Ubiquitin Targeting and Differential Accumulation of Mutant Huntingtin 公开

Wade, Brandy Elizabeth (2014)

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

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

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 injections..................................... 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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