The synaptic vesicle glycoprotein 2C regulates vesicular dopamine transport Restricted; Files Only
Hoffman, Carlie (Fall 2018)
Published
Abstract
Regular packaging of dopamine into synaptic vesicles is required for the survival of dopamine neurons. Ineffective sequestration of dopamine leads to dopaminergic cell death and can contribute to neurodegenerative disease progression. Indeed, dopamine vesicle function is impaired in patients with Parkinson’s disease (PD). Proper vesicle function relies on synaptic vesicle glycoprotein 2 (SV2), which facilitates responsiveness to Ca2+ and transports with synaptotagmin to the synapse. Further, the expression of one isoform in the SV2 family, SV2C, is enriched in dopaminergic brain regions and alterations in the expression of SV2C have been linked to PD. SV2C expression is disrupted in the basal ganglia of PD brain and variations in the human SV2C gene are correlated with responsiveness to levodopa and the protective effect of nicotine on PD risk. In the experiments described herein, I examined how alterations in SV2C expression mediate dopamine vesicle packaging. To this end, I developed a series of fluorescent in vitro assays to visualize vesicle packaging mediated by the dopamine-packaging protein the vesicular monoamine transporter 2 (VMAT2), and to measure changes in vesicle packaging arising from pharmacological, toxicological, and genetic manipulations. I first used a fluorescent plate reader assay to test a representative group of pharmacological compounds and environmental toxicants for their effect on vesicle function and dopamine packaging. I show significant reduction in vesicular storage of a dopamine analog after treatment with tetrabenazine, reserpine, methylphenidate, and methamphetamine, and modest reduction in vesicular storage after treatment with select pesticides and halogenated toxicants. Seeking to determine how SV2C affects dopamine vesicle function, I then used the plate reader assay to determine how SV2C expression affected vesicle packaging in vitro. I demonstrate SV2C expression leads to increased uptake of a dopamine analog. I then overexpressed SV2C in the substantia nigra of C57BL/6 mice and observed enhanced retention of dopamine over time and prolonged presence of dopamine in the synaptic cleft after electrically-stimulated release. These results identify SV2C as a mediator of dopamine vesicle packaging, retention, and release and indicate that SV2C represents an exciting target for further understanding and treating dopaminergic pathologies.
Table of Contents
Chapter 1- Introduction: Rationale, hypothesis, and scope.......................................... 1
Section I: Environmental and genetic contributors to Parkinson’s disease
pathogenesis.................................................................................................................... 2
Environmental exposures and Parkinson’s disease..................................................... 2
Genetic mutations and Parkinson’s disease................................................................ 3
Alpha-synuclein and Parkinson’s disease pathology.................................................. 5
Section II: Vesicle handling and the dopamine system................................................... 6
Synaptic vesicles: From vesicle pools to vesicle recycling......................................... 6
Monoamines, VMAT2, and dopamine handling......................................................... 9
Section III: The synaptic vesicle glycoprotein 2 C, dopamine, and
Parkinson’s disease....................................................................................................... 11
Structure and function of the synaptic vesicle glycoprotein 2 (SV2)....................... 11
The synaptic vesicle glycoprotein 2 C (SV2C)......................................................... 14
Section IV: Rationale and hypothesis............................................................................ 15
Rationale.................................................................................................................... 15
Hypothesis and scope................................................................................................ 15
Chapter 2- Assessing vesicular monoamine transport using false fluorescent neurotransmitters 21
Abstract......................................................................................................................... 22
Introduction................................................................................................................... 23
Materials and Methods.................................................................................................. 25
Results........................................................................................................................... 28
Discussion..................................................................................................................... 33
Chapter 3- The role of SV2C in dopamine vesicle function........................................ 58
Abstract......................................................................................................................... 59
Introduction................................................................................................................... 60
Materials and Methods.................................................................................................. 62
Results........................................................................................................................... 69
Discussion..................................................................................................................... 74
Chapter 4- Discussion, future directions, and concluding remarks......................... 101
Summary of findings.................................................................................................... 102
Working hypothesis of the mechanism of action of SV2C........................................... 103
Discussion of unexpected results................................................................................. 108
Future directions......................................................................................................... 109
Conclusion................................................................................................................... 113
References ..................................................................................................................... 124
Appendix I- SV2C expression mediates alpha-synuclein pathology........................ 150
Abstract....................................................................................................................... 151
Introduction................................................................................................................. 152
Materials and Methods................................................................................................ 154
Results......................................................................................................................... 156
Discussion................................................................................................................... 159
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