes were strategically sectioned to allow for simultaneous probing of multiple
proteins, usually the TRPC subtypes (top portion) and the control protein, β-actin or α-
tubulin (bottom portion). For TRPC3 detection, membranes were blocked with 0.5%
EWA in PBS with 1% Tween (PBST), whereas membranes probed for TRPC5 and
TRPC6 were blocked with LI-CORTM blocking buffer and β-actin and α-tubulin
membranes were blocked with 5% skimmed milk in PBST on a shaker for 1 hour at
room temperature. After blocking, the membranes were incubated with primary
antibodies at 1:500 in their respective blocking solution for TRPC3, TRPC5 and TRPC6,
1:1000 in 5% skimmed milk for β-actin, and 1:15 000 in 5% skimmed milk for α-tubulin,
on a shaker at 4C overnight. Following incubation with primary antibody, the
membranes were rinsed 3 times and washed 3 times for 5 minutes in PBST. The
membranes were then incubated with secondary antibodies, 1:1000 HRP-conjugated
with protein A in PBST for TRPC3, 1:5000 goat anti-rabbit IRDye 800CW antibody in
PBST with 0.1% SDS for TRPC5 and TRPC6, 1:7500 goat anti-mouse IRDye 680CW
antibody in PBST with 0.1% SDS for β-actin and 1:35 000 anti-mouse antibody in PBST
for α-tubulin, on a shaker for 1 hour at room temperature. The membranes were then
rinsed 3 times and washed 3 times for 5 minutes in PBST. At this point, the TRPC5,
TRPC6 and β-actin membranes were visualized using the Odyssey Infrared Imaging
System and quantified using the accompanying Odyssey V3.0 software (LI-COR
Biosciences, Lincoln, NE). Enhanced chemiluminesence by ECLPlus kits was used to
56
visualize the TRPC3 and α-tubulin on blots detected using a STORM 860
phosphoimager system using LED (excitation 450nm, emission 520nm) and quantified
using ImageQuant 5.2 (Amersham/GE Life Sciences, England). The optimal
immunoblotting conditions of each target protein are summarized in Table 2.1.
57
Table 2.1 Immunoblotting conditions for TRPC3, TRPC5, TRPC6, α-tubulin and β-actin.
Protein of
Interest
Blocking Solution
1 Antibody 2 Antibody Imaging Method
TRPC3 0.5% EWA in PBST
1:500 in blocking solution
1:1000 HRP-protein A
ECL detected by Storm 860
TRPC5 LI-COR blocking buffer
1:500 in blocking solution
1:5000 goat anti rabbit IRDye 800CW
IR800 detected by LI-COR Odyssey
TRPC6 LI-COR blocking buffer
1:500 in blocking solution
1:5000 goat anti rabbit IRDye 800CW
IR800 detected by LI-COR Odyssey
α-tubulin 5% skimmed milk
1:15000 in blocking solution
1:15000 HRP-anti-mouse
ECL detected by Storm 860
β-actin 5% skimmed milk
1:1000 in blocking solution
1:5000 goat anti rabbit IRDye 800CW
IR680 detected by LI-COR Odyssey
58
2.6 Quantitative Real-time PCR
2.6.1 RNA Extraction
RNA was isolated from RCN cultures, approximately 3.5 x 106 cells, using the RNeasy
Plus Mini kit. As per the manufacturer’s recommended protocol, an on-column DNA
digestion was performed using deoxyribonuclease (DNase I) (Qiagen, Mississauga,
ON). Subsequent RNA purity and quality was confirmed using the NanoDrop
Spectrophotometer (ThermoScientific, Wilmington, DE) (ratio absorbance at 260 and
280, A260/280, is approximately 2) and RNA gel electrophoresis.
2.6.2 First Strand Synthesis
cDNA was synthesized by reverse transcription of total RNA extracted as above.
Synthesis was carried out using the SuperScript III RT kit. All incubations were
performed using the MyCycler Thermal Cycler (Bio-Rad, Mississauga, ON). The
reaction products were aliquoted and stored at -30C. Quantification of cDNA was
conducted using the Quant-iT High-Sensitivity DNA Assay kit. Fluorescence (excitation
502 nm, emission 523 nm) was detected and quantified using the Fluoroskan Ascent
microplate reader (ThermoScientific, Fremont, CA).
2.6.3 Primer Design and Optimization
Gene-specific forward and reverse primer pairs for rat TRPC5 and TRPC6 were
designed using PrimerExpress 3.0 (Applied Biosystems, Streetsville, ON). Sequences
for primer pairs for rat TRPC3, heme oxygenase-1 (HO-1), and glyceraldehydes 3-
phosphate dehydrogenase (GAPDH) were obtained from Angela Roedding. Prior to
59
synthesis, the primer specificity was verified by a nBLAST search of the National Centre
for Biotechnology Information (NCBI) and the primer products to cross intron-exon
junctions was confirmed by a Primer-BLAST search of the NCBI. All primer pairs were
synthesized by ATCG (Toronto, ON) and subsequently reconstituted in DNase, RNase-
free water to a stock concentration of 100 µM. Primers were stored at -30C. Table 2.2
lists the accession numbers, primer sequences and predicted amplicon size of each
target gene.
A set of preliminary experiments were performed to confirm primer specificity,
optimal primer concentration, and efficiency of primer pairs. Primer specificity was
ensured by conducting a dissociation curve using the ABI7300 Real-Time PCR systems
(Applied Biosystems, Streetsville, ON) and an agarose gel electrophoresis of the qrt-
PCR product. Optimal primer concentration and efficiency of primers were tested by
performing reactions at a range of primer and cDNA template concentrations,
respectively.
2.6.4 Polymerase Chain Reaction
Quantitative real-time polymerase chain reaction using the SYBR green method was
performed to measure relative gene expression using the ABI7300 Real-Time PCR
systems (Applied Biosystems, Streetsville, ON). SYBR green is a fluorescent dye which
binds to double stranded DNA. A reaction mixture of 25 µl consisted of 12.5 ul of SYBR
dye mixture (Applied Biosystems, Streetsville, ON), 1.5 µl of each primer (forward and
reverse), 5 µl of template cDNA, and 4.5 µl of DNase, RNase-free water. The reactions
underwent the following thermal cycling conditions: 95C for 10 minutes and 40 cycles
60
of 95C for 15 seconds and 60C for 1 minute. The expression levels of TRPC3,
TRPC5, TRPC6, and HO-1 were quantified relative to GAPDH using the comparative
threshold (CT) method [311].
61
Table 2.2 Accession number, sequence and expected product length of primer pairs used for
TRPC3, TRPC5, TRPC6, HO-1, and GAPDH
Gene Accession No.
Sequence (5' to 3') Fragment Length
TRPC3 NM_021771 (F) CTGGATTGCACCTTGTACCAGG 98
(R) GCAGACCCAGGAAGATGATGAA
TRPC5 NM_080898 (F) GGCAATCAAATATCACCAGAAAGA 84
(R) GGGAAGCCATCGTACCACAA
TRPC6 NM_053559 (F) TCGCTGTCGCCATTGGA 74
(R) CTGCAAGGAGCACACCAGTATATG
HO-1 NM_012580 (F) GCCTGCTAGCCTGGTTCAAG 87
(R) AGCGGTGTCTGGGATGAACTA
GAPDH NM_017008 (F) GACTCTACCCACGGCAAGTTCA 93
(R) TCGCTCCTGGAAGATGGTGAT
62
2.7 Live Cell Calcium Kinetics Imaging
2.7.1 Preparation of cells
On the 11th DIV, media was gently aspirated from cultures grown in 96-well optical
imaging microplate wells and washed 3 times with Fura-2 assay buffer. The neurons
were then incubated in Fura-2 dye solution (5 uM Fura-2 AM in Fura-2 assay buffer)
following the manufacturer’s recommendations for 30 minutes at 37C with 5%
CO2/95% air. After incubation, the cells were washed 3 times with loading assay buffer
alone and incubated in assay buffer for another 30 minutes at 37C in a 5% CO2/95%
air humidified atmosphere to allow for hydrolysis of the Fura-2 AM ester. To minimize
dye degradation and photobleaching, all steps were done in the dark. Plates were then
transferred to the bioimager system (Pathway 855 Bioimager, BD Biosciences,
Mississauga, ON) for fluorescence image acquisition and analysis.
2.7.2 Imaging
All live cell calcium kinetic assays were performed in a temperature controlled
environmental chamber at 37C under subdued ambient light. Images of 50 to 200 cell
bodies or regions of interest (ROIs) were acquired through a 10x objective. Manual and
laser-automated focusing mechanisms were used to obtain optimally resolved images.
Segmentation of ROIs was based on basal Fura-2 fluorescence (excitation 340/380 nm,
emission 510 nm) using the whole cell polygon algorithm within the BD Attovision
software. An exposure setting of 0.07s for the numerator (340 nm) and 0.15s for the
denominator (380 nm) was used. The same settings were used for all experiments. To
63
monitor OAG activation of Ca2+ mobilization in the neurons, a baseline was determined
by using the fluorescence ratio from 5 images in approximately 10 seconds prior to
adding drug. OAG and vehicle controls were then manually added to the well in a 0.5µl
injection volume using a P10 micropipette and mixed 6 times using the same pipette
and volume. Fura-2 fluorescence ratio intensities were monitored for 4 minutes after
drug injection with images captured at 1.9 seconds intervals. For Pyr3 inhibition
experiments, Pyr3 was added to the wells at the desired concentration at least 10
minutes prior to imaging and stimulation with OAG. Fluorescence ratiometric data,
including the maximum intensity, average intensity, rate of rise, and area under the
curve, were analyzed using the BD Attovision and the BD IDE (Image Data Explorer)
software provideing the data output for mean normalized relative intensity, percentage
of responding regions of interest (ROIs), rate of rise, and area under the curve (BD
Biosciences, Mississauga, ON).
64
2.8 Data Analysis
Statistical analysis was conducted using SPSS 17.0 software (IBM, New York, USA).
Differences in mRNA levels and immunoreactivity of TRPC subtypes following chronic
rotenone and/or lithium treatment were tested by repeated measures one-way analysis
of variance (ANOVA). Post hoc comparisons were carried out using the Bonferroni
pairwise test. Quantitative real-time RT-PCR results were analyzed using ΔCT values
where ΔCT = CT (target) – CT (GAPDH) and protein levels were tested after normalizing
to a loading control (β-actin or α-tubulin) or against the vehicle treatment [311].
Fluorescence data from live cell calcium assays were normalized to baseline,
calculated from 5 images taken prior to drug addition, using the BD IDE program.
Values for mean maximal intensity, area under the curve (AUC), rate of rise, and
percentage of responding ROIs were obtained using the same program. The
percentage of responding cells was determined based on the ratio of ROIs with maximal
intensities within two standard deviations of the mean maximal intensity of vehicle
treated wells. Differences in these four parameters defining “responding cells” between
treatment conditions were tested using repeated measures one-way ANOVA with
Tukey’s HSD test for post hoc comparison. Differences with p ≤ 0.5 were considered
statistically significant.
65
3 Results
66
3.1 Optimal conditions for real-time RT-PCR
Optimal conditions for real-time RT-PCR was established by a series of initial
experiments designed to measure the quality of extracted RNA, the specificity and
efficiency of primers used, and to determine optimal primer concentration and template
cDNA.
The quality of RNA extracted was confirmed based on the criterion of 260nm to
280nm (A260/280) absorbance ratio >1.8 and well resolved ribosomal 28S and 16S bands
on 1% agarose gels (Figure 3.1).
The specificity of primers for the genes of interest was confirmed by dissociation
analyses and electrophoresis of PCR products. Figure 3.2 displays the derivative
melting curves of TRPC3, TRPC5, TRPC6, HO-1 and GAPDH. The single sharp peak in
the dissociation curve represents the amplification of only one species which signifies
the specificity of the primer pairs. Moreover, DNA gel electrophoresis of amplicons
revealed the presence of respective single dense bands at the expected sizes (Figure
3.3) further supporting the specificity of the primer pairs used.
The efficiency and dynamic ranges of the primers were determined. The
similarity of the dynamic range between the gene of interest (TRPC5 or TRPC6) and the
reference gene (GAPDH) was determined by comparing their amplification efficiencies
at a range of template concentrations and is demonstrated by a horizontal line with
absolute slope of < 0.1. Both TRPC5 and TRPC6 primers demonstrated amplification
efficiencies similar to GAPDH (Figure 3.4).
67
Finally, through a series of amplification experiments, the optimal primer
concentration was determined by using concentrations of primer from 250 nM to 750
nM. Similarly, a range of template concentrations from 0.1 ng and 1 ng was used to
determine the optimal cDNA concentration for the PCR reaction. A summary of optimal
primer and template concentrations are listed in Table 3.1.
68
Figure 3.1 Gel electrophoresis of RNA extracted from RCN and BLCL. The presence of two crisp
bands representing the 28S and 16S rRNA is indicative of high RNA quality. RNA was extracted using the
RNeasy Plus Mini kit from Invitrogen with an on column DNA digestion using DNase I. Following
extraction 15 ng of RNA in 15 μL of RNA loading buffer was pipetted into wells of a 1% agarose gel. The
gel was visualized under UV light with the ChemDoc gel imaging system. BLCL – B-lymphocyte cell line,
RCN – rat cortical neuron.
69
Figure 3.2 Dissociation curves of TRPC3, TRPC5, TRPC6, HO-1 and GAPH primers. Real time PCR
melting point analysis of amplicons derived from respective primer sets yielded single sharp peaks in the
derivative melting curves in all cases under the optimized PCR conditions determined. The average
melting point temperature for (a) TRPC3, (b) TRPC5, (c) TRPC6, (d) HO-1, and (e) GAPDH was
determined to be 79.4C, 78.1C, 77.4C, 79.7C, and 80.4C, respectively.
70
Figure 3.3 Agarose gel electrophoresis of TRPC5, TRPC6 and GAPDH amplicons. Reaction
products were prepared in DNA loading buffer and run on a 1% agarose gel. The gel was visualized with
the ChemDoc gel imaging system. Single bands migrating at the predicted sizes (refer to Table 2.2)
denoting primer specificity were found for each respective cDNA species.
100 bp
71
Figure 3.4 Relative efficiency curves of TRPC5 and TRPC6 primers. To ensure similar dynamic
ranges between TRPC5 and TRPC6 and the reference gene, GAPDH, the amplification efficiencies of the
TRPC primers were compared to the amplification efficiencies of the GAPDH primers. Similar dynamic
range was demonstrated by a horizontal line with absolute slope <0.1.
72
Table 3.1 Optimal primer concentration and template concentration for the amplification of TRPC3,
TRPC5, TRPC6, HO-1 and GAPDH
Gene Primer Concentration Template Concentration
TRPC5 600nM 3ng
TRPC6 300nM 3ng
GAPDH 600nM 3ng
*TRPC3 600nM 3ng
*HO-1 600nM 3ng
* Optimal conditions for TRPC3 and HO-1 amplification were determined by Angela Roedding (in
preparation 2012)
73
3.2 Detection of TRPC5 and TRPC6 protein levels in RCN
Using standard immunoblotting techniques (refer to section 2.5), all three TPRC
subtypes, TRPC3, TRPC5 and TRPC6 were detected in the primary rat cortical neurons
migrating at the expected masses of 97 kDa, 108 kDa, and 122 kDa, respectively
(Figure 3.5a, 3.5c, 3.5e). Interestingly, TRPC6 appeared as a double band. Specificity
of TRPC6 antibodies was confirmed by the presence of bands of similar molecular
weight in positive control sample, rat brain lysates, and the absence of immunoreactive
species in negative control, BLCL. While the TRPC5 specificity was determined
similarly, no suitable negative control was found due to its ubiquitous expression [287].
In addition, a standard curve was generated to determine the linear dynamic range of
detection (Figure 3.5b, 3.5d, 3.5f). These experiments were replicated thrice, with
coefficient of variations less than 0.2, to ensure reproducibility.
74
Figure 3.5 Western blot assay and linearity of detection vs lysate protein concentration of TRPC3,
TRPC5, and TRPC6 in primary rat cortical neurons. Representative immunoblots of (a) TRPC3, (c)
TRPC5, and (e) TRPC6 are shown with the amount of protein sample loaded per lane noted. Standard
curves of (b) TRPC3, (d) TRPC5, and (f) TRPC6 immunolabeling versus lysate protein concentration
were determined using pooled lysate samples. In each case, linear regression analysis demonstrated
strong linear relationships with R2 > 0.90.
TRPC
3
TRPC
6
TRPC
5
75
3.3 Effect of Oxidative Stress on Primary Rat Cortical Neurons
The neuronal enrichment of rat primary cortical cultures was achieved using cytosine
arabinoside, a mitotic inhibitor, initiated cultures for 24 hours and confirmed by
immunocytochemistry. For this purpose, MAP2, CNPase and GFAP were used as
immunocytological markers for neurons, oligodendrocytes, and astrocytes, respectively.
As shown in Figure 3.6, dual labelling with either MAP2 and CNPase or MAP and
GFAP, revealed that over 90% of the total population possessed neuronal phenotype
with little or no contaminating oligodendritic or astroglial populations.
The effect of rotenone-induced oxidative stress was evaluated in several
capacities over the time course of oxidative stress treatment including visual monitoring
of gross cell morphology using phase-contrast microscopy. Images were taken every 24
hours for each of the treatment groups, 0 nM, 15 nM or 30 nM of rotenone (Figure 3.7).
It was observed that oxidative stress resulted in the truncation and reduction of neuronal
projections and cell bodies over time, in a dose-dependent manner.
In addition, transcript levels of HO-1, an oxidative stress response gene
measured to confirm the induction of oxidative stress in the RCN preparations
(Roedding et al., under review 2011), increased significantly in a dose-dependent
manner with rotenone treatment (Figure 3.8a) [F(2,7) = 14.43, p < 0.001]. Moreover the
viability of RCN after chronic oxidative stress treatment decreased in a dose-dependent
manner to approximately 50% in cultures treated with 15nM rotenone and 25% in
cultures treated with 30nM rotenone (Figure 3.8b). Cell viability was determined every
24 hours of the 4 day stress treatment period using propidium iodide staining.
76
Figure 3.6 Representative dual labelled immunocytochemistry images of primary rat cortical
neuron cultures. (a) Staining with anti-MAP2, a marker for neurons, revealed a prominent neural
population within rat cortical neuronal cultures. Labelling cultures with only secondary anti-bodies, (b) α-
rabbit and (c) α-mouse, served as negative controls. Further double immunocytochemistry with (d, e, f)
MAP2 and CNPase, a marker for oligodendrocytes and (g, h, i) MAP2 and GFAP, a marker for astrocytes,
revealed primary rat cortical neuron cultures to be highly enriched in cells of a neuronal phenotype.
77
Figure 3.7 Representative phase contrast microscopy images of primary rat cortical neuron
cultures over 4 day rotenone-induced oxidative stress treatment. Images were acquired 10x
magnification before and every 24 hours during 4 day rotenone treatment (images taken after 48 hours
and 72 hours not shown) at 0 nM, 15 nM and 30 nM. Neuronal cultures show reductions in the densities
of projections and cell bodies over time in a dose-dependent manner.
78
Figure 3.8 Effects of rotenone on HO-1 expression and cell viability in primary rat cortical
neurons. (a) HO-1 mRNA levels were measured using relative quantitative RT-PCR after 24 hour or 4
day treatment with rotenone at 0 nM (vehicle), 15 nM and 30 nM. Levels of HO-1 mRNA were significantly
higher in the 30nM treatment group compared to 0 nM and 15 nM (n=7, *p < 0.05). (b) Cell viability was
determined using propidium iodide assay every 24 hours during 4 day rotenone treatment (n=11). Data
are expressed as mean ±SD. All data shown in this figure was acquired and compiled by Angela
Roedding and reprinted with her permission.
*
79
3.4 The Effect of Acute and Chronic Oxidative Stress on TRPC5 and TRPC6
Previous work by Roedding et al. (under review 2011) has shown that TRPC3 protein (p
< 0.001) and mRNA (p < 0.001) are significantly reduced in cultures undergoing
rotenone induced oxidative stress compared to vehicle treated samples in a dose-
dependent manner after 4 days, but not 24 hours, of stressor exposure (Figure 3.9).
Based on these findings, TRPC5 and TRPC6 protein and mRNA transcript levels were
examined in primary rat cortical neuron preparations exposed to oxidative stress
inducing agent, rotenone, for 24 hours (acute) and 4 days (chronic). TRPC protein and
mRNA levels were quantified as described in section 2.5 and 2.6, respectively.
After 24 hours of rotenone treatment, no differences were found in the protein
levels of TRPC5 in 15 nM and 30 nM groups compared to vehicle as tested by repeated
measure ANOVA [F (2,8) = 1.148, n = 5, p = 0.345] (Figure 3.10a). However, rotenone
dose-dependently reduced the levels of TRPC5 mRNA at 24 hours [F (2,8) = 17.498, n
= 5, p < 0.001]; transcript levels showed a 1.87 fold reduction in the 30 nM rotenone
treatment group compared to vehicle controls (p < 0.05 Bonferroni pairwise test)
(Figure 3.10b). Although an approximately 15% reduction inTRPC5 mRNA was seen in
the 15 nM rotenone treatment group, this did not reach significance, likely due to small
sample size and large variance. After 4 days, rotenone significantly lowered the protein
levels of TRPC5 [F (2,6) = 19.044, n =4, p = 0.003] in the 15 nM groups, 69% relative to
controls (p < 0.01), and reduction was also found in the 30 nM group, 45% compared to
vehicle treatment (p < 0.05) (Figure 3.10c). Interestingly, no statistically significant
80
differences were found in the mRNA levels of the TRPC5 transcript after 4 day
treatment with rotenone [F (2, 8) = 2.528, n = 5, p = 0.185] (Figure 3.10d).
In regards to TRPC6 expression, as shown in Figure 3.11a and 3.11b, no
significant changes were found in either the protein levels [F (2,8) = 3.061, n = 5, p =
0.103] or the mRNA levels [F (2,8) = 1.687, n = 5, p = 0.245] in primary rat cortical
neurons exposed to 15nM and 30nM of rotenone for 24 hours compared to vehicle
treated controls. In contrast, 4 day stressor treatment significantly decreased TRPC6
protein levels [F (2, 8) = 14.728, n = 5, p = 0.002]. At 15 nM, rotenone decreased
TRPC6 protein levels by 55%, whereas the protein levels were decreased by 76% at 30
nM (Figure 3.11c). In the low concentration (15nM) group, rotenone reduced protein
levels by 55% compared to healthy controls (p < 0.05). Similar to TRPC5, no differences
were found in TRPC6 mRNA levels [F (2, 8) = 1.691, n = 5, p = 0.244] in rotenone
treated samples after 4 days of oxidative stress treatment (Figure 3.11d).
81
Figure 3.9 The effect of acute and chronic rotenone treatment on TRPC3 protein and mRNA levels
in rat primary cortical neurons. When neurons were exposed to 0 nM, 15 nM and 30 nM of rotenone for
24 hours, no significant differences in (a) protein [n = 3] or (b) mRNA [n = 3] levels were detected. In
contrast, a statistically significant dose-dependent decreases in TRPC3 (c) protein (60% ±28, Friedman
statistic=16.22, p<0.001, n=9) and (d) mRNA [31% ±12, F(2,7)=25.18, p<0.001] were observed upon
chronic rotenone treatment. Error bars represent SD. * denotes p < 0.05 and • denotes outliers. From
Roedding et al, under review 2012.
82
Figure 3.10 The effect of acute and chronic rotenone treatment on TRPC5 protein and mRNA
levels in rat primary cortical neurons. Neurons exposed to 0 nM, 15 nM and 30 nM of rotenone for 24
hours showed no significant differences in (a) protein levels [n = 5] but (b) mRNA levels decreased [F (2,
8) = 17.498, n = 5, p < 0.001], with a marked reduction at 30 nM rotenone relative to 0 nM controls (p <
0.05, Bonferroni test). After 4 days of rotenone treatment, there were also a significant effect on protein
levels (c) [F (2, 8) = 19.044, n= 4, p = 0.003] which were reduced at both 15 nM (p < 0.01) and 30 nM (p <
0.05) compared to vehicle controls. No changes were detected in the (d) mRNA levels [n = 5] of
chronically treated primary rat cortical neurons. Data are expressed as the means (bars) + SD for n=5
independent cell culture preparations. * denotes p < 0.05 relative to vehicle treated controls.
83
Figure 3.11 The effect of acute and chronic rotenone treatment on TRPC6 protein and mRNA
levels. Rat primary cortical neurons were exposed to 0 nM, 15 nM and 30 nM of rotenone for 24 hours or
4 days. Rotenone exposure did not affect (a) protein or (b) mRNA levels at 24 hours but significantly
reduced (c) TRPC6 protein level [F(2,8) = 14.728, n= 5, p = 0.002] at 15 nM (p < 0.05, Bonferroni test)
and 30 nM (p < 0.05) rotenone compared to vehicle controls but not mRNA levels after 4 days treatment.
Data are expressed as the means ± SD for n = 5 independent cell culture preparations. * denotes p <
0.05 relative to vehicle treated controls.
84
3.5 OAG Induces a TRPC3-mediated Ca2+
Response in Primary Rat Cortical Neurons
Given the profound effects of chronic oxidative stress on the mRNA and/or protein
levels of the TRPC channels examined, I next explored whether there were likely to be
associated reductions in the function of these channels. To this end, I assessed the
functionality of TRPC3 channels as a representative of the diacyglyerol activated TRPC
channels subgroup and for which there is a known agonist, 1-oleoyl-2-acetyl-sn-
glycerol (OAG) and an established specific antagonist, ethyl-1-(4-(2,3,3-
trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (pyr3) [312].
To confirm functional activation of TRPC3 channels in rat primary cortical neuron
cultures, representative cultures were loaded with 1 µM fura-2, cultures and stimulated
with OAG. Although the OAG dose-response was highly variable for what was identified
as technical solvation problems because of its hydrophobicity, it was found that
response was maximized at 100 μM as has been reported in a number of studies [313-
317] and increasing the concentration did not result in a corresponding increase in
response. As such, a stimulatory concentration of 100 µM was used. To confirm the
OAG stimulated Ca2+ response was mediated at least in part by TRPC3, the inhibition of
the response by the specific TRPC3 inhibitor, pyr3 was examined. Comparison of the
effects of pretreatment with increasing concentrations of pyr3 (vehicle, 0.5, 1, 2.5, or 5
μM for 10 minutes) on the OAG activated Ca2+ response showed a maximal inhibitory
effect at 2.5 µM pyr 3 (Figure 3.12). One-way repeated measures ANOVA revealed
statistically significant differences in the mean maximum intensities (normalized to
85
baseline) [F (4, 12) = 3.895, n = 4, p = 0.029] (Figure 3.12a), rate of rise [F (4, 12) =
4.133, n = 4, p = 0.024] (Figure 3.12c), and area under the curve [F (4, 12) = 6.121, n =
4, p = 0.006] (Figure 3.12d) in the different pyr3 treatments. Tukey post-hoc
comparisons indicated that 2.5 mM pyr3 treatment had significantly lower mean
maximum intensity, rate of rise, and area under the curve of the OAG-activated Ca2+
response, p < 0.05, relative to vehicle controls. The area under the curve of 1 mM pyr3
inhibition treatment was also significantly lower than that of vehicle controls (p < 0.05).
The percentage of responding ROIs, defined as ROIs with a maximum intensity which
were within 2 standard deviations of the mean maximum intensity of vehicle treated
OAG-induced cultures, was not significantly affected by pyr3 treatment [F (4, 12) =
2.185, n = 4, p = 0.132] (Figure 3.12b) supporting that the inhibitory effect of pyr3 was
generalized to all cells in the imaged field. For the purposes of clarity, Figure 3.13
presents a representative OAG induced Ca2+ response in RCN compared with vehicle
(0.5% DMSO) and 2.5 µM pyr3 inhibition curves. There was over 60% reduction of the
magnitude of the OAG-induced response by 2.5 µM of pyr3 pretreatment compared with
the vehicle.
86
Figure 3.12 Pry3 has a significant effect on mean normalized maximal intensity, rate of rise, and
area under the curve but not the percentage of OAG activated responding ROIs in primary rat
cortical neurons. (a) Repeated measures one way ANOVA revealed that pyr3 treatment had a
statistically significant effect on the mean normalized (to baseline) maximal intensity [F (4, 12) = 3.895, n
= 4, p = 0.029]. Tukey post-hoc analysis found that mean normalized maximal intensity was reduced after
2.5 mM (1.963, 95% CI [1.482-2.045]) pyr3 treatment compared to 0 nM (3.171, 95% CI [1.822-4.520])
but not at other concentrations. (b) There were no statistically significant differences in the percentage of
responding ROIs between pyr3 treatment groups. (c) Pyr3 attenuated the rate of rise [F (4, 12) = 4.133, n
= 4, p = 0.024] in the 2.5 mM (2.952 x 10-3
, 95% CI [2.284 x 10-3
-3.620 x 10-3
]) pyr3 treatment group
compared to 0 nM treated controls (7.315 x 10-3
, 95% CI [3.425 x 10-3
-11.21 x 10-3
]). (d) The area under
the curve was also significantly reduced by pyr3 treatment [F (4, 12) = 6.121, n = 4, p = 0.006] and was
significantly less in the 1 mM (77.34, 95% CI [33.32-121.4]) and 2.5mM (85.46, 95% [38.07-132.8]) pyr3
treatment groups compared to vehicle controls (225.9, 95% CI [108.9-343.0]). Data are expressed as the
means (bars) + SD for n = 4 independent cell culture preparations. *p < 0.05 vs. vehicle control. D –
DMSO.
87
Figure 3.13 OAG induced a Ca2+
response in rat primary cortical neurons is mediated in part by
TRPC3. Primary rat cortical neurons were cultured and loaded with 1µM fura-2. Cultures were treated
with 2.5nM pyr3, a specific inhibitor of TRPC3, or vehicle control for ten minutes before imaging. Baseline
340/380 ratio was determined by 5 data points measured prior agonist stimulation. When treated with
DMSO vehicle (grey), neurons did not demonstrate any response with or without pyr3. In comparison, a
strong Ca2+ response was observed when cultures were stimulated with OAG (blue). Treatment with
2.5nM pyr3 for 10 minutes resulted in a substantial decrease in response in primary rat cortical cultures
(red).
88
3.6 The Effect of Chronic Rotenone Induced Oxidative Stress on the Mobilization of Calcium in Primary Rat Cortical Neurons
In light of the differences found in TRPC3 protein and mRNA levels due to oxidative
stress, we sought to determine if chronic rotenone treatment also had an impact on the
regulation of Ca2+ flux through TRPC3. After 4 days of rotenone exposure, primary rat
cortical neurons were loaded with 1µM fura-2 and stimulated with 100 µM OAG.
As shown in Figure 3.14 rotenone reduced the magnitude of OAG-induced Ca2+
response in a dose-dependent manner. Further, repeated measures, one-way ANOVA
found that there were significant main effect of rotenone on the mean maximum
intensity [F (2, 6) = 31.787, n = 4, p = 0.001] (Figure 3.15a), percentage of responding
ROIs [F (2, 6) = 47.895, n = 4, p = 0.011] (Figure 3.15b), rate of rise [F (2, 6) = 31.645,
n = 4, p = 0.001] (Figure 3.15c), and area under the curve [F (2, 6) = 16.395, n = 4, p =
0.004] (Figure 3.15d). Post-hoc analysis using Tukey’s test for multiple comparisons
found that 15nM and 30nM of rotenone decreased the mean maximum response by
20% and 40%, percentage of responding ROIs by 34% and 64%, rate of rise by 55%
and 76%, and area under the curve by 70% and 84% in primary rat cortical neurons
relative to vehicle treated samples (p’s < 0.05).
89
Figure 3.14 Chronic rotenone treatment reduces OAG-induced Ca2+
response in primary rat
cortical neurons. Primary rat cortical neurons were treated with 0nM, 15nM, or 30nM of rotenone for 4
days before loaded with 1µM Fura-2. Baseline 340/380 ratio was determined by 5 data points measured
over 10 seconds prior OAG stimulation. A very robust Ca2+
response was observed in vehicle-control
cultures stimulated with OAG (blue). In comparison, the magnitude of OAG-induced Ca2+ response was
attenuated in a dose-dependent manner by rotenone (green - low dose, red – high dose).
90
Figure 3.15 Rotenone treatment significantly attenuates calcium responses in rat primary cortical
neurones stimulated with OAG. (a) Repeated measures one way ANOVA revealed that rotenone
treatment had a statistically significant effect on the mean normalized (to baseline) maximal intensity [F
(2, 6) = 31.787, n = 4, p = 0.011]. Tukey post-hoc analysis found that mean normalized maximal intensity
was decreased after 15nM (20% reduction) and 30nM (40% reduction) rotenone treatment compared to
0nM. (b) The percentage of responding ROIs was significantly reduced [F (2, 6) = 47.895, n = 4, p =
0.011] in the low dose (34%) and the high dose (64%) relative to vehicle controls. (c) Rotenone similarly
attenuated the rate of rise [F (2, 6) = 31.645, n = 4, p = 0.001] in 15nM (55%) and 30nM (76%) treatment
groups compared to 0nM treated controls. (d) The area under the curve was also significantly reduced by
rotenone treatment [F (2, 6) = 16.395, n = 4, p = 0.004]. Post-hoc analysis reveal that the area under the
curve was significantly less in the low dose (70%) and high dose (84%) rotenone treatment groups
relative to vehicle controls. Data are expressed as the means (bars) + SD for n = 4 independent cell
culture preparations. *p < 0.05 relative to vehicle control.
91
3.7 Effect of Chronic Lithium on the Levels of TRPC3, TRPC5, and TRPC6 Protein and mRNA under Chronic Rotenone-induced Oxidative Stress
Following evidence of oxidative modulation on these TRPC channels, the effect of
lithium, a prototypical mood stabilizer with neuroprotective properties, on rotenone-
mediated regulation of TRPC3, TRPC5, and TRPC6 was investigated (reviewed in
Section 1.4, page 27). Protein and mRNA levels of TRPC subtypes were measured in
primary rat cortical neurons pre-treated with 1 mM of lithium for 8 days and exposed to
15nM or 30nM rotenone or vehicle together with lithium for 4 days. Protein levels were
quantified by immunoreactivity levels and normalized to loading control (β-actin).
Transcript levels were analyzed using the comparative ΔCt method using GAPDH as a
housekeeping gene and mRNA levels are displayed as a fold change of the vehicle
control.
Repeated measures two-way ANOVA with lithium treatment and rotenone
treatment concentration as factors revealed a statistically significant main effect of
rotenone [F (2, 6) = 5.881, n = 4, p = 0.039] but not lithium [F (1, 3) = 0.418, n = 4, p =
0.564] on TRPC5 protein levels (Figure 3.16a). A trend interaction [F (2, 6) = 4.141, n =
4, p = 0.074] was also detected between these two factors. In contrast, TRPC5
transcript levels were not affected by rotenone treatment [F (2, 8) = 2.507, n = 5, p =
0.143] or lithium treatments [F (1, 4) = 0.539, n = 5, p = 0.504], nor was there an
interaction between rotenone and lithium [F (2, 8) = 3.613, n = 5, p = 0.076] on TRPC5
mRNA levels (Figure 3.16b).
92
Examination of TRPC6 protein levels (Figure 3.17a) also showed a significant
effect of rotenone [F (2, 6) = 9.560, n = 4, p = 0. 014]. Further statistical analysis
revealed that 30nM rotenone groups showed a trend in the reduction (51% in no lithium
and 42% in lithium treated groups) of TRPC6 protein levels compared to respective
vehicle controls (p = 0.073, Bonferroni pairwise test). However, there was no significant
effect of lithium treatment [F (1, 3) = 0.296, n = 4, p = 0.624], on TRPC6 protein levels
(Figure 3.17a) or an interaction effect [F (2, 6) = 0.989, n = 4, p = 0.425] between the
two treatment factors. There also were no significant effects of these treatments on
TRPC6 mRNA levels (Figure 3.17b) (rotenone [F (2, 8) = 1.512, n = 5, p = 0.277],
lithium [F (1, 4) = 1.757, n = 5, p = 0.256], lithium x rotenone [F (2, 8) = 3.103, n = 5, p =
0.437]).
Based on earlier work, under review, demonstrating rotenone-induced alterations
in TRPC3 expression, the effect of lithium on the TRPC3 subtype within this framework
was also examined. To reduce the effect of inter-animal variation, immunoreactivity
measurements for TRPC3 were normalized to the respective vehicle treatment groups
(0nM Li and 0nM rotenone treatments) as it was found that normalizing to a loading
control increased variance. As such, a repeated measure two-way ANOVA conducted
on TRPC3 protein levels (Figure 3.18a) only compared the low-dose rotenone and high
dose rotenone groups. It was shown that both rotenone [F(1, 4) = 14.607, n = 5, p =
0.019] and lithium [F(1, 4) = 75.949, n=5, p < 0.001] had a significant main effect on
protein levels, however, there was no significant interaction between these treatment
factors on protein levels [F(1, 4) = 0.687, n = 5, p=0.687]. Post hoc pairwise
comparisons revealed a statistically significantly decrease in protein levels between
15nM and 30nM rotenone treatment groups (p < 0.05, Bonferroni test) and a statistically
93
significant increase in protein levels between 0mM and 1mM lithium treatment groups (p
< 0.001). To determine whether there was an effect of lithium in the rotenone vehicle
treatment condition, rotenone vehicle immunoreactivity levels were normalized against
the 15nM rotenone, lithium vehicle group to control for inter-blot variability. No
statistically significant differences were found between the two groups (t = 0.367, df = 4,
p = 0.732, paired t-test). With regards to TRPC3 mRNA levels (Figure 3.18b), there
was a statistically significant effect of rotenone [F (2, 6) = 5.720, n = 4, p = 0.41] but not
lithium [F (1, 3) = 0.807, n = 4, p = 0.435] on TRPC3 transcript levels and no statistically
significant interaction between the two [F (2, 6) = 0.737, n = 4, p = 0.517]. Investigation
of TRPC3 protein and mRNA levels were initiated by Angela Roedding and Masoumeh
Emamghoreishi and completed by me thus representing a collaborative effort.
Masoumeh Emamghoreishi contributed an n of 3 to the quantification of TRPC3 mRNA
levels and Lydia Zhou contributed to an n of 3 to the quantification of TRPC3 protein
and I contributed an n of 1 to the quantification of both TRPC3 protein and TRPC3
mRNA, and did all of the analyses of the effect of lithium on TRPC5 and 6 as presented
above.
94
Figure 3.16 The effect of lithium pretreatment on the downregulation of TRPC5 by chronic
rotenone treatment. (a) Rat primary cortical neuron cultures were pretreated with lithium (1 mM) for 8
days and exposed to 4 day oxidative stress in the continued presence of lithium. Repeated measures
two-way ANOVA found a statistically significant main effect of rotenone [F (2, 6) = 5.881, n = 4, p = 0.039]
but no statistically significant effect of lithium or an interaction between lithium and rotenone were found.
(b) No significant effects of rotenone, lithium or an interaction of lithium and rotenone was found in
TRPC5 transcript levels. Data are expressed as the means (bars) ± SD of four cell cultures treated in
independent experiments.
95
Figure 3.17. The effect of lithium pretreatment on the downregulation of TRPC6 by chronic
rotenone treatment. (a) Rat primary cortical neuron cultures were pretreated with lithium (1 mM) for 8
days and exposed to 4 day oxidative stress in the continued presence of lithium. Repeated measures
two-way ANOVA found a statistically significant main effect of rotenone [F (2, 6) = 9.560, n = 4, p = 0.
014]. Bonferroni post-hoc tests revealed that groups treated with 30 nM of rotenone had a trend of
reduced levels of TRPC6 protein compared to vehicle controls (• p = 0.073). However, no statistically
significant effect of lithium or an interaction between lithium and rotenone were found. (b) No significant
effects of rotenone, lithium or an interaction of lithium and rotenone was found in TRPC6 transcript levels.
Data are expressed as the means (bars) ± SD of 4 cell cultures treated in independent experiments.
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Figure 3.18 The effect of lithium pretreatment on the downregulation of TRPC3 by chronic
rotenone treatment. (a) Rat primary cortical neuron cultures were pretreated with lithium (1 mM) for 8
days and exposed to 4 day oxidative stress in the continued presence of lithium. Repeated measures
two-way ANOVA comparing the 15 mM to the 30m M rotenone groups found a statistically significant
main effect of rotenone [F(1, 4) = 14.607, n = 5, p = 0.019] and lithium [F(1, 4) = 75.949, n=5, p < 0.001]
but not an interaction between the two factors. Bonferroni post-hoc tests revealed that the groups treated
with 30 nM of rotenone had reduced levels of TRPC3 protein compared to 15 nM groups (p < 0.05) and
that both groups had increased protein levels when treated with lithium compared to non-lithium treated
groups (p < 0.001). However, no statistically significant effect of an interaction between lithium and
rotenone was found. (b) With regards to TRPC3 transcript, rotenone had a statistically significant main
effect on mRNA levels [F (2, 6) = 5.720, n = 4, p = 0.41] although no effect of lithium or an interaction of
lithium and rotenone was found. Data are expressed as the means (bars) ± SD of 4 cell cultures treated in
independent experiments.
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4 Discussion
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The principal objectives of this study were to investigate the effects of oxidative stress
on the expression and/or functionality of TRPC3, TRPC5 and TRPC6 in primary rat
cortical neurons. As such, the effects of acute and chronic oxidative stress on mRNA
and protein levels of TRPC5 and TRPC6 were examined. Based on Roedding et al’s.
finding on the effects of chronic rotenone treatment on TRPC3, that is, downregulation
of its expression and protein levels, I hypothesized that TRPC5 and TRPC6 mRNA and
protein levels would also be decreased after chronic but not acute oxidative stress.
Additionally, I hypothesized that such reductions in TRPC3 expression or protein levels
would lead to a corresponding decrease in TRPC3 function, which had not been
examined by Roedding et al. The results revealed that while TRPC5 and TRPC6 protein
levels were decreased significantly, TRPC5 and TRPC6 mRNA levels did not seem to
be affected by chronic oxidative stress. The reduction of TRPC3-mediated Ca2+ flux in
neurons exposed to oxidative stress corroborates a functionally significant effect of
oxidative stress on the TRPC3 channels in rat primary cortical neuron cells.
While the obtained results suggest that TRPC3, TRPC5, and TRPC6 are
regulated, in part, by chronic oxidative stress, the degree and level of regulation
(expression, protein turnover and function) seem to vary between subtypes indicating a
complex regulatory system which may have important physiological implications in
cellular Ca2+ homeostasis linked to the pathogenesis of bipolar disorder.
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4.1 Acute and Chronic Oxidative Stress on TRPC5 and TRPC6
Based on the observed levels of TRPC3, TRPC5, and TRPC6 mRNA and protein levels
following chronic or acute oxidative stress, the primary findings of this study are first,
that the expression or protein levels of this group of TRPC channels is affected by
oxidative stress and, second, that there are differences in the regulation of TRPC3,
TRPC5 and TRPC6 by oxidative stress, both acutely and/or chronically. As the effects
of rotenone on the mRNA and/or protein levels of these TRPC channels were time
dependent, these changes are likely to reflect the effect of other mechanisms linked to
the redox state of the cell that govern their expression and/or turnover.
At the transcriptional level, compared to vehicle controls there was a marked
reduction in TRPC3 mRNA after 4 day treatment (Roedding et al. 2011, submitted) and
in TRPC5 mRNA after 24 hours in rotenone treated samples, but no changes were
detected in TRPC6 mRNA levels. The decreased TRPC3 and TRPC5 mRNA levels are
unlikely to represent a global reduction in mRNA stability or expression as expression of
TRPC6 and the housekeeping gene, GAPDH, were constant across treatment groups.
More likely, the changes in TRPC3 and TRPC5 mRNA levels reflect the influence of
ROS on the expression and/or mRNA stability of these two channel transcripts. As the
structure of the TRPC3 and TRPC5 promoters are not well elucidated, the exact
mechanisms and pathways involved in regulating TRPC gene expression are currently
unclear. For instance, the transcription of multiple antioxidant genes is activated through
sequences known as antioxidant response elements (ARE) mediated by transcription
factors Nrf1 and Nrf2. However, there is as yet no published evidence to support the
existence of ARE in the promoters of TRPC channels [318]. In one study, the
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involvement of mitogen-activated protein kinases (MAPKs), extracellular signal
regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) as well as the ubiquitously
expressed transcription factor, nuclear factor-κB (NF-κB) have been implicated at least
in the case of thrombin-mediated upregulation of TRPC3 in astrocytes [319]. However,
conflicting reports demonstrate both dependence and independence of MAPK activation
on Ca2+ signalling, suggesting that molecular pathways modulating TRPC3 expression
may be more complicated [320-322]. In addition, erythropoiethin, known to initiate
signalling pathways including MAPK, has been demonstrated to upregulate TRPC5
mRNA in cultured endothelial cells after 24 hours [323]. The ERK pathway, on the other
hand, has been shown to increase TRPC1 expression following stimulation by TNF-α
[324]. Most importantly, these intracellular cascades are known to be involved in
physiological responses to a variety of stimuli including ROS and thus may provide
possible links between oxidative stress and TRPC gene regulation.
Another line of evidence implicates the involvement of TRPC subtypes 3 and 6 in
the activation of calcineurin-nuclear factor activated T cells (NFAT) signalling cascade in
cardiac models, permitting the possibility of biological feedback of NFAT on TRPC
channel expression [325-327]. Indeed, Kuwahara et al. found that in mice TRPC6 was
upregulated in response to calcineurin and that the promoter of mice TRPC6 contained
two conserved NFAT consensus sequences [328]. Activation of PKC signalling, another
intricate intracellular pathway linked to both ROS and NFAT, has been shown to down-
regulate TRPC6 expression after 2 days in cultured mesangial cells [329]. Therefore, it
is possible that a reduction in TRPC channel functionality leading to decreased NFAT-
mediated Ca2+ signalling could result in the further downregulation of TRPC3 through
feedback inhibition. Taken together, available data suggest that the regulation of TRPC
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gene expression by rotenone is the product of complex interactions between several
intracellular signalling pathways which could vary among cell types, mechanism of ROS
production, and duration of stress.
At the protein level, all three channels, TRPC3, TRPC5 and TRPC6,
demonstrated significant dose-dependent decreases to rotenone-induced oxidative
stress treatment after 4 days but not 24 hours. Similar to TRPC gene expression, there
is currently not a clear understanding of the modulation of TRPC protein level by
oxidative stress. However, the differential changes in TRPC channel expression at the
transcriptional and translation level hint that these channels may be regulated through
different mechanisms. In the case of TRPC3, the reduction in protein levels most likely
reflects the significant decrease in mRNA levels after 4 day rotenone exposure.
Although TRPC5 mRNA was not reduced significantly following chronic rotenone
treatment, a decrease in its mRNA levels was seen in the acute treatment group
suggesting that TRPC5 gene expression may be affected by ROS, at least acutely.
However, as TRPC6 mRNA levels did not show any response to either acute or chronic
rotenone treatment, a different mechanism of regulation must exist in its case. For
instance, ROS may regulate these channels through post-translational modifications
that alter the protein turnover [330]. Protein oxidation can result in protein aggregation,
fragmentation, loss of function and increased degradation through mechanisms such as
the ubiquitin-proteasome pathway and lysosomes [331, 332]. It is important to note that
while TRPC3 and TRPC5 may be affected by a downregulation in expression, these
channels may also be susceptible to post-translational modification/degradation.
Interestingly, as TRPC3 and TRPC6 are very closely related, the different non-
homologous coded regions of these two proteins may encapsulate the targeted post-
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translation modification or proteolytic sites. As currently knowledge in this area is very
limited, further work into the structural motifs of TRPC channels, by means of proteomic
bioinformatics for example, are necessary to better understand the differential regulation
observed.
In addition, safeguards and controls were implemented to ensure the validity of
the findings observed. First, although TRPC5 and TRPC6 expression were only
measured within the context of rotenone-induced oxidative stress, Roedding et al 2012
(under review) found that TRPC3 expression was reduced using both rotenone and
paraquat, another inhibitor of complex I of the mitochondria, suggesting that the
decrease in TRPC3 channel expression is not a result of chemical specific effects of
rotenone. Moreover, the consistent expression of the housekeeping gene GAPDH and
loading control β-actin across vehicle and rotenone treatment groups implied that the
reductions in TRPC channel expression and protein levels are not attributable to global
impairments in gene transcription and protein synthesis mechanisms caused by
oxidative stress. Lastly, though these changes occur against the backdrop of cell death,
the differential pattern of changes in expression and protein levels of these TRPC
species and the control housekeeping proteins is inconsistent with an explanation that
the observed TRPC changes can be simply ascribed to cell death.
A survey of the literature revealed a range of TRPC channel responses to
oxidative stress in several different models and using different mechanisms of ROS
generation. For example, Graham et al. found a significant decrease in TRPC6 protein
levels in cultured mesangial cells after treatment with high glucose and H2O2 after 2
days and 6 hours, respectively [329]. In contrast, Wuensch et al. found an increase in
TRPC1, TRPC3, TRPC5 and TRPC6 in monocytes after 4 hours in response to high
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glucose [333]. In platelets, Liu et al. found that high glucose treatment also increased
TRPC6 protein levels [334]. Interestingly, both TRPC3 and TRPC5 channels have been
shown to be activated by reactive oxygen species [335]. However, the effects of long-
term oxidative stress on their expression were largely unknown until now. These
findings of a strong reduction in TRPC3, TRPC5 and TRPC6 protein levels after chronic
but not acute oxidative stress highlight the importance of cell-type specific and channel
subtype specific responses to ROS and the temporal aspect of oxidative regulation on
these channels.
Overall, this study found, for the first time, that chronic but not acute oxidative
stress significantly reduces TRPC5 protein, and TRPC6 protein levels, and confirmed
TRPC3 mRNA and protein reductions, although acute stress seems to decrease
TRPC5 mRNA. Most importantly, this study highlights the importance of the duration of
oxidative stress in the regulation of these channels.
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4.2 The Functional Significance of Chronic Oxidative Stress on TRPC3/6
As there are few known specific, direct acting agonists and antagonists to assess the
function of TRPC3 and 6 individually. Within these limitations, the best characterized
agonist of these two channels, OAG, an analogue of endogenous DAG, has been
widely used and shown to directly gate these channels and stimulate Ca2+ entry (see
Section 1.6.3, page 37). Thus, OAG was used in this study to stimulate both TRPC3
and TRPC6 to evaluate whether the reduced levels were accompanied by a net
reduction in their function. A pharmacological strategy was then applied in order to
estimate at least that fraction of the total OAG stimulated response (TRPC3 and 6) Ca2+
response attributable to TRPC3. This was accomplished by measuring the OAG
stimulated response in the presence and absence of the TRPC3 specific inhibitor pyr3
in the RCN preparation [312]. In preliminary dose ranging inhibition experiments,
interestingly, a concentration of 2.5mM pyr3 was found to give the greatest inhibition
across the range of concentrations tested (0.5 - 5 μM). Thus, the inhibition curve was U-
shaped. Regardless of the complex inhibition curve, pyr3 pretreatment significantly
reduced the levels of OAG-activated Ca2+ flux, maximally to 62% above basal (a 38%
reduction) (Figure 3.13), compared to non-inhibited vehicle controls. These results are
consistent with the findings of Kiyonaka et al. that pyr3 inhibits TRPC3 specifically in the
low µM range (IC50 value of 0.8 µM) and support that TRPC3 mediates a significant
fraction of the OAG activated response [312]. Moreover, they are in agreement with
reports of inhibition of TRPC3 by pyr3 through the ranges from 1 µM to 10 µM with
maximal inhibition at ≈3 µM depending on the cell model studied [313, 336-340].
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Although it remains to be tested directly, the fraction of the OAG response that was
uninhibited by pyr3 may represent the contribution by TRPC6.
In the absence of TRPC6 specific inhibitor the degree of contribution of TRPC6
to the OAG-mediated response could not be discerned using a similar pharmacological
strategy. Although aminoethoxydiphenyl borate (2-APB) and N-(p-amylcinnamoyl)
anthranilic acid (ACA) inhibit TRPC6, these compounds lack specificity for TRPC6 and
have been reported to block the activity of members of the TRPC and TRPM families,
calcium release-activated calcium modulator channels (CRACM), and TRPV1 channels
[341-343]. An alternative approach to evaluating TRPC6 functionality would have been
contingent on the availability of a specific agonist to determine the effect of rotenone on
the agonist induced Ca2+ flux rather than the reduction in agonist induced activation.
Hyperforin, a bicyclic polyprenylated acylphloroglucinol derivative and the main active
ingredient in St. John’s Wort extracts, is a direct activator of TRPC6 [343]. Studies in
keratinocytes, however, question its specificity to TRPC6 as hyperforin was reported to
activate TRPC1, TRPC3, TRPC4, TRPC5 and TRPC7 [344]. Therefore, the lack at this
time of pharmacological modulators of established specificity to TRPC6 indicates
alternative strategies, such as TRPC6 knockdown models or direct electrophysiological
methodologies, must be used to study its function. As these techniques were not
available to be used in this thesis research, the effect of chronic oxidative stress on
TRPC6 function was not examined further.
Following chronic rotenone treatment, it was observed that there were significant
dose-dependent reductions in OAG-mediated Ca2+ flux. Rotenone-treated cultures
stimulated with OAG were not pretreated with pyr3 due to the cytotoxic properties of
DMSO, which solvated both OAG and pyr3. However, preliminary pyr3 inhibition
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experiments support a major role of TRPC3 in OAG-mediated Ca2+ flux. Thus, these
results indicate that there is a significant decline in TRPC3 functionality following
oxidative stress. Moreover, as there were statistically significant reductions in TRPC3
and TRPC6 protein levels and TRPC3 mRNA levels after chronic oxidative stress, the
dramatic reduction in expression may be the basis of the decreased functionality
observed.
The physiological importance of the observed reduced TRPC3 function and
whether it represents a cellular response to chronic oxidative stress or a consequence
of increased ROS, however, is not well understood. While data on TRPC3 function in a
neuronal context is sparse, based on earlier work by Jia et al., TRPC3 and TRPC6
channels have been recognized to possess neural protective properties through BDNF-
induced Ca2+ elevations [345]. On the other hand, at least two studies have found
reduced TRPC3 expression and TRP-mediated Ca2+ influx to promote cellular survival
[346, 347]. While these reports seem contradictory, the observation that TRPC3 can
function in a store-dependent or independent manner depending on the expression
system and protein expression levels may suggest that the physiological role of TRPC3
is cell-type specific [348, 349]. Although evidence is sparse, a reduction in TRPC3 could
be a cellular response to control NFAT-mediated genes, as discussed earlier, and
possibly other Ca2+ dependent expression profiles. Therefore, further knowledge of the
physiological roles of TRPC3 and TRPC6 channels and the consequences of their
altered expression and function is necessary to appreciate the molecular mechanism(s)
which induce the observed functional decline and how it may contribute to pathogenesis
of bipolar disorder. Despite this, this study found, for the first time, a significant
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reduction of OAG-induced TRPC3/6-mediated Ca2+ flux following chronic rotenone
treatment.
