Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease

Abstract

Huntington's disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAG/polyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2/HSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently transfecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43-74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2/HSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2/HSDJ in protein folding.

Figure 1

Time and polyQ repeat dependency of inclusion formation. (a) SH-SY5Y (human neuroblastoma cells; *, inclusions are occasionally present (<1% of EGFP-positive cells). The decrease of FP-positive cells showing inclusions is possibly because of cell death for EGFP-HDQ₇₄ at 72 h. (b) COS-7 (African green monkey cells). (c) PC12 (rat pheochromocytoma cells).

Figure 2

HD exon 1 protein aggregates in SH-SY5Y and COS-7 cells. (a) SH-SY5Y cells expressing EGFP-HDQ₇₄ (green). Note that the cell with multiple inclusions shows weak EGFP staining in the cytoplasm compared with the other transfected cells. Nuclei were stained with 4′,6-diamidino-2-phenylindole. (b) COS-7 cell transfected with HA-HDQ₇₄ showing a single inclusion.

Figure 3

Ubiquitin, HSP40, HSP70, and the 20S proteasome colocalize with inclusions. Colocalization was determined by using confocal image analysis. SH-SY5Y cells were transfected with EGFP-HDQ₇₄. In a, c, e, and g, the cells were microphotographed by using the FITC filter to show the inclusions formed by HD exon 1 constructs. In b, d, f, and h, the same cells are shown by using the Texas red filter to detect the other proteins sequestered into the inclusions. Cells were analyzed with specific antibodies: (a and b) anti-ubiquitin; (c and d) anti-HSP40 (HDJ-2/HSDJ); (e and f) anti-HSP70; (g and h) anti-20S proteasome.

Figure 4

Cells expressing pEGFP-HDQ₇₄ develop multiple inclusions when they coexpress HDJ-2/HSDJ, when treated with heat shock or lactacystin. Comparisons of typical inclusion phenotypes of COS-7 cells expressing pEGFP-HD₇₄ when (a) cotransfected with pFLAG-CMV-2 (empty vector); (b) cotransfected with wt HDJ-2/HSDJ; (c) treated with heat shock; (d) treated with lactacystin (10 μM).

Figure 5

Proportion of COS-7 cells showing nuclear fragmentation in EGFP-HDQ₂₃-expressing cells and EGFP-HDQ₇₄-expressing cells with or without inclusions (n = 50). These data are from one experiment that was representative of three independent experiments.