Analysis of the role of heat shock protein (Hsp) molecular chaperones in polyglutamine disease

Abstract

Polyglutamine (polygln) diseases are a group of inherited neurodegenerative disorders characterized by protein misfolding and aggregation. Here, we investigate the role in polygln disease of heat shock proteins (Hsps), the major class of molecular chaperones responsible for modulating protein folding in the cell. In transfected COS7 and PC12 neural cells, we show that Hsp40 and Hsp70 chaperones localize to intranuclear aggregates formed by either mutant ataxin-3, the disease protein in spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD), or an unrelated green fluorescent protein fusion protein containing expanded polygln. We further demonstrate that expression of expanded polygln protein elicits a stress response in cells as manifested by marked induction of Hsp70. Studies of SCA3/MJD disease brain confirm these findings, showing localization of Hsp40 and, less commonly, Hsp70 chaperones to intranuclear ataxin-3 aggregates. In transfected cells, overexpression of either of two Hsp40 chaperones, the DNAJ protein homologs HDJ-1 and HDJ-2, suppresses aggregation of truncated or full-length mutant ataxin-3. Finally, we extend these studies to a PC12 neural model of polygln toxicity in which we demonstrate that overexpression of HDJ-1 suppresses polygln aggregation with a parallel decrease in toxicity. These results suggest that expanded polygln protein induces a stress response and that specific molecular chaperones may aid the handling of misfolded or aggregated polygln protein in neurons. This study has therapeutic implications because it suggests that efforts to increase chaperone activity may prove beneficial in this class of diseases.

Fig. 1.

Hsp40 and Hsp70 chaperones localize to intranuclear inclusions formed by mutant ataxin-3. Shown are immunofluorescence images of representative transfected PC12 neural cells containing intranuclear aggregates formed by truncated, HA-tagged ataxin-3 (HA-Q78) (A) or full-length, myc-tagged NLS-ataxin-3 (Q78) (B). Fixed cells were colabeled with anti-HA or anti-myc antisera to label ataxin-3 (green, left panels) and the indicated anti-Hsp antisera to label the chaperones Hsc70, Hsp70, HDJ-1, or HDJ-2 (red, middle panels). All four chaperones localize to polygln aggregates, as indicated by the merged images (right panels). DAPI staining of nuclei (blue) is included in the left andright panels of A andB.

Fig. 2.

The polygln-mediated stress response: aggregate-containing cells show marked induction of the inducible chaperone Hsp70. Shown in A are immunofluorescence images of COS7 cells transfected with empty vector, pcDNA3-HAQ27, or pcDNA3-HAQ78 as indicated. Colabeling with anti-HA (green, left panels) and anti-Hsp70 (red, middle panels) antisera demonstrates that cells with aggregates show marked upregulation of Hsp70 with colocalization of induced Hsp70 to these intranuclear aggregates (arrows, middle panel ofbottom row). In contrast, levels of Hsp70 remain low in cells transfected with control vector or cells expressing nonpathogenic HA-Q27. DAPI staining of nuclei (blue) is shown in theleft and right panels. Immunoblot analysis (B) confirmed that Hsp70 levels were increased in cells expressing HA-Q78. The arrowindicates Hsp70.

Fig. 3.

Colocalization of Hsp chaperones to NI in SCA3 brain. Shown are representative views of tissue sections from the ventral pons of an SCA3 brain immunohistochemically stained for Hsp70, HDJ-1, and HDJ-2. HDJ-1 and HDJ-2 commonly localize to NI (arrows). Hsp70 induction and colocalization to NI, however, is uncommon, as illustrated in the right panel in which only a single neuron shows increased Hsp70, although several neurons in this field are predicted to contain NI based on ubiquitin staining of neighboring sections (data not shown). The inset shows a higher power view of this NI-containing neuron with markedly increased Hsp70 staining (NI indicated by arrow). Scale bars: left,middle, 5 μm; right, 10 μm.

Fig. 4.

The misfolded polygln stress response in PC12 neurons: Hsp70 induction is correlated with the presence of aggregates.A, Immunofluorescence analysis of Hsp70 induction in transfected PC12 neurons expressing unmodified GFP or GFP-fusion proteins with Gln repeats of 19 or 80 residues (GFP-Q19, GFP-Q80). Hsp70 levels are markedly elevated in neurons containing GