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T HE E XPRESSION AND R OLE OF
S PECIFIC TRP C HANNELS IN D OPAMINERGIC N EURONS OF THE S UBSTANTIA N IGRA
A thesis submitted in partial fulfilment of the requirements
Kenny Kwok Hin CHUNG
A BSTRACT
Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease, and
although can be treated symptomatically, it remains incurable. The main pathological feature of this disease is the death of dopaminergic neurons in the substantia nigra pars compacta (SNc).
It is the loss of dopamine release that causes the debilitating motor symptoms experienced by PD patients. Understanding the function of SNc neurons during normal and diseased state is critical for the development of effective treatments and preventative measures.
This study focused on specific TRP (transient receptor potential) channels that may influence the function of SNc neurons. Members of the TRP channel superfamily are highly diverse in terms of activation modality, gating mechanism and expression pattern. Although many of these channels are expressed in the brain, their pathophysiological role in neurons, particularly in SNc neurons, is largely unknown. Review of channel properties led to the hypothesis that a number of TRP channels are functionally expressed in SNc neurons. Activation of TRPM2 and TRPM7 channels can lead to cell death; TRPM2 is sensitive to oxidative stress whereas TRPM7 is regulated by intracellular ATP and/or magnesium. TRPV3 and TRPV4 are temperature‐gated channels; however, their expression in the brain, where temperature does not fluctuate
significantly, suggests they have an additional role unrelated to temperature sensing. The aim of this study was to assess the expression and functional significance of these four channels (TRPM2, TRPM7, TRPV3 and TRPV4) in SNc neurons and astrocytes.
A combination of molecular, electrophysiological and ion imaging techniques was used in acute brain slices obtained from rats. Evidence of mRNA expression in the SNc was obtained for all four TRP channels using reverse transcription‐polymerase chain reaction. Immunolabelling
revealed TRPM2 protein expression in SNc neurons, whereas TRPV4 was detected in astrocytes. The protein expression of TRPM7 and TRPV3 was not tested.
Intracellular application of ADPR, an activator of TRPM2 channels, evoked a dose‐dependent inward current and [Ca2+]i rise in SNc neurons. The response was inhibited by non‐selective TRPM2 channel blockers clotrimazole and flufenamic acid. Bath application of H2O2 evoked a complex response that was mediated in part by KATP channels. TRPM2 channels were also activated, as demonstrated by partial inhibition of the response with clotrimazole and N‐(p‐
amylcinnamoyl)anthranilic acid (ACA). The response was also shown to be dependent on [Ca2+]o and resting [Ca2+]i, providing additional evidence for the activation of these channels at the plasma membrane.
SNc neurons demonstrated strong sensitivity to temperature changes (24–39 °C): warming induced an increase in firing frequency, [Ca2+]i and an inward whole‐cell current. These responses were comparable to those observed by others with activation of TRPV3 or TRPV4 channels in heterologous expression systems. Although the current response was partially inhibited by ruthenium red, a non‐selective blocker of TRP channels, the lack of selective antagonists hindered identification of the specific channels activated. SNc astrocytes displayed similar temperature sensitivity, but were unresponsive to hypoosmolarity. TRPV4 channels expressed in heterologous systems are typically responsive to changes in both temperature and osmolarity.
Data obtained in this study provide novel information on the expression and role of the four TRP channels in the SNc of the rat, and form a basis for further investigations into the role of these channels in the pathophysiological processes leading to PD.
D EDICATION
This thesis is dedicated to my parents for their unconditional love, support and understanding.
They taught me the value of patience and persistence, and to strive for the best. I thank them for shaping me into the person I am today.
A CKNOWLEDGEMENTS
First and foremost I need to thank my supervisor, Prof. Janusz Lipski, who introduced me to the field of scientific research. He generously shared his wisdom and experience, and provided guidance and support throughout the course of this study.
I also need to thank a number of laboratory members. I am particularly grateful to Dr John Lin and Tharu Bowala, who readily offered advice and assistance when I first started. I also need to thank Dr Dennis de Castro, Dr Ji‐Zhong Bai and Dr Michael Grammer for their advice and help. I need to thank our technicians, Dong Li and Kritika Gopal, who ensured smooth day‐to‐day running of the laboratory.
I need to especially thank Dr. Peter Freestone, who was also a doctoral student in the
laboratory. We shared five years working together, and I greatly appreciate his friendship, and his help and support as a research peer. I also need to thank Lin‐Chien Huang for her friendship and help.
I would also like to acknowledge the students who have passed though the laboratory during my course of study, for their companionship: Joanne Davidson, Carthur Wan, Nirubhana Arunthavasothy, Alexander Trevarton, Stanley Lee, Thomas Park, Nishani Dayaratne and Rashi Karunasinghe.
To our newest laboratory members, James McKearney and Andrew Yee, I wish them the best of luck with their projects.
T ABLE OF C ONTENTS
Abstract ... ii
Dedication ... iv
Acknowledgements ... v
Table of Contents ... vi
List of Figures ... x
List of Tables ... xii
List of Abbreviations ... xiii
1.
Introduction ... 1
1.1. Dopaminergic neurons of the substantia nigra ... 1
1.1.1. Dopamine as a neurotransmitter in the mesencephalon ... 1
1.1.2. Physiological role of the substantia nigra pars compacta ... 2
1.1.3. Properties of nigral dopaminergic neurons ... 5
1.2. Parkinson’s disease ... 8
1.2.1. Clinical symptoms and treatments ... 8
1.2.2. Pathology ... 9
1.2.3. Aetiology and pathogenesis ... 10
1.2.3.1. Aging ... 10
1.2.3.2. Genetic factors ... 11
1.2.3.3. Environmental factors ... 12
1.2.3.4. Proteolytic stress ... 13
1.2.3.5. Oxidative stress ... 14
1.2.3.6. Calcium dyshomeostasis ... 16
1.2.3.7. Neurotoxins ... 17
1.2.3.8. Mitochondrial dysfunction ... 18
1.2.3.9. Activity‐dependent survival and KATP channels ... 19
1.3. TRP channels – Significance for neuronal cell death ... 21
1.3.1. General properties and classification of TRP channels ... 21
1.3.2. Cellular stress‐sensitive TRP channels ... 24
1.3.2.1. TRPM2 ... 24
1.3.2.2. TRPM7 ... 28
1.3.3. Temperature‐sensitive TRP channels ... 30
1.3.3.1. TRPV3 ... 31
1.3.3.2. TRPV4 ... 33
1.4. TRP channels in the nigrostriatal system ... 36
2.
Aims of the Study ... 38
3.
Methods ... 40
3.1. Brain tissue preparation ... 40
3.1.1. Fresh slices ... 40
3.1.2. Fixed sections ... 41
3.2. Analysis of mRNA expression ... 42
3.2.1. RNA extraction ... 42
3.2.2. Reverse transcription‐polymerase chain reaction (RT‐PCR) ... 42
3.2.3. Immunohistochemistry ... 46
3.3. Electrophysiology ... 49
3.3.1. Single‐unit extracellular recording ... 49
3.3.2. Whole‐cell patch‐clamp recording ... 49
3.4. Calcium imaging ... 51
3.5. Changes of temperature ... 55
3.6. Drug application ... 56
3.7. Solutions ... 57
3.7.1. Artificial cerebral spinal fluid (ACSF) ... 57
3.7.2. Extracellular microelectrode solution ... 58
3.7.3. K+‐based patch pipette solution ... 58
3.7.4. Cs+‐based patch pipette solution ... 59
3.8. Data analysis ... 61
4.
