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

Abstract

Huntington's disease is caused by CAG trinucleotide expansions in the gene encoding huntingtin. Nterminal 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 immnuocytochemistry 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 endosomelysosomal 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 endosomelysosomal system, and that this contributes to huntingtin proteolysis.Keywords: endosomes; HSP70 heat-shock proteins; Huntington disease; lysosome IntroductionHuntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by progressive chorea leading to severe debilitation and death within 15-20 years. Selective neuronal loss in the neostriatum and cortex causes choreic movement and dementia (Ross et al., 1997). The gene defective in HD is located on chromosome 4p16.3 (Lesperance et al., 1995) and is translated into huntingtin, a 350 kDa ubiquitous protein (Ide et al., 1995;Sharp et al., 1995). Moreover, increased CAG trinucleotide repeats in exon 1 lead to polyglutamine expansion in the N-terminus of huntingtin and are responsible for neurodegeneration in the HD brain (MacDonald et al., 1993, The Huntington's Disease Collaborative Research Group). The intracellular aggregates observed in the HD human brain and in transgenic mice are mainly formed by proteolysis of the N-terminus region in mutant huntingtin (Davies et al., 1997;DiFiglia et al., 1997), and are related to cellular toxicity (Cooper et al., 1998;Zala et al., 2005;Benchoua et al., 2006;Scheibel and Buchner, 2006).In dying neurons of the HD brain, huntingtinHeat shock protein 70 alters the endosome-lysosomal localization of huntingtin Huntingtin and HSP70 39 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 ...

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