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The Contribution of Calcium-Activated Potassium Channel Dysfunction to Altered Purkinje Neuron Membrane Excitability in Spinocerebellar Ataxia

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The Contribution of Calcium-Activated Potassium Channel Dysfunction to Altered Purkinje Neuron Membrane Excitability in Spinocerebellar Ataxia The Contribution of Calcium-Activated Potassium Channel Dysfunction to Altered Purkinje Neuron Membrane Excitability in Spinocerebellar Ataxia by David D. Bushart A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular and Integrative Physiology) in The University of Michigan 2018 Doctoral Committee: Professor Geoffrey G. Murphy, Co-Chair Associate Professor Vikram G. Shakkottai, Co-Chair Professor William T. Dauer Professor W. Michael King Professor Andrew P. Lieberman Professor Malcolm J. Low David D. Bushart [email protected] ORCiD: 0000-0002-3852-127X © David D. Bushart 2018 Acknowledgements I would like to acknowledge three groups which provided me with support and motivation to complete the studies in this dissertation, and for helping me keep my research efforts in perspective. First, I would like to acknowledge my friends and family. Their emotional support, and the time they have invested in supporting my growth as both a person and a scientist, cannot be overstated. I am eternally grateful to have a network of such caring people around me. Second, I would like to acknowledge cerebellar ataxia patients for their perseverance and positive outlook in the face of devastating circumstances. My ability to interact with patients at the National Ataxia Foundation meetings, along with the positive messages that my research efforts were met with, was my greatest motivating factor throughout the final years of my dissertation studies. I encourage other researchers to seek out similar interactions, as they will make for a more invested and focused scientist. Third, I would like to acknowledge the research animals used in the studies of this dissertation. It is without a voice that their sacrifice was made. These studies would have truly been impossible without the use of these animals, and it is essential that we researchers continue to provide research animals with as much compassion and care as possible. While understanding and curing disease is a noble cause, we must keep in mind the sacrifices that must be made in order to find these cures. ii Table of Contents Acknowledgements. .ii List of Figures. vii List of Tables. ix Abstract. .x Chapter 1: Introduction. .1 1.1 Purkinje neuron dysfunction in spinocerebellar ataxia: a common feature of disease 1.1.1 Overview of spinocerebellar ataxia. .1 1.1.2 Unique physiology promotes enhanced cerebellar Purkinje neuron susceptibility in spinocerebellar ataxia. 2 1.1.3 Channelopathies in spinocerebellar ataxia. .4 1.1.4 Ion-channel dysfunction is associated with mouse models of polyglutamine spinocerebellar ataxia. .7 1.2 Calcium-activated potassium channels are important therapeutic targets in spinocerebellar ataxia. .9 1.2.1 Calcium-activated potassium channel expression and function in cerebellar Purkinje neurons. .9 1.2.2 Calcium channel-mutant mice display abnormal Purkinje neuron membrane excitability and motor impairment. 11 1.2.3 Calcium-activated potassium channel modulators improve motor performance in mouse models of polyglutamine spinocerebellar ataxia. 12 1.2.4 Human clinical trials with calcium-activated potassium channel modulators indicate potential efficacy. 14 1.3 Summary and aims of dissertation. .16 Chapter 2: Potassium channel activation improves Purkinje neuron physiology and motor impairment in a mouse model of spinocerebellar ataxia type 1. 