Age-dependent, region-specific mechanisms contribute to Spinocerebellar Ataxia 17 pathogenesis. Open Access

Yang, Su (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/z603qz10f?locale=en
Published

Abstract

Spinocerebellar Ataxia 17 (SCA17), caused by an expansion of polyglutamine (polyQ) tract (> 42 repeats) in the N-terminus of TATA-box binding protein (TBP), belongs to a family of polyQ expansion-mediated neurodegenerative diseases. SCA17 is characterized by specific neurodegeneration in the cerebellum, despite the fact that mutant TBP is globally expressed. Aging is believed to be the primary risk factor for SCA17 and other polyQ expansion diseases, as symptoms associated with such diseases predominantly manifest during middle or late stages of life. However, how aging contributes to neurodegeneration remains unclear. To investigate aging-related molecular alterations and their involvement in SCA17 pathogenesis, we established SCA17 knock-in (KI) mice that inducibly express one copy of mutant TBP at different ages by tamoxifen-mediated Cre recombination. We find that mutant TBP, when expressed in aged mice, leads to accelerated onset of neurodegeneration and neurological symptoms, compared with young mice. Furthermore, the level of accumulated mutant TBP is higher in older SCA17 inducible KI mouse brains than younger ones, and correlates with age-related decline in chaperone activity. Mutant TBP reduces the expression of mesencephalic astrocyte derived neurotrophic factor (MANF), a protein that is enriched in the Purkinje cell layer of the cerebellum. Overexpression of MANF ameliorates mutant TBP-mediated toxicity both in vitro and in vivo. Lastly, we provided evidence that MANF increases protein kinase C (PKC) phosphorylation, which could underlie the neuroprotective effect. Taken together, these findings suggest that the age-related decline in chaperone activity affects polyQ protein functioning that is important for the viability of specific types of neurons.

Table of Contents

TABLE OF CONTENTS

Chapter 1: General Introduction 1

1.1 The polyglutamine diseases 2

1.2 Aging and polyglutamine diseases 4

1.3 Protein homeostasis and polyglutamine diseases 6

1.4 Neurotrophic factors and polyglutamine diseases 10

1.5 Spinocerebellar Ataxia 17 12

1.6 TATA-box binding protein 16

1.7 Summary of aims and results of the study 18

Chapter 2: Aging exacerbates SCA17 neuropathology by decreasing Hsc70 28

2.1 Abstract 29

2.2 Introduction 29

2.3 Materials and Methods 31

2.4 Results 36

2.5 Discussion 42

Chapter 3: Age dependent decrease of MANF contributes to region specific neurodenegeration in SCA17 63

3.1 Abstract 64

3.2 Introduction 64

3.3 Materials and Methods 66

3.4 Results 71

3.5 Discussion 78

Chapter 4: MANF alleviates SCA17 neuropathology through protein kinase C (PKC) signaling 106

4.1 Abstract 107

4.2 Introduction 107

4.3 Materials and Methods 108

4.4 Results 110

4.5 Discussion 112

Chapter 5: Conclusions and Future directions 124

5.1 Summary and conclusions 125

5.2 Remaining questions and future directions 128

References 135

FIGURES AND TABLES

Chapter 1: General Introduction

Table 1-1 The polyglutamine diseases 21

Figure 1-1 Schematic representation of human MANF 22 Figure 1-2 Schematic representation of human TBP 24

Figure 1-3 Previous knowledge about SCA17 pathogenesis and unanswered questions 26

Chapter 2: Aging exacerbates SCA17 neuropathology by decreasing Hsc70

Figure 2-1 Charaterization of mutant TBP expression in TBP105Q inducible knock-in mouse model 45 Figure 2-2 Characterization of body weight loss in TBP105Q inducible knock-in mouse model 47

Figure 2-3 Characterization of motor impairment in TBP105Q inducible knock-in mouse model 49

Figure 2-4 Characterization of Purkinje cell degeneration in TBP105Q inducible knock-in mouse model 51

Figure 2-5 Loss of calbindin in the cerebellum of TBP105Q inducible knock-in mouse model 53

Figure 2-6 Age-related increase of mutant TBP in TBP105Q inducible knock-in mice 55

Figure 2-7 Age-related decrease of wild-type TBP in TBP105Q inducible knock-in mice 57

Figure 2-8 Autophagy activity in differently aged wild-type mouse brain 59

Figure 2-9 Age-dependent decrease in chaperone activity and Hsc70 level in wild-type mouse brain 61

Chapter 3: Age dependent decrease of MANF contributes to region specific neurodenegeration in SCA17

Figure 3-1 Age-dependent decrease of MANF occurs specifically in the cerebellum of TBP105Q inducible knock- in mice 80

Figure 3-2 MANF is enriched in the Purkinje cell layer and the enrichment is lost in TBP105Q inducible knock-in mice

82

Figure 3-3 Reduced MANF mRNA level in the cerebellum of TBP105Q inducible knock-in mice 84

Figure 3-4 Reduced occupancy of XBP1s on MANF promoter in the presence of mutant TBP 86

Figure 3-5 Expanded polyQ impairs the binding between TBP and XBP1s 88

Figure 3-6 Luciferase assay showed that mutant TBP impairs XBP1s mediated MANF transcription 90

Figure 3-7 Hsc70 overexpression rescues MANF reduction in the presence of mutant TBP 92

Figure 3-8 Generation of lentiviral MANF vector 94

Figure 3-9 MANF overexpression by lentiviral infection ameliorates mutant TBP toxicity in vitro 96

Figure 3-10 MANF overexpression by lentiviral infection ameliorates mutant TBP toxicity in vivo 98

Figure 3-11 Generation of MANF transgenic mouse model

100

Figure 3-12 Improvement of behavioral phenotypes in SCA17 KI mice with MANF overexpression 102

Figure 3-13 Improvement of Purkinje cell survival in SCA17 KI mice with MANF overexpression 104

Chapter 4: MANF alleviates SCA17 neuropathology through protein kinase C (PKC) signaling

Figure 4-1 Low levels of activiated caspase-3 in the cerebellum of SCA17 KI and KI/MANF mice 114

Figure 4-2 Purified MANF protein activates PKC signaling in PC12 cell culture 116

Figure 4-3 Reduction of phosphorylated PKC in the cerebellum of SCA17 KI mice 118

Figure 4-4 Inhibition of PKC signaling exacerbated motor impairment in SCA17 KI mice 120

Figure 4-5 Overexpression of PKCγ ameliorated Purkinje cell degeneration in the cerebellum of SCA17 KI mice 122

Chapter 5: Conclusions and Future directions

Figure 5-1 Proposed model for the age-related, region- specific neurodegeneration in SCA17 133

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