Results ... 62
4.1. Identification of dopaminergic neurons in the SNc ... 62
4.2. mRNA and protein expression of specific TRP channels in the SN ... 69
4.2.1. mRNA expression of specific TRP channels ... 69
4.2.2. Protein expression of TRPM2 and TRPV4 channels ... 71
4.3. Functional expression of specific TRP channels in SNc neurons ... 76
4.3.1. Effects of adenosine diphosphate ribose (ADPR) ... 76
4.3.2. Effects of oxidative stress evoked by application of H2O2 ... 79
4.3.2.1. Extracellular recording ... 79
4.3.2.2. Whole‐cell patch‐clamp recording ... 81
4.3.3. Effects of endogenous H2O2 ... 95
4.3.4. Effects of temperature ... 100
4.3.4.1. Extracellular recording ... 100
4.3.4.2. Whole‐cell patch‐clamp recording ... 101
4.4. Functional expression of TRPV4 channels in SNc astrocytes ... 106
5.
Discussion ... 108
5.1. mRNA and protein expression of specific TRP channels in the SN ... 112
5.1.1. TRPM2 ... 113
5.1.2. TRPM7 ... 115
5.1.3. TRPV3 ... 115
5.1.4. TRPV4 ... 115
5.2. Functional expression of specific TRP channels in SNc neurons ... 117
5.2.1. Functional expression of TRPM2 channels ... 117
5.2.1.1. Response to ADPR ... 117
5.2.1.2. Response to application of H2O2 ... 119
5.2.1.3. Response to endogenous H2O2 ... 124
5.2.1.4. Comparison between ADPR‐ and H2O2‐evoked responses ... 125
5.2.2. Functional expression of temperature‐sensitive TRP channels ... 128
5.3. Functional expression of TRPV4 channels in SNc astrocytes ... 131
6.
Conclusions ... 133
7.
Future Directions ... 135
8.
List of References ... 135
L IST OF F IGURES
Figure 1. Motor control circuitry of the basal ganglia. ... 4
Figure 2. Phylogenetic tree of the TRP protein superfamily ... 23
Figure 3. TH, TRPM2 and TRPM7 mRNAs and primers. ... 44
Figure 4. TRPV3 and TRPV4 mRNAs and primers. ... 45
Figure 5. Calcium calibration curve for fura‐2. ... 54
Figure 6. SNc region in rat midbrain sections in the coronal plane. ... 65
Figure 7. SNc dopaminergic neurons in whole‐cell patch‐clamp recording. ... 67
Figure 8. SNc dopaminergic neurons in extracellular recording. ... 68
Figure 9. mRNA expression of specific TRP channels in the SN. ... 70
Figure 10. TH, TRPV4 and GFAP immunoreactivity in the SNc region. ... 74
Figure 11. TH and TRPM2 immunoreactivity in the SNc region and the striatum. ... 75
Figure 12. Effects of intracellular loading with ADPR. ... 77
Figure 13. Effects of CLT and FFA on ADPR‐evoked responses ... 78
Figure 14. Changes in firing frequency in response to H2O2 application... 80
Figure 15. Changes in whole‐cell current and [Ca2+]i in response to H2O2 application... 83
Figure 16. Activation of KATP channels in response to H2O2 application. ... 84
Figure 17. Effects of intracellular caesium substitution on H2O2‐induced responses. ... 85
Figure 18. Dependence of H2O2‐induced responses on extracellular calcium. ... 87
Figure 19. Dependence of H2O2‐induced responses on intracellular calcium. ... 88
Figure 20. Effects of CLT on H2O2‐induced responses. ... 91
Figure 21. Effects of CLT and ACA pre‐incubation on H2O2‐induced responses. ... 92
Figure 22. Effects of 2‐APB and inhibition of ionotropic glutamatergic receptors on H2O2‐ induced responses. ... 93
Figure 23. Effects of DPQ on H2O2‐induced responses. ... 94
Figure 24. Effects of MCS and exogenous SOD... 97
Figure 25. Effects of ATZ and MCS. ... 98
Figure 26. Effects of CLT on ATZ‐evoked whole‐cell current. ... 99
Figure 27. Changes in firing frequency in response to temperature stimuli. ... 103
Figure 28. Changes in whole‐cell current in response to temperature stimuli. ... 104
Figure 29. Changes in whole‐cell current and [Ca2+]i in response to temperature stimuli. ... 105
Figure 30. Responses of SNc astrocytes to warming and to hypoosmotic ACSF... 107
Figure 31. Cellular mechanisms considered in the current study. ... 109
Figure 32. Enzymatic pathways involved in the production and degradation of H2O2. ... 127
L IST OF T ABLES
Table 1. Primary antibodies used for immunolabelling. ... 48
Table 2. Secondary antibodies used for immunolabelling. ... 48
Table 3. Composition of the standard ACSF. ... 58
Table 4. Composition of the extracellular microelectrode solution. ... 58
Table 5. Composition of the K‐methanesulfonate based patch pipette solution. ... 59
Table 6. Composition of the Cs‐methanesulfonate based patch pipette solution. ... 60
L IST OF A BBREVIATIONS
2‐APB 2‐aminoethoxydiphenyl borate
4α‐PDD 4α‐phorbol 12,13 didecanoate
5,6‐EET 5’,6’‐epoxyeicosatrienoic acid
6‐OHDA 6‐hydroxydopamine
Aβ amyloid β‐peptide
ACA N‐(p‐amylcinnamoyl)anthranilic acid
ADHD attention‐deficient hyperactivity disorder
ADP adenosine diphosphate
ADPR adenosine diphosphate ribose
ALS‐G Guamanian amyotrophic lateral sclerosis
AMP adenosine monophosphate
AP5 2‐amino‐5‐phosphonopentanoic acid
ATP adenosine triphosphate
ATZ 3‐amino‐1,2,4‐triazole
BAA bisandrographolide A
β‐NAD+ β‐nicotinamide adenine dinucleotide
[Ca2+]i intracellular calcium concentration
cADPR cyclic adenosine diphosphate ribose
CDS coding sequence
CLT clotrimazole
CNQX 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione
CNS central nervous system
COMT catechol‐O‐methyltransferase
DA dopamine
DAB 3,3'‐diaminobenzidine
DAT dopamine transporter
DLB dementia with Lewy bodies
DNA deoxyribonucleic acid
DPQ 3,4‐Dihydro‐5‐[4‐(1‐piperidinyl)butoxyl]‐1(2H)‐isoquinolinone
ER endoplasmic reticulum
ETC electron transport chain
FFA flufenamic acid
GABA γ‐aminobutyric acid
GFAP glial fibrillary acidic protein
GIRK G protein‐coupled inwardly‐rectifying potassium
GPe external globus pallidus
GPi internal globus pallidus
GPx reduced glutathione
GSSG oxidised glutathione
HCN channels hyperpolarisation‐activated cyclic nucleotide gated channels
HEK cell human embryonic kidney 293 cell
IR‐DIC infrared‐differential interference contrast
IS‐SD initial segment‐somatodendritic area
LB Lewy body
L‐DOPA L‐3,4‐dihydroxyphenylalanine
MagNuM magnesium‐nucleotide‐regulated metal ion currents
MAO‐B monoamine oxidase B
MCS mercaptosuccinate
MIC magnesium‐inhibited current
MPTP 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine
NAC non‐Aβ component
NCBI National Center for Biotechnology Information
NGF nerve growth factor
NSAID non‐steroidal anti‐inflammatory drugs
OGD oxygen glucose deprivation
PARG poly ADPR glycohydrolase
PARP poly ADPR polymerase
PD Parkinson’s disease
PD‐G Guamanian Parkinson‐dementia
PPN pedunculopontine nucleus
RNS reactive nitrogen species
ROS reactive oxygen species
RR ruthenium red
RRF retrorubral field
RT‐PCR reverse‐transcription‐polymerase chain reaction
SN substantia nigra
SNc substantia nigra pars compacta
SNr substantia nigra pars reticulata
SOD superoxide dismutase
SR101 sulforhodamine 101
STN subthalamic nucleus
TH tyrosine hydroxylase
TNF‐α tumor necrosis factor‐α
TRP transient receptor potential
UPS ubiquitin proteasome system
VTA ventral tegmental area