4.3 The Effect of Chronic Lithium Treatment on Chronic Rotenone-induced Changes in TRPC3, TRPC5, and TRPC6
If the effect of chronic oxidative stress to downregulate TRPC3, TRPC5, and TRPC6 is
relevant to the pathophysiology of bipolar disorder, it would be expected that mood
stabilizer pretreatment of RCN would prevent or minimize the effect chronic rotenone
treatment. In this regard, lithium’s neuroprotective effects have been linked to its ability
to abrogate oxidant damage in both clinical and pre-clinical trials (refer to Section 1.4,
page 27) [256, 258]. Of note, chronic lithium treatment of rat primary cortical neuron
cultures at a therapeutically relevant concentration of 1 mM partially rescued the effect
of rotenone to downregulate TRPC3 protein but not mRNA levels. In contrast, there was
no appreciable effect of lithium to mitigate the effects of chronic rotenone on TRPC5
and 6, either mRNA or protein levels.
The observation that there was no difference in the levels of TRPC3 mRNA and
the protein levels of the other related TRPC channels, TRPC5 and TRPC6, with and
without lithium treatment implies that the effect of lithium was specific for TRPC3
degradation and/or clearance. Lithium is considered the standard pharmacological
treatment for BD and has been shown to possess neuroprotective properties in various
cellular and animal models [111]. Lithium’s neuroprotective effects have been
associated with several actions including the induction of survival molecules in brain
(i.e. Bcl-2, BDNF, and VEGF), induction of autophagy and induction of neurogenesis
[239, 240, 249, 350, 351]. In fact, findings by Jia et al. implicate TRPC3 and TRPC6 as
mediators of BDNF-induced Ca2+ elevation and subsequent signalling in neuronal
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survival [345]. Therefore, lithium may regulate TRPC channel expression indirectly
through molecular targets such as BDNF. However, if this were the case, then a
separate mechanism must exist to target specifically TRPC3 over TRPC5 and TRPC6
based on the findings of this study.
The rescue like effect of chronic lithium treatment on the rotenone induced
reduction in TRPC3 protein levels in the RCN contrasts with the effect of chronic lithium
at similar therapeutic concentrations to reduce TRPC3 immunoreactivity (but not
mRNA) levels in BLCLs from bipolar and healthy subjects [307] and to reduce TRPC3
mediated Ca2+ responses in human astroglial cell lines (U87) (Takuji 2011, unpublished
data). While the directionality of the changes is discrepant between these cell models,
the confinement to the TRPC3 protein levels is the same. This suggests a common
effect of lithium on the disposition and clearance of the TRPC3 at the protein level. The
simplest explanation for the divergence of TRPC3 protein modulation by lithium among
these cell models is that they are cell-type dependent, RCN being an excitable cell type
whereas U87 and BLCLs are non-excitable. Not to be excluded is the possibility of
species differences in the cell models, rat versus human. Another explanation may
reside with the fact that RCN are primary cell lines whereas BLCLs and U 87 cells lines
are transformed and immortalized by viruses. Notwithstanding these possibilities, a
more interesting question arising from these cell model discrepancies is whether they
reflect responses of unique physiological significance within the excitable as compared
to the non-excitable cell type. Knowledge of the cell physiology of TRPC3 in many
respects is still rudimentary.
The specificity of an effect by lithium to TRPC3 and not TRPC6 is surprising.
Unlike TRPC5, TRPC6 sequence homology and response to DAG and its analogues
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places it in the TRPC3/6/7subfamily grouping. Thus, one would have expected similar
effects of lithium on it as for TRPC3. In fact, given the greater reduction in TRPC6 as
compared with TRPC3 in the same cells exposed to chronic rotenone treatment, a more
prominent lithium rescue effect was anticipated. That this did not occur may reflect that
in primary rat cortical neurons, modulation of TRPC6 is more sensitive to intracellular
ROS production than TRPC3. Coupled with the caveat that rotenone treatment likely
resulted in a highly oxidative environment relative to ROS generated under
physiological conditions, lithium treatment at therapeutic levels may not have been able
to rescue TRPC6 protein expression in comparison to TRPC3. TRPC5, on the other
hand, falls into the TRPC4/5 grouping of TRPC channels, therefore, the dissimilarity to
TRPC3 in response to lithium again could be related to difference in mechanisms that
regulate the former. Though the exact explanation for the effect of chronic lithium on
TRPC3 remains to be elucidated, the fact that it exerts effects across several different
cell models and in human cell lines argues that the observed effects of lithium on
TRPC3 may still be relevant to its therapeutic spectrum of action.
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4.4 Methodological Considerations and Limitations
4.4.1 Cell Model and Stressor
In the investigation of the effect of oxidative stress on canonical TRP expression and
function and its relevance to BD pathogenesis, the translational potential hinges on the
cell model and stressor used. In general, findings from in vitro models may be cell-type
and species specific and they may not fully represent a true physiological state where
cells are exposed to numerous regulatory signals and cell-cell interactions. Additionally,
being primary cell lines, inter-litter or inter-batch variation may increase the range of
responses seen to drug treatment and must be taken into consideration in interpreting
these results. However, such models serve hypothesis-generating roles in respect to
extrapolating to an in vivo state. Therefore, while there are limitations in using primary
rat cortical neuron and further work needs to be done in human cells and models, they
are cell-type relevant and relatively easy to acquire.
Rotenone was chosen for its ability to generate ROS intracellularlly by inhibiting
the complex I of the electron transport chain within the mitochondria. However, the
degree of cell death seen implies that concentrations used in this study were likely
supraphysiological. Although a low dose rotenone treatment was included to represent
a condition with a limited or milder degree of cell death, there was a significant reduction
in cell viability at both 15nM and 30nM rotenone treatment groups, approximately 25%
and 50%, respectively, compared to vehicle treatment controls. However, the differential
reductions in expression and function between TRPC subtypes support that these
findings are unlikely to be simply artifacts of cell death. Future studies using more
physiologically relevant intracellular stressors of ROS, such as mercaptosuccinate, an
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inhibitor of glutathione peroxidase, may provide insight into the cellular response to
oxidative stress in the context of TRPC channels [352, 353].
4.4.2 mRNA Quantification: Real-time RT-PCR
Messenger RNA levels were determined by real-time RT-PCR using SYBR green
fluorescence using the comparative CT method for relative quantitation [311]. Amongst
the different fluorescence monitoring systems available, the SYBR green method is
relatively simple technologically and inexpensive, and as such was used in this study.
The disadvantages of this system are the requirement for extensive optimization and an
additional dissociation follow-up analysis to ensure amplicon specificity. As the
comparative Ct method eliminates the need for a standard by quantifying relative
expression, the dynamic range of both the target and the reference gene (GAPDH)
must to be similar. A nearly horizontal line (slope < 0.1) in the plot of log input versus
ΔCt (Figure 3.4) demonstrates that the efficiencies of the two amplicons are
approximately equal. While TRPC6 fulfilled this requirement, TRPC5 showed a slight
deviation from this slope value. The TRPC5 primers were still used because previous
responses to oxidative stress in TRPC3 mRNA levels were so large that it was expected
that an effect would still be detectable despite a small decrease in efficiency. As show in
Figure 3.2, the appearance of a single strong peak at the corresponding melting point
demonstrates the specificity of the reactions.
4.4.3 Protein Quantification: Western Blot
Immunoblotting, being the current standard for protein detection, was used to visualize
and quantify levels of TRPC subtypes. Prior to experimentation, significant optimization
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was performed to maximize the accuracy of the procedure, including ensuring the
reproducibility of the procedure for each TRPC subtype and establishing a linear
dynamic range of detection by a 6 point standard curve. All samples were analyzed in
triplicate and the amount of protein homogenates loaded were within the linear range of
the standard. Despite this, the data presented must be taken within consideration of
logistical and procedural limitations. First, while BLCL lysates were used as a negative
control for TRPC6, no similar controls for TRPC5 were available. However, in rat frontal
cortex, a control tissue, homogenates and rat primary cortical neurons, for both TRPC5
and TRPC6 were distinct, well resolved immunoreactive bands that migrated with the
expected molecular mass. Second, it was logistically difficult to include all treatment
groups on one acrylamide gel and thus controls were required to adjust for inter-gel/blot
variance. Although TRPC5 and TRPC6 were normalized to loading control β-actin to
account for this, TRPC3 was not as Roedding (2010) found normalizing against β-actin
increased variance of TRPC3 relative immunoreactivity. Third, TRPC3 protein levels
were quantified using chemifluorescence detection with horseradish peroxidise tagged
secondary antibodies whereas TRPC5 and TRPC6 were detected with a more
contemporary near infrared scanning system. The near infrared system was adopted in
TRPC5 and TRPC6 experiments as it became available since it afforded higher
sensitivity and wider linear dynamic assay range than the STORM detection platform.
Lastly, immunoblotting using whole cell lysates does not provide insight into the
disposition of the protein within the cell. Given that ROS significantly reduces TRPC
protein levels, localization of these channels, by fractionating the preparations, before
and after chronic stress could provide insight into the mechanism of the oxidative
regulation such as through increased protein degradation or decreased expression.
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4.4.4 Functional Assay: Calcium Kinetics
The function of TRPC3 and TRPC6 channels were assessed by measuring Ca2+
mobilization after agonist stimulation using ratiometric Ca2+ dye, fura-2 AM in primary rat
cortical neurons after chronic rotenone treatment. Intracellular Ca2+ flux was measured
in lieu of electrophysiological assays, which is the gold standard for evaluating TRP ion
channel function, because of the lack of equipment and technical expertise to perform
such procedures during the course of this work. In general, techniques such as patch-
clamping allow direct measurements of channel activity, resolution of the opening of
single ion channels, and detection of current signals spanning three orders of
magnitude. However, these procedures require physical contact with the tissue/cells
and standard techniques cannot be used to quantify multiple cells simultaneously.
