Ubiquitin Targeting and Differential Accumulation of Mutant Huntingtin Open Access

Wade, Brandy Elizabeth (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/7h149q66x?locale=en


Huntington's disease (HD) is a rare and fatal neurodegenerative disease caused by expansion of a polyglutamine (polyQ) tract in the N-terminus of the gene encoding the huntingtin (Htt) protein. PolyQ expansion of 37 or more causes neurodegeneration that is particularly severe in the striatum and cortex compared to other brain regions. Expansion of the polyQ tract causes the protein to misfold and selectively accumulate in an age dependent manner. Htt is expressed throughout the entire body; however, it only accumulates in the brain. The mechanism underlying the selective accumulation and toxicity observed in HD is unknown. In this study we observed that in HD knock-in mice mHtt expression is higher in the brain. However, expression of mHtt at the same level via injection of viral vectors results in greater accumulation in the striatum than in the muscle. Development of an in vitro degradation assay revealed that mHtt is more stable in the brain. Striatal and cortex tissues also promote the formation of high molecular weight (HMW) mHtt. Protein stability is tightly linked to ubiquitination, a process requiring the catalytic activity of three enzymes to. Ubiquitination requires an E1, E2 and E3 to activate, and subsequently link an ubiquitin (Ub) moiety to a target protein. Using a pyrazone compound, PYR41, which targets and inactivates the Ubiquitin E1 enzyme (Ube1) we were able to observe an increase in formation of HMW mHtt complexes even in tissues that are relatively unaffected in HD. Importantly, Ube1 protein levels are lower in brain tissues than peripheral tissues and decline in the nucleus with age. This is correlated with the increase accumulation of mHtt that is observed in the brain with aging. These finding suggest that decreased Ub targeting may contribute to differences in stability between tissues which leads to the preferential accumulation of toxic forms of mHtt in the brain. Thus, we have discovered a novel mechanism that contributes to the age related accumulation of mHtt and the selective toxicity that characterizes HD.

Table of Contents

Chapter 1 General Introduction 1

1.1 Poly Glutamine Diseases 2

1.2 Huntington's Disease 4

1.3 Huntingtin Protein and N-Terminal Fragments of Huntingtin Protein 6

1.4 Proteostatic disruption in Huntington's disease 8

1.5 Aggregation of Expanded Poly Glutamine Protein 13

1.6 Hypothesis 15

Table 1.1. PolyQ Diseases 16

Figure 1.1. Huntingtin Protein 18

Figure 1.2. Ubiquitination Cascade 19

Figure 1.3. Autophagy Pathway 21

Figure 1.4. The Proteasome Degradation Pathway 23

Figure 1.5. Huntingtin protein lifecycle 25

Chapter 2 Materials and Methods 27

2.1. Animals 28

2.2 Plasmids and antibodies 28

2.3 Antibodies and Western Blotting 28

2.4 Immunohistochemistry 29

2.5 Subcellular Fractionation 30

2.6 Formic Acid Solubilization 30

2.7 Cell cultures 31

2.8 In vitro degradation assay (IVDA) 32

2. 9 qRT-PCR and RT-PCR 33

2.10 Viral Injection: Stereotaxic and muscle injection 36

2.11 Rotarod assay 36

2.12 Densitometry and Statistical Analysis 37

Chapter 3 Ubiquitin-activating enzyme activity contributes to differential accumulation of mutant huntingtin in brain and peripheral tissues 38

3.1 Abstract 39

3.2 Introduction 40

3.3 Results 42

3.4 Discussion 51

Figure 3.1. Differential levels of mutant huntingtin in brain and peripheral tissues of HD CAG140 KI mice 57

Figure 3.2. Formation of Htt aggregates by N-terminal mHtt fragments in HD KI mouse brain 59

Figure 3.3. In vitro degradation assay of mHtt stability 61

Figure 3.4. Stability and toxicity of N-terminal mHtt fragments in vivo 63

Figure 3.5. In vitro degradation of N-terminal mHtt fragments 65

Figure 3.6. Promotion of the formation of HMW mHtt and accumulation by inhibiting ubiquitin-activation enzyme E1 67

Figure 3.7. Differential levels of Ube1 in brain and peripheral tissues 69

Figure 3.8. A proposed model for the differential accumulation of mHtt in affected brain regions 71

Chapter 4 Conclusions and Future Directions 72

4.1 Summary 73

4.2 Remaining Questions and Future Directions 76

4.3 Conclusions 82

Figure 4.1. A model for the age dependent decline in nuclear localization of Ube1 and its effect on mHtt aggregation 84

References 85

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