Neurochemical, neuroprotective, and behavioral effects of enhanced vesicular storage of dopamine 公开
Lohr, Kelly (2015)
Abstract
The vesicular monoamine transporter 2 (VMAT2; SLC18A2) is responsible for both packaging monoamine neurotransmitters and for sequestering neurotoxic species in the cytosol, thereby protecting neurons. Recently, reduced vesicular filling via decreased VMAT2 function has been demonstrated in Parkinson's disease brains. These findings, along with work from mouse models of reduced VMAT2 levels, suggest that increasing vesicular filling may be beneficial to the health of the dopamine system. To assess this, we generated a mouse model of increased vesicular function via bacterial artificial chromosome-mediated overexpression of VMAT2. VMAT2-overexpressing mice show an enhanced vesicular capacity for dopamine storage (56% increase), dopamine vesicle volume (33%), and total striatal dopamine content (21%). This elevated vesicular capacity also leads to increased stimulated dopamine release (84%) and extracellular dopamine levels (44%) in the striatum. Additionally, the VMAT2-HI mice exhibit altered dopamine handling following treatment with L-DOPA, the neurochemical precursor to dopamine. These findings suggest that elevated vesicular storage may allow more efficient use of L- DOPA, a common Parkinson's disease therapeutic. Perhaps most importantly, it also appears that VMAT2 overexpression does not cause negative behavioral side effects. VMAT2-overexpressing mice show improved outcomes on anxiety and depressive-like behaviors and only a mild increase in locomotor activity during their active period (41%). Finally, we examined whether elevated VMAT2 levels altered the vulnerability of the nigrostriatal pathway to toxicant exposure. The VMAT2-HI mice show significant protection from the dopaminergic toxicants 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine in the nigrostriatal pathway. Together, the VMAT2- overexpressing mice show that enhanced vesicular capacity is indeed possible and that these changes are capable of augmenting dopamine output without compensation in the rest of the dopamine system. The increased dopamine release, improved L-DOPA efficacy, and neuroprotection in the VMAT2-overexpressing mice suggest that interventions aimed at enhancing vesicular capacity may be of therapeutic benefit in disorders of reduced dopamine like Parkinson's disease.
Table of Contents
CHAPTER I: INTRODUCTION 1
DOPAMINE NEUROMODULATION 2
SYNAPTIC VESICLE FUNCTION 3
VESICULAR NEUROTRANSMITTER FILLING 4
NEUROTOXIC MECHANISMS OF CYTOSOLIC DOPAMINE 5
VMAT2 AS A NEUROPROTECTIVE MECHANISM 7
PHARMACOLOGICAL MANIPULATIONS OF VESICULAR FILLING 7
PRESYNAPTIC MACHINERY AND PARKINSON'S DISEASE 9
PHENOTYPES OF VMAT2-DEFICIENT MICE 13
RESEARCH OVERVIEW: VMAT2-OVEREXPRESSING MICE 14
FIGURE 1.1. THE VESICULAR MONOAMINE TRANSPORTER 2 (VMAT2; SLC18A2).
17
FIGURE 1.2. MODEL OF ALTERED VESICULAR FUNCTION. 19
FIGURE 1.3. THE VESICULAR FUNCTION CONTINUUM. 21
CHAPTER II: BAC-MEDIATED VMAT2 OVEREXPRESSION INCREASES VMAT2
PROTEIN CONTENT AND DOPAMINE OUTPUT IN THE VMAT2-HI MICE 22
INTRODUCTION 23
Limitations to vesicular filling 23
BAC transgenic overexpression of VMAT2 25
METHODS 27
Generation of Transgenic Mice 27
Animals 27
Western blotting 27
Immunohistochemistry 28
Stereological Analysis 28
HPLC determination of catecholamines and metabolites 28
Fast-scan cyclic voltammetry in striatal slice 29
Statistical analysis 29
RESULTS 30
VMAT2 overexpression in VMAT2-HI mice 30
Increased vesicular capacity for dopamine increases striatal
dopamine content in VMAT2-HI mice 31
Increased synaptic dopamine release in VMAT2-HI mice 31
DISCUSSION 32
VMAT2-HI mice reveal an unexpected enhancement of vesicular storage
capacity 32
VMAT2 overexpression increases dopamine release and
neurotransmission without compensatory changes to other presynaptic
functions 32
VMAT2-HI phenotypes contrast with vesicular transporter
overexpression in nonmonoamine transmitter systems 33
FIGURE 2.1. THE BACTERIAL ARTIFICIAL CHROMOSOME. 36
FIGURE 2.2. INCREASED VMAT2 EXPRESSION IN THE VMAT2-HI MICE.
38
FIGURE 2.3. VMAT2 OVEREXPRESSION OCCURS IN ALL MONOAMINERGIC BRAIN
REGIONS IN THE VMAT2-HI MICE. 40
FIGURE 2.4. DAT AND TH LEVELS ARE UNCHANGED IN THE VMAT2-HI MICE.
42
FIGURE 2.5. INCREASED DOPAMINE LEVELS IN THE STRIATUM OF VMAT2-HI
MICE. 44
FIGURE 2.6. INCREASED STIMULATED DOPAMINE RELEASE IN VMAT2-HI MICE.
46
CHAPTER III: VMAT2-OVEREXPRESSING (VMAT2-HI) MICE SHOW ALTERED
OUTCOMES IN MONOAMINE-MEDIATED BEHAVIORS 47
INTRODUCTION 48
METHODS 51
Animals 51
Circadian locomotor activity 51
Amphetamine-stimulated locomotor activity 51
Forced-Swim Test 51
Marble-Burying Assay 52
Tail suspension test 52
Sleep latency 52
Sucrose preference 53
Olfactory discrimination (social odor) 53
Dot test (tactile stimulation) 54
Quinine taste aversion 54
Statistical analysis 55
RESULTS 56
Increased locomotor activity in VMAT2-HI mice. 56
Enhanced amphetamine-stimulated locomotor activity in VMAT2-HI
mice. 56
Reduced anxiety-like and depressive-like behaviors in VMAT2-HI
mice. 56
DISCUSSION 57
VMAT2 overexpression increases locomotor activity 57
VMAT2 overexpression improves outcomes in measures of
depressive-like and anxiety-like behaviors 58
FIGURE 3.1. INCREASED LOCOMOTOR ACTIVITY IN THE VMAT2-HI MICE.
61
FIGURE 3.2. VMAT2-HI MICE SHOW A SMALL INCREASE IN
AMPHETAMINE-STIMULATED LOCOMOTOR ACTIVITY. 63
FIGURE 3.3. IMPROVED OUTCOMES ON MEASURES OF DEPRESSIVE- AND
ANXIETY-LIKE BEHAVIORS. 65
FIGURE 3.4. NON-SIGNIFICANT BEHAVIORAL DIFFERENCES BETWEEN THE
VMAT2-HI MICE AND WILDTYPE LITTERMATES. 67
CHAPTER IV: VMAT2-OVEREXPRESSING (VMAT2-HI) MICE ARE PROTECTED FROM
MPTP TOXICANT EXPOSURE 68
INTRODUCTION 69
METHODS 71
Animals 71
MPTP injection schedules 71
Western blotting 71
Immunohistochemistry 71
Stereological Analysis 72
Statistical analysis 72
RESULTS 73
Reduced MPTP neurotoxicity in VMAT2-HI mice following a
terminal-targeting MPTP lesion. 73
Reduced MPTP neurotoxicity in VMAT2-HI mice following a cell
body-targeting MPTP lesion. 73
VMAT2 overexpression protects against MPTP-induced terminal and
cell loss in the substantia nigra. 74
DAT to VMAT2 expression ratio as a determinant of vulnerability to
MPTP in midbrain dopamine pathways. 75
VMAT2 overexpression as a mediator of cytosolic toxic burden in
dopaminergic neurons. 75
FIGURE 4.1. SCHEMATIC OF MPTP TOXICITY IN A DOPAMINERGIC NEURON
TERMINAL. 78
FIGURE 4.2. VMAT2-HI MICE ARE PROTECTED FROM TERMINAL TOXICITY
FOLLOWING 2 X 15 MG/KG MPTP. 80
FIGURE 4.3. REDUCED TOXICITY IN VMAT2-HI MICE FOLLOWING 5 X 20
MG/KG MPTP. 82
FIGURE 4.4. VMAT2-HI MICE ARE SIGNIFICANTLY PROTECTED FROM NIGRAL
CELL LOSS FOLLOWING 5 X 20 MG/KG MPTP. 84
CHAPTER V: VMAT2-OVEREXPRESSING (VMAT2-HI) MICE ARE PROTECTED FROM
METHAMPHETAMINE TOXICANT EXPOSURE 85
INTRODUCTION 86
METHODS 89
Animals 89
Methamphetamine injection schedule 89
Core body temperature 89
Western blotting 89
Immunohistochemistry 90
Isolectin B4 90
Statistical analysis 90
RESULTS 91
Increased VMAT2 protects against gliosis in the striatum. 91
Preferential targeting of the striosomes following METH treatment.
92
Increased VMAT2 level does not alter the hyperthermic response
following METH treatment. 92
Elevated VMAT2 is also neuroprotective at a lower METH dose.
93
DISCUSSION 94
Elevated VMAT2 protects against METH toxicity. 94
Increased VMAT2 reduces the neuroinflammatory response to METH.
95
Elevated VMAT2 does not change the hyperthermic response to METH.
95
Elevated VMAT2 does not increase the rewarding potential for METH.
96
Potential mechanisms for METH neuroprotection in VMAT2-HI mice.
98
VMAT2 inhibitors as a treatment for addiction. 99
Neuroprotection via elevated VMAT2 function. 100
FIGURE 5.1 SCHEMATIC FOR METHAMPHETAMINE TOXICITY IN A DOPAMINERGIC
TERMINAL. 102
FIGURE 5.2. VMAT2-HI MICE ARE SIGNIFICANTLY PROTECTED FROM METH
TOXICITY. 104
FIGURE 5.3. HIGHER MAGNIFICATION OF TH-POSITIVE TERMINALS IN
METH-TREATED MICE OF BOTH GENOTYPES. 106
FIGURE 5.4. VMAT2-HI MICE ARE PROTECTED FROM THE INFLAMMATORY
CASCADE FOLLOWING METH TREATMENT. 108
FIGURE 5.5. PREFERENTIAL LOSS OF DOPAMINE MARKERS IN THE STRIOSOMES
OF BOTH GENOTYPES. 110
FIGURE 5.6. INCREASED VMAT2 LEVEL DOES NOT ALTER THE HYPERTHERMIC
RESPONSE FOLLOWING METH TREATMENT. 112
FIGURE 5.7. VMAT2-HI MICE ARE ALSO PROTECTED FROM METHAMPHETAMINE
TOXICITY AT A LOWER DOSE 4 X 5 MG/KG METH. 114
FIGURE 5.8. HYPOTHETICAL INTERACTION OF VMAT2 FUNCTION AND
METHAMPHETAMINE. 116
CHAPTER VI: NEURONAL VULNERABILITY AND LEVODOPA HANDLING IN VMAT2
GENOTYPES 117
INTRODUCTION 118
METHODS 120
VMAT2-HI mice 120
MPTP injection schedules 121
Western blotting 121
Immunohistochemistry 121
Fast-scan cyclic voltammetry in striatal slice 122
L-DOPA fast-scan cyclic voltammetry 122
[3H]-WIN binding 123
Statistical analysis 123
RESULTS 124
Reduced VMAT2 causes loss of dopamine terminal integrity in aged
mice on a C57BL/6 genetic background. 124
VMAT2 level modulates MPTP toxicity in mice on a C57BL/6 genetic
background. 124
VMAT2 level modifies stimulated dopamine release following MPTP
treatment. 125
VMAT2 LEVEL MODIFIES STIMULATED DOPAMINE RELEASE FOLLOWING MPTP
TREATMENT. 125
VMAT2 LEVEL MODIFIES L-DOPA INDUCED INCREASES IN DOPAMINE RELEASE.
125
VMAT2 LEVEL MODIFIES MOVEMENT OF EXTRACELLULAR DOPAMINE. 126
No change in total DAT levels in the VMAT2-HI mice. 127
DISCUSSION 128
VMAT2 as a mediator of toxicity 128
VMAT2 as a modifier of L-DOPA handling 129
Implications for L-DOPA-induced dyskinesias 130
Possible mechanisms of for altered extracellular dopamine uptake
131
FIGURE 6.1. C57BL/6 VMAT2-LO MICE HAVE SIGNIFICANTLY REDUCED
VESICULAR UPTAKE. 135
FIGURE 6.2. C57BL/6 VMAT2-LO MICE SHOW PROGRESSIVE LOSS OF THE
DOPAMINE TERMINAL MARKER DAT WHEN AGED. 137
FIGURE 6.3. VMAT2 LEVEL MODIFIES VULNERABILITY TO DOPAMINE TERMINAL
MARKER LOSS FOLLOWING MPTP. 139
FIGURE 6.4. VMAT2 LEVEL MODIFIES VULNERABILITY TO DOPAMINE TERMINAL
MARKER LOSS FOLLOWING MPTP. 141
FIGURE 6.5. FAST-SCAN CYCLIC VOLTAMMETRY EXPERIMENTAL DESIGN.
143
FIGURE 6.6. VMAT2 LEVEL MODIFIES STIMULATED DOPAMINE (1P) RELEASE
WITH AND WITHOUT MPTP LESIONING. 145
FIGURE 6.7. VMAT2 LEVEL ALTERS DOPAMINE DYNAMICS FOLLOWING L-DOPA
APPLICATION IN UNLESIONED AND MPTP-LESIONED MICE. 147
FIGURE 6.9. VMAT2 LEVELS ALTER NEUROCHEMICAL OUTPUT FOLLOWING
L-DOPA APPLICATION IN UNLESIONED MICE EVEN WITH A MORE INTENSE
4-PULSE STIMULATION. 151
FIGURE 6.11. THERE ARE NO CHANGES IN DAT EXPRESSION IN SYNAPTOSOMES
PREPARED FROM WILDTYPE AND VMAT2-HI MICE. 155
FIGURE 6.12. THERE IS NO CHANGE IN SYNAPTOSOMAL DAT BINDING BETWEEN
WILDTYPE AND VMAT2-HI MICE. 157
CHAPTER VII: SUMMARY AND FUTURE DIRECTIONS 158
CONCLUSIONS 159
FUTURE DIRECTIONS 165
To screen for a compound that positively modulates vesicular
function 165
To investigate the ability of VMAT2 levels to rescue PD pathology
in an α-synuclein mouse model 167
To examine the interaction of the vesicle with other proteins of
interest 168
FINAL THOUGHTS 169
APPENDIX 170
1. IN VIVO FAST-SCAN CYCLIC VOLTAMMETRY 171
Introduction 171
Methods 171
Results 171
Discussion 172
Figure 7.1. VMAT2-HI mice show increased stimulated dopamine
release in vivo. 174
2. AMPHETAMINE-INDUCED REDISTRIBUTION OF VMAT2 175
Introduction 175
Methods 175
Results 175
Discussion 176
Figure 7.2. VMAT2-HI mice exhibit similar VMAT2 redistribution
following amphetamine treatment. 178
REFERENCES 179
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