Muscarinic receptor antagonists as therapeutics for DYT1-TOR1A dystonia Restricted; Files Only

Abstract

Trihexyphenidyl (THP), a nonselective muscarinic receptor antagonist, is the preferred small molecule treatment for DYT1-TOR1A dystonia. A better understanding of the mechanism(s) of action of THP is needed for the development of improved treatments. DYT1-TOR1A dystonia, caused by a mutation in the TOR1A gene (Tor1a(ΔE)), is associated with both abnormal cholinergic and dopaminergic neurotransmission in the striatum. In this thesis, we investigated two distinct but interrelated hypotheses. First, we hypothesized that the reduction in DA release in Tor1a^+/ΔE knockin mice is caused by abnormal function at nerve terminals and/or abnormal receptor-mediated regulation of DA release. Second, we hypothesized that THP normalizes striatal DA release by modulating striatal cholinergic signaling. We generated mice that selectively express mutant Tor1a(ΔE) in either DA neurons or striatal cholinergic interneurons (ChIs). Using ex vivo fast scan cyclic voltammetry, we found that the reduction in DA release is caused by expression of Tor1a(ΔE) specifically in DA neurons but not ChIs, which demonstrates that intrinsic effects of Tor1a(ΔE) on DA neuron function cause the deficit in DA release. Next, we systematically investigated changes in DA terminal function that may mediate the reduced DA release observed in Tor1a^+/ΔE knockin mice. The exact mechanisms mediating the deficit in DA release remain unknown, but it is not the result of changes in terminal excitability, calcium signaling, vesicle recycling, D2 DA autoreceptor function, nicotinic receptor function, or GABAB receptor function. THP normalized the deficit in DA release both ex vivo and in vivo in Tor1a^+/ΔE knockin mice. Using cell-type specific M1 and M4 mAChR knockout mice along with newly developed M1 and M4 subtype-selective mAChR antagonists, we determined that the effect of THP was mediated primarily by M4 mAChR expressed on striatal ChIs. Taken together we have demonstrated that the dopaminergic dysfunction observed in mouse models of DYT1-TOR1A dystonia is caused by intrinsic effects of the Tor1a(ΔE) on DA neurons. Further, we have identified M4 mAChR on striatal ChIs as a key therapeutic target for normalizing DA release in Tor1a^+/ΔE knockin mice and propose that M4 subtype selective mAChR antagonists may be potential therapeutics for DYT1-TOR1A dystonia.

Table of Contents

Table of Contents

Chapter 1. Introduction. 1

DYSTONIA: GENERAL FEATURES. 1

Dystonia. 1

DYT1-TOR1A dystonia. 2

DYT1-TOR1A dystonia and TorsinA function. 3

PATHOMECHANISMS OF DYSTONIA. 6

Anatomy of dystonia. 6

Dopamine and dystonia. 10

STRIATAL DOPAMINE AS A PATHOMECHANISM IN DYT1-TOR1A DYSTONIA. 12

Dopamine: Basic function and regulation of release. 15

Regulation of DA by TH.. 17

Regulation of DA vesicular storage capacity. 18

Regulation of synaptic vesicle fusion. 18

Regulation of DA uptake. 21

Regulation of DA release by presynaptic receptors. 22

DA autoreceptors. 22

Nicotinic acetylcholine receptors. 23

Other presynaptic receptors. 26

MUSCARINIC RECEPTORS AS THERAPEUTIC TARGETS IN DYSTONIA. 27

Current treatments for dystonia. 27

Treatments for DYT1-TOR1A dystonia. 29

Muscarinic Receptors: Basic Pharmacology and pharmaceutical compounds. 30

Localization and function of muscarinic receptors in the periphery. 33

Muscarinic receptors in the CNS: General functional themes. 35

Muscarinic receptor localization and function in hippocampus and neocortex. 38

Cholinergic dysfunction in dystonia. 38

mAChR localization and function in the local striatal circuitry. 39

SUMMARY AND GUIDING QUESTIONS. 47

Chapter 2. Cell-intrinsic effects of the Tor1a(ΔE) mutation attenuate striatal dopamine release in a mouse model of DYT1 dystonia. 49

ABSTRACT. 49

INTRODUCTION.. 50

Animals. 51

Fast scan cyclic voltammetry. 54

Tissue monoamines. 54

Immunohistochemistry. 55

Laser capture microdissection. 56

RNA isolation and sequencing. 57

Image collection. 58

Statistical analysis. 58

Compounds. 58

RESULTS. 59

Conditional expression of Tor1a(ΔE) in striatal ChIs does not affect striatal DA release. 59

Conditional expression of Tor1a(ΔE) in DA neurons causes abnormal DA release. 64

Effects of the Tor1a(ΔE) mutation on the excitability of DA terminals. 68

Sensitivity to extracellular Ca²⁺ concentration in Tor1a^+/ΔE KI mice. 68

Vesicle utilization in Tor1a^+/ΔE mice. 71

D2 DA autoreceptor function in Tor1a^+/ΔE mice. 73

GABAB receptor function in Tor1a^+/ΔE mice. 73

TorsinA localization in DA in neurons in Tor1a^+/ΔE mice. 75

DISCUSSION.. 77

Chapter 3: Trihexyphenidyl rescues the deficit in dopamine neurotransmission in a mouse model of DYT1-TOR1A dystonia. 80

ABSTRACT. 80

INTRODUCTION.. 81

MATERIALS AND METHODS. 82

Animals. 82

Brain Slice Preparation. 82

Fast Scan Cyclic Voltammetry. 83

In Vivo Microdialysis. 83

Monoamine Detection. 84

Acetylcholinesterase Activity Assay. 84

Quantitative real-time PCR.. 85

Compounds. 85

Statistical Analysis. 85

RESULTS. 86

DA release is reduced in Tor1a^+/ΔE mice. 86

Trihexyphenidyl enhances DA release. 88

THP normalizes extracellular DA in Tor1a^+/ΔE mice. 91

Functional nAChRs are required for the THP-induced increase in DA release. 93

The THP-induced increase in DA release is not dependent on glutamatergic signaling. 93

Tor1a^+/ΔE mice are more sensitive to nAChR antagonists. 96

DISCUSSION.. 101

Chapter 4. Blockade of M4 muscarinic receptor on striatal cholinergic interneurons normalizes striatal dopamine release in a mouse model of DYT1-TOR1A dystonia. 104

ABSTRACT. 104

INTRODUCTION.. 105

MATERIALS AND METHODS. 106

Animals. 106

Fluorescent in situ hybridization. 107

Fast san cyclic voltammetry. 108

Compounds. 109

Statistical analysis. 109

RESULTS. 110

M4 mAChRs in striatal cholinergic cells are necessary for the effects of THP. 110

A selective M4 mAChR antagonist is sufficient to recapitulate the effects of THP. 115

Blockade of M4 mAChRs in ChIs but not dSPNs recapitulates the effects of THP. 117

M1 mAChR selective antagonists enhance DA release. 119

M1 mAChRs on dSPNs and iSPNs are required for the effects of THP in Tor1a^+/+ but not Tor1a^+/ΔE KI mice. 121

Knockout of M1 mAChR from either dSPNs or iSPNs alone does not inhibit the effects of the M1 subtype selective mAChR antagonist 125

DISCUSSION.. 128

Chapter 5. Overall discussion and future studies. 132

SUMMARY OF FINDINGS. 132

CONTRIBUTIONS OF THE DYT1-TOR1A MUTATION TO REDUCED DOPAMINE NEUROTRANSMISSION.. 133

STRIATAL DOPAMINE NEUROTRANSMISSION AS A THERAPEUTIC TARGET IN DYT1-TOR1A DYSTONIA. 136

MECHANISMS OF ACTION OF MUSCARINC RECEPTOR ANTAGONISTS IN DYT1-TOR1A DYSTONIA. 138

Literature cited. 143

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Biology, Molecular
Keyword	Dopamine Movement Disorders Dystonia Pharmacology Neurology Acetylcholine
Committee Chair / Thesis Advisor	Ellen Hess, Emory University
Committee Members	Stephen Traynelis, Emory University Yoland Smith, Emory University Hyder Jinnah, Emory University

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