20 2.1 Abstract. .20 2.2 Introduction. .21 2.3 Methods. 23 2.3.1 Mice. 23 2.3.2 Patch-clamp electrophysiology. .23 iii 2.3.2.1 Patch-clamp electrophysiology: solutions. .23 2.3.2.2 Patch-clamp electrophysiology: reagents. .24 2.3.2.3 Acute slice preparation for electrophysiological recordings. 25 2.3.2.4 Patch-clamp recordings. 25 2.3.2.5 Analysis of intrinsic dendritic excitability. .26 2.3.3 Phenotype analysis. .26 2.3.4 Water bottle delivery of pharmacologic agents. .28 2.3.5 Mass spectrometry of brain tissue and blood plasma. .28 2.3.5.1 SKA-31 mass spectrometry. 28 2.3.5.2 Baclofen mass spectrometry. .29 2.3.5.3 Chlorzoxazone mass spectrometry. .30 2.3.6 Molecular layer thickness measurements. .31 2.3.7 Review of patient charts. 31 2.3.8 Statistical analysis. .33 2.4 Results. 33 2.4.1 ATXN1[82Q] Purkinje neurons display both and absence of repetitive spiking and dendritic hyperexcitability. .33 2.4.2 Potassium channel-activating compounds restore spiking in non-firing ATXN1[82Q] Purkinje neurons. 34 2.4.3 KCa activators and baclofen enhance the AHP and repolarize the membrane potential of ATXN1[82] Purkinje neurons. 36 2.4.4 Chlorzoxazone and baclofen, but not SKA-31 and baclofen, sustains improvement in motor dysfunction in ATXN1[82Q] mice. 37 2.4.5 KCa activator and baclofen co-administration does not affect dendritic degeneration in ATXN1[82Q] mice. .39 2.4.6 Chlorzoxazone and baclofen reduce dendritic hyperexcitability in ATXN1[82Q] mice by activating subthreshold-activated potassium channels. 40 2.4.7 Chlorzoxazone and baclofen co-administration is tolerated in SCA patients and improves symptoms. .42 2.5 Discussion. 44 2.6 Acknowledgements . .49 Chapter 3: Potassium channel dysfunction and disrupted calcium homeostasis contributes to Purkinje neuron dysfunction in a mouse model of spinocerebellar ataxia type 7. 61 3.1 Abstract. .61 3.2 Introduction. .62 3.3 Methods. 64 3.3.1 Mice. 64 3.3.2 Phenotype analysis: Rotarod. 65 3.3.3 Patch-clamp electrophysiology. .65 3.3.3.1 Patch-clamp electrophysiology: solutions. .66 3.3.3.2 Patch-clamp electrophysiology: reagents. .66 3.3.3.3 Acute slice preparation for electrophysiological recordings. 66 iv 3.3.3.4 Patch-clamp recordings. 67 3.3.3.5 Capacitance measurements. .67 3.3.3.6 Analysis of firing properties. .68 3.3.3.7 Analysis of intrinsic dendritic excitability. .69 3.3.3.8 AHP decay. 69 3.3.4 Transcriptome analysis. .69 3.3.5 Real-time quantitative RT-PCR. .70 3.3.6 Immunohistochemistry. 71 3.3.6.1 Sample preparation. .71 3.3.6.2 Fluorescence intensity measurements. 72 3.3.6.3 Confocal microscopy. .72 3.3.7 Stereotaxic cerebellar delivery of adeno-associated virus. 72 3.3.8 Vestibular phenotype testing. .73 3.3.8.1 Surgical implant of IMU. .73 3.3.8.2 Vestibular testing procedure. .74 3.3.8.3 Vestibular testing: Statistical analysis. .74 3.3.9 Analysis of RNA sequencing and microarray datasets. .74 3.3.10 Statistical analysis. .75 3.4 Results. 75 3.4.1 Purkinje neuron dysfunction begins in the posterior cerebellum of fxSCA7 92Q mice and progresses globally. 75 3.4.2 Purkinje neuron dysfunction is present in the posterior cerebellar lobules, but not anterior cerebellar lobules, of fxSCA7 92Q mice. 76 3.4.3 Dendritic hyperexcitability is present in Purkinje neurons from the posterior cerebellar lobules of fxSCA7 92Q mice. 78 3.4.4 Genes necessary for Purkinje neuron function show reduced expression in fxSCA7 92Q cerebellum. 79 3.4.5 Impaired BK channel function results from decreased calcium availability and contributes to irregular Purkinje neuron spiking in fxSCA7 92Q mice. ..
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