Differential Proteolysis of Mutant Huntingtin in Astrocytes and Neurons Open Access
Zhao, Ting (2017)
Published
Abstract
Huntington's disease (HD) is a neurodegenerative disorder caused by the expression of misfolded mutant huntingtin protein (mHtt). HD involves the preferential accumulation of mHtt in neuronal cells and selectively kills neurons. The mechanisms underlying the selective neurodegeneration in HD remain unknown. My study addresses two issues: 1) why mHtt preferentially aggregates in neurites; 2) why astrocytes are more resistant than neurons to mHtt and other misfolded proteins. Given that mHtt aggregates in neurites are prevalent in the HD brains, I hypothesize that the degradation of mHtt is inefficient in neurites. To test this hypothesis, I combined the "optical-pulse chase assay" with brain slice and primary culture models. I found that mHtt is slowly cleared in neurites compared with neuronal cell bodies, indicating compartment-dependent degradation of mHtt in neurons. In contrast, mHtt is rapidly degraded in both the cell bodies and processes of astrocytes.
My live-cell imaging results also show that mHtt is cleared faster by astrocytes than by neurons, which explains a well-known phenomenon that astrocytes are affected to a much lesser extent than neurons in the HD brains. Importantly, I found that C-terminus of Hsp70 interacting protein (CHIP), a co-chaperone of Hsp70, is more active in astrocytes than in neurons, which is evidenced by increased mono-ubiquitinated CHIP in astrocytes. CHIP binds to mHtt selectively in astrocytes, promoting ubiquitination and proteasomal degradation of mHtt. Active CHIP accelerates degradation of various misfolded proteins, in addition to mHtt, in astrocytes. It is known that the heat-shock response is induced in astrocytes, but not in neurons, however the mechanism causing this differential response is poorly understood. I found that differential CHIP activity in astrocytes and neurons leads to a distinct heat-shock response. Moreover, active CHIP also increases basal levels of Hsp70 in astrocytes, which in turn promotes the association of CHIP with mHtt. Furthermore, I found that deficiency of HspBP1 in astrocytes accounts for the high activity of CHIP, and overexpression of HspBP1 inhibits CHIP activation and causes the accumulation of mHtt in astrocytes. Finally, knocking down HspBP1 in neurons with CRISPR/Cas9 in a HD knock-in mouse model reduced mHtt aggregates and ameliorated neuropathology. Collectively, my results provide an explanation for the higher resistance to deleterious effects of misfolded proteins in astrocytes compared to neurons in neurodegenerative diseases, and suggest that HspBP1 is a potential therapeutic target for HD treatment.
Table of Contents
Chapter 1: General Introduction
1.1 Huntington's disease 2
1.2 Proteolysis of mutant huntingtin 4
1.3 Aggregation of mutant huntingtin in the neuropil 7
1.4 Selective neurodegeneration in Huntington's disease 10
1.5 "Optical-pulse chase" assay 11
1.6 Roles of C-terminus of Hsp70-interacting protein in clearance of misfolded proteins 13
1.7 Dissertation goals 15
Chapter 2: Degradation of mHtt is compartment- and cell-type-dependent
2.1 Abstract 17
2.2 Introduction 18
2.3 Results 19
2.4 Discussion 25
2.5 Materials and Methods 28
Chapter 3: Differential HspBP1 expression accounts for the greater vulnerability of neurons than astrocytes to misfolded proteins
3.1 Abstract 51
3.2 Introduction 51
3.3 Results 53
3.4 Discussion 61
3.5 Materials and Methods 63
Chapter 4: General conclusions and future directions
4.1 General conclusions 98
4.2 Future directions 101
References 103
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