Heat shock protein 70 alters the endosome-lysosomal localization of huntingtin

Bong Sun Kang,Lami Kang,Jin-Young Ahn,Jae-Seon Lee,Hun-Chang Lim,Choong Ik Cha,Seung U Kim,Manho Kim,Hyun-Jeong Kim,Jeong-Sun Seo,Min Ky Kim,Yoon Jeong Park,Kyung-Sook Park

doi:10.1038/emm.2007.5

Bong Sun Kang, Lami Kang + Show 11 more

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https://doi.org/10.1038/emm.2007.5

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Abstract

Huntington's disease is caused by CAG trinucleotide expansions in the gene encoding huntingtin. N- terminal fragments of huntingtin with polyglutamine produce aggregates in the endosome-lysosomal system, where proteolytic fragments of huntingtin is generated. Heat shock protein 70 (HSP70) prevents the formation of protein aggregates, but the effect of HSP70 on the huntingtin in the endosome-lysosomal system is unknown. This study was to determine whether HSP70 alters the distribution of huntingtin in endosome-lysosomal system. HSP70 expressing stable cells (NIH/3T3 or cerebral hybrid cell line A1) were generated, and mutant [(CAG)(100)] huntingtin was transiently overexpressed. Analysis of subcellular distribution by immunocytochemistry or proteolysis cleavage by Western blotting was performed. 18 CAG repeat wild type [WT; (CAG)(18)] huntingtin was used as a control. Cells with huntingtin showed patterns of endosome-lysosomal accumulation, from a "dispersed vacuole (DV)" type into a coalescent "perinuclear vacuole (PV)" type over time. In WT huntingtin, HSP70 increased the cells with the PV types that enhanced the proteolytic cleavage of huntingtin. However, HSP70 reduced cells of the DV and PV types expressing mutant huntingtin, that result in less proteolysis than that of control. In addition, intranuclear inclusions were formed only in mutant cells, which was not affected by HSP70. These results suggest that HSP70 alters the distribution of huntingtin in the endosome- lysosomal system, and that this contributes to huntingtin proteolysis.

Highlights

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder characterized by progressive chorea leading to severe debilitation and death within 15-20 years
The purpose of this study was to determine whether the distribution of huntingtin in the endosomelysosomal system is altered by Heat shock protein 70 (HSP70), and whether HSP70 affects the generation of proteolytic fragments
The over-expressed wild type huntingtin resides in cytoplasmic vacuoles, and HSP70 enhanced the endosome-lysosomal degradation process mainly by increasing the proportion of coalescent vacuoles in the perinuclear region

Summary

Introduction

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder characterized by progressive chorea leading to severe debilitation and death within 15-20 years. In dying neurons of the HD brain, huntingtin aberrantly accumulates in perinuclear regions and in numerous punctate cytoplasmic structures that resemble the endosomal-lysosomal system (Sapp et al, 1997). In an in vitro model of HD, using a clonal mouse striatal cell line transiently transfected with human huntingtin (Kim et al, 1999), exogenous wild-type and mutant huntingtins accumulated diffusely in the cytoplasm and formed dispersed cytoplasmic vacuoles, which was followed by the formation of a coalescent large complex in the perinuclear region. Huntingtin labeled vacuoles show the ultrastructural features of early or late autophagosomes, suggesting endosomal-lysosomal system, which is known to contribute to huntingtin proteolysis and to the autophagic process of cell death (Kegel et al, 2000; Qin et al, 2003; 2004; Rubinsztein et al, 2005; Kim et al, 2006; Pal et al, 2006; Suopanki et al, 2006)

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