Abstract
Huntington Disease (HD) is caused by an abnormal expansion of polyQ tract in the protein named huntingtin (Htt). HD pathology is featured by accumulation and aggregation of mutant Htt in striatal and cortical neurons. Aberrant Htt degradation is implicated in HD pathogenesis. The aim of this study was to investigate the regulatory role of chaperone-mediated autophagy (CMA) components, heat shock protein cognate 70 (Hsc70) and lysosome-associated protein 2A (LAMP-2A) in degradation of Htt fragment 1-552aa (Htt-552). A cell model of HD was produced by overexpression of Htt-552 with adenovirus. The involvement of CMA components in degradation of Htt-552 was determined with over-expression or silencing of Hsc70 and LAMP-2A. The results confirmed previous reports that both macroautophagy and CMA were involved in degradation of Htt-552. Changing the levels of CMA-related proteins affected the accumulation of Htt-552. The lysosomal binding and luminal transport of Htt-552 was demonstrated by incubation of Htt-552 with isolated lysosomes. Expansion of the polyQ tract in Htt-552 impaired its uptake and degradation by lysosomes. Mutation of putative KFERQ motif in wild-type Htt-552 interfered with interactions between Htt-552 and Hsc70. Endogenous Hsc70 and LAMP-2A interacted with exogenously expressed Htt-552. Modulating the levels of CMA related proteins degraded endogenous full-length Htt. These studies suggest that Hsc70 and LAMP-2A through CMA play a role in the clearance of Htt and suggest a novel strategy to target the degradation of mutant Htt.
Highlights
Abnormal accumulation of misfolded and aggregated proteins in neurons is a hallmark of several neurodegenerative diseases
chaperone-mediated autophagy (CMA) is involved in degradation of Htt containing 552 amino acids (Htt-552) The estimated rate of infection of adenovirus Htt-552 in PC12 or HeLa cells was over 95% (Figure S1 A)
The results showed that ammonium chloride significantly inhibited the degradation of Htt-552
Summary
Abnormal accumulation of misfolded and aggregated proteins in neurons is a hallmark of several neurodegenerative diseases. The mutant proteins of neurodegenerative diseases can cause dysfunction and death of neurons. Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by an abnormal expansion of polyQ tract in the N-terminal huntingtin (Htt). HD is characterized by the accumulation and aggregation of mutant N-terminal Htt proteins in diseased neurons [1]. When the polyQ repeat expands above 35, disease will manifest, typically striking in the late 40 s [2]. Htt552 with an expanded polyQ repeats causes an aggressive HD-like disease in animal and cell models [3]. The chaperone-mediated autophagy (CMA) has been proposed to be involved in degradation of Htt, the molecular process and regulatory mechanisms have not yet been fully characterized
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