Imaging techniques, on the other hand, do not interfere with neuronal function, provide
spatial resolution, and are able to simultaneously measure responses in multiple cells.
That said, Ca2+ imaging assays which are based on the use of Ca2+ binding dyes are
subject to such factors as dye loading efficiencies, compartmentalization and
photobleaching that can impact quantification.
For this study, the ratiometric fura-2 Ca2+ indicator was preferred over single
wave-length dyes such as fluo-4. In general, ratiometric dyes provide a number of
advantages including minimizing the effects of uneven dye distribution (i.e. dye loading
variations) and photobleaching since these factors would affect both measurements. To
prevent effects such as partial AM ester hydrolysis, the dye was loaded according to
manufacturer’s recommendations in regards to concentration, duration and time
required to hydrolyze. To minimize dye compartmentalization, assays were performed
within 60 minutes following dye loading. However, other factors that are difficult to
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control for include uneven illumination intensity and optical path length, which may vary
across the field of view.
Another important aspect of OAG was its hydrophobicity, requiring solvation in
organic solvents with limited aqueous solubility in their own right and potential cytotoxic
effects at concentrations greater than 0.5 to 1%, such as the case for DMSO, which has
been widely used for this purpose. Although DMSO-vehicle controls were included to
account for any DMSO-mediated responses, difficulties in the delivery of hydrophobic
molecule such as OAG into aqueous medium are well known [354]. Moreover, OAG
undergoing micelle formation in aqueous solutions may result in lower than expected
free OAG concentrations to stimulate TRPC3 channels of cultured cells under assay. To
mitigate this characteristic of OAG, a standardized methodology was employed over all
treatment conditions.
In addition, there were several limitations and technical difficulties that were
encountered with the BD Pathway imaging system. First, although using a 20x objective
allowed for greater resolution, images were acquired using a 10x lens to ensure
approximately 100 cells or regions of interest (ROIs) were captured within the field of
view in control wells. Despite maximizing the field of view, cultures exposed to rotenone
exhibited significant cell death resulting in fewer ROIs per imaged field. Further, the
segmentation algorithm in the BD Attovision software used to define ROIs, the whole
cell polygon algorithm, captured only the neuronal soma and not the axonal or dendritic
projections. Lastly, due to the cytotoxic properties of DMSO, OAG was added at high
concentrations but low volumes. However, the robotics system of the Pathway does not
allow for accurate delivery of volumes less than 2μl into 96-well plates. As a result,
manual addition and mixing of OAG or DMSO was performed.
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4.5 Future Studies
The results of this study provides evidence of ROS-mediated regulation of intracellular
Ca2+ signalling dynamics through TRPC channels which have been implicated in BD in
other work.
Within the scope of the current model, further replication of drug treatment would
be valuable in increasing the sample size and in turn statistical power of the study,
especially within the context of variables that are difficult to control such as inter-animal
variation. As mentioned in the previous section, use of a perturbation agent that induces
ROS at more physiologically relevant levels would be helpful to further understanding
the effect of chronic ROS on TRPC channel regulation and support the findings in this
study. Although inhibitors of mitochondrial complexes may be more suitable for long-
term studies compared to short lived species such as H2O2, these compounds are often
toxic and induce cell death in cell cultures. Additionally, testing a greater range of drug
concentrations could yield further insight into any concentration-dependent alterations in
expression and function. For instance, the findings in this study show a dose-dependent
reduction in TRPC channel protein expression and function. Similarly, expanding the
range of treatment duration could result in a better understanding of time-dependent
drug effects although it is limited by the toxic effects of ROS generation. Indeed, the
significant reductions in TRPC3, TRPC5, and TRPC6 protein levels after chronic but not
acute rotenone treatment support a model of time-dependent responses to oxidative
stress which aligns with the hypothesis that BD is a chronic relapsing illness involving
cellular pathophysiology developed over long periods of time.
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The availability of RNAi procedures have also created an opportunity to generate
transient siRNA-mediated knockdown in cell lines which may compensate for a lack of
TRPC channel specific inhibitors and agonists. For example, transient TRPC6
knockdowns provide a potentially useful strategy to dissect the extent to which TRPC6
contributes to the rotenone induced changes in OAG activated Ca2+ influx. Despite the
flexibility in experimental design provided by RNAi strategies, they do not represent a
panacea and have several limitations including off-target effects, inefficacy, instability,
and may alter the physiological state of cells. However, in light of the lack of suitable
and specific pharmaceutical modulators, knockdown models provide a means to
circumvent this limitation. All together, primary rat cortical neuronal models can still
provide insight into possible pathways, targets of interaction, mechanisms, and impact
on cellular functions of chronic ROS exposure. Subsequently, greater understanding of
intricate cellular process may be useful in dissecting the underlying process in BD
pathophysiology.
In addition to primary rat cortical neurons, other potential human cellular models
can be explored within an oxidative stress environment. In the past, the non-invasive
procedures and relative ease in obtaining peripheral blood cells, and subsequent
transformed cell lines such as BLCLs, from patients and healthy controls made these
models preferable to post-mortem tissue. The obvious advantage to using BLCLs is the
ability to compare cells reporting aspects of the disease phenotype to cells from healthy
controls and further stratification of BD lines based on treatment response or various
cellular measurements can reduce the inherent disease heterogeneity. With huge
developments in the field of stem cell research, differentiated human neuronal lineages
such as olfactory-derived neural epithelial cells (ONes) or induced pluripotent stem cells
118
(iPS) are prospective models that may possess the greatest translational value due to
their species and cell-type relevance. Examining the expression profile and function of
TRPC channels after chronic oxidative stress in these models may provide further
insight into the mechanisms on the regulation of these channels by ROS in a cell and
disease relevant context. As suggested above, this can be achieved through the single
candidate approach, like in this study, or via high-throughput methodologies.
Beyond the expression and functional changes presented, a logical next step
would be to examine the mechanisms underlying regulation of TRPC gene expression,
especially TRPC3 and TRPC5 whose mRNA levels were found to be modulated by
ROS exposure in this study. One approach to this would be analyzing regions within
TRPC promoters for potential binding sites of known transcriptional factors that may be
responsive to ROS such as NF-κB and intracellular Ca2+ concentration including NFAT
in silico. Follow up assays utilizing partial promoter deletions, transfecting neurons with
promoter-reporter constructs and/or ChIP may confirm these results. A greater
understanding of these signal transduction cascades may shed light on the mechanisms
underlying the changes observed.
The question of the relationship between regulation of TRPC function and
expression should also be explored. For instance, is the reduced response to OAG after
chronic rotenone treatment a result of the decrease in TRPC3 and TRPC6 protein levels
or are the two alterations independent? Indeed, the findings that long-term oxidative
stress exposure modulates TRPC3 mRNA levels but not TRPC5 and TRPC6 levels may
imply that ROS-mediated regulation occurs at several levels and/or is TRPC subtype
dependent. The importance of de novo protein synthesis in contribution to the OAG
induced response can be assessed by inhibiting global protein synthesis. Protein
119
turnover is also another process that can be studied to understand the mechanism of
ROS regulation.
Finally, the idea of neuroprotective properties of TRPC3 and TRPC6 could be
further addressed in subsequent studies. As described earlier, TRPC knockdown and
overexpression systems can be a model to evaluate these properties. For instance,
comparing cell viability after oxidative stress among cell lines with a specific TRPC
subtype knockdown or overexpressed to control cell lines would be a simple way to
determine if TRPC channels impact cell viability. Concurrently, functional and
expression assays such as Ca2+ influx and immunoblotting can confirm the genetically
engineered state of these cell lines. However, these results must be taken into account
with limitations of RNAi approach such as possible alterations in the physiological state
of the cell.
120
4.6 Conclusions
The results of this study provide some novel insights into the regulation by chronic
oxidative stress of several TRPC channels thought to be involved in Ca2+
dyshomeostasis implicated in the pathophysiology of BD. It was found that chronic
rotenone treatment of primary rat cortical neurons resulted in differential regulation of
TRPC3, TRPC5 and TRPC6 at the mRNA level while significant dose-dependent
reductions in the protein levels were seen for all three subtypes. The observation of
downregulation of TRPC3 and TRPC6 protein levels following chronic but not acute
oxidative stress implies that a sequence of molecular events, possible adaptive to
increased ROS production, are activated from the transcriptional level that then reduces
expression in the case of TRPC3 or activates the expression of proteins involved in
protein degradation like in TRPC6. The reduction of TRPC3 and TRPC6 protein levels
was confirmed to impact the function of these channels when the functionality of at least
TRPC3 was found to be significantly reduced in a similar dose-dependent manner
following chronic rotenone treatment. Overall, these findings highlight a role of oxidative
stress on the regulation and/or function of these channels and that this regulation is
dependent on the duration of exposure to oxidative stressors. Finally, it was found that
a therapeutically relevant concentration of lithium was able to partially and selectively
rescue the rotenone-induced reductions in TRPC3 protein levels after chronic exposure
by lithium treatment. Whether this contributes to the therapeutic effect of lithium or is
simply an epiphenomenon side effect of lithium is largely unknown. Despite this, the
effect of lithium on TRPC3 protein levels following chronic rotenone treatment is a novel
121
finding and adds to the breadth of cellular actions of lithium and may be relevant to its
spectrum of action in the treatment of mood disorders.
In summary, the findings of this work support an important mechanistic link
between abnormal oxidative stress in neurons, these TRPC channels and the disruption
of Ca2+ homeostasis implicated in the pathophysiology of BD.
122
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