Impaired vesicular storage of norepinephrine and serotonin in Parkinson's disease-related pathogenesis Open Access

Alter, Shawn Patrick (2015)

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The vesicular monoamine transporter 2 is essential for the storage and release of dopamine, norepinephrine, and serotonin. While the etiology of Parkinson's disease remains obscure, it is known that vesicular monoamine function is disrupted in Parkinson's disease, and these neurotransmitter systems degenerate or are disrupted. In addition to the profound motor symptoms of PD, patients also experience a host of nonmotor symptoms, including psychiatric and autonomic disturbances. I hypothesized that disrupting vesicular storage of norepinephrine and serotonin would lead to dysfunction and degeneration of the respective neuronal systems. Using histological, pharmacological, and behavioral analyses, I assessed the integrity of the noradrenergic and serotonergic systems of mice with drastically reduced expression of VMAT2. In addition to nigrostriatal degeneration, VMAT2 deficient mice undergo severe and progressive loss of the norepinephrine-producing cells of the locus ceruleus, as well as reductions in noradrenergic innervation of the brain. We also assessed neurochemical innervation of the hearts of VMAT2 deficient mice and observed drastic reductions in norepinephrine and reduced expression of the norepinephrine transporter, which is consistent with the pathology of orthostatic hypotension in Parkinson's disease. Despite preservation of the dorsal raphe and efferent innervation, VMAT2 deficient mice exhibited neurochemical depletion of serotonin that resulted in aberrant response to serotonergic agonists. Collectively, these data indicate that reduced vesicular monoamine function wreaks havoc on the noradrenergic and serotonergic systems, causing general monoamine depletion, degeneration of the locus ceruleus, and disruptions in serotonergic function, all of which can drive the nonmotor symptoms of Parkinson's disease.

Table of Contents

Chapter 1: Introduction and Background:...1

Synaptic vesicle function...5
Monoaminergic brain regions affected in Parkinson's disease...8
Cytoplasmic monoamine toxicity and VMAT2...11
Pharmacologic blockade of VMAT2...16
Toxicological blockade of VMAT2...17
Genetic variability of SLC18A2 in PD...18
VMAT2 imaging in PD...20

Chapter 2: Reduced vesicular storage of catecholamines causes progressive degeneration in the locus ceruleus...52

Materials and Methods...56

Chapter 3: Reduced vesicular monoamine function drives noradrenergic depletion in the hearts of mice and men...89

Materials and Methods...94

Chapter 4: Reduced vesicular storage disrupts serotonin signaling but does not cause degeneration of serotonin neurons...112

Materials and Methods...118

Chapter 5: Conclusions and Reflections...152

On the staging of LC and SN loss and the relative toxicities of cytosolic DA and NE...154
A challenge to the cytosolic catecholamine hypothesis: the invincible VTA...157
Serotonergic resilience...159
Concluding Remarks...163
APPENDIX: Adaptation of CLARITY: Hydrogel tissue clearing on a shoestring...168

Figure 1-1. Biosynthesis, transport, and catabolism of monoamines...24
Figure 1-2. Neuronal pathways for the biosynthesis and metabolic breakdown of monoamines...27
Figure 1-3. Mechanisms of vesicular disruption in Parkinson's disease...29
Figure 2-1. Immunohistochemical analysis of VMAT2 expression in the substantia nigra and locus ceruleus...70
Figure 2-2. VMAT2 LO mice have reduced brain catecholamines and increased catecholamine turnover at 2 months of age...72
Figure 2-3. VMAT2 LO mice have reduced expression of the norepinephrine transporter...74
Figure 2-4. VMAT2 LO animals undergo progressive catecholaminergic cell loss...76
Figure 2-5. Quantitative analysis of catecholaminergic cell loss reveals degeneration of LC precedes that of SNpc...78
Figure 3-1. Reduced vesicular function disrupts sympathetic norepinephrine homeostasis...102
Figure 3-2. VMAT2 LO mice have impaired uptake and storage of 6-FDA...104
Figure 3-3. VMAT2 LO mice have reduced abundance of cardiac norepinephrine transporter...106
Figure 3-4. Cardiac neurochemistry in PD patients and VMAT2 LO mice...108
Figure 4-1: Preservation of dorsal raphe in aged VMAT2 LO mice...133
Figure 4-2: Serotonergic Innervation is preserved in VMAT2 LO mice...135
Figure 4-3. VMAT2 LO mice have reduced levels and increased catabolism of serotonin and dopamine...137
Figure 4-4. VMAT2 hypomorphy causes reduced synaptic release of serotonin...139
Figure 4-5: VMAT2 LO mice have an ablated hypothermic response to the 5HT1A agonist 8-OH-DPAT...141
Figure 4-6. VMAT2 LO mice have increased sensitivity to the 5-HT2 agonist DOI...143
Figure A-1. Glove box constructed for de-gassing CLARITY hydrogel...190
Figure A-2: Electrophoretic tissue clearing chamber...193
Figure A-3. Clarified hydrogel mouse brain...194
Figure A-4. Imaging chamber for 1 mm slice of clarified tissue...196
Figure A-5. TPH2 immunolabeling of the dorsal and median raphe in hydrogel...199
Figure A-6. TH+ neurons in the arcuate hypothalamic nucleus...201
Figure A-7. IHC of dopamine transporter in clarified FSCV tissue...203

Table A-1. CLARITY hydrogel ingredients...186
Table A-2. CLARITY tissue clearing solution ingreadients...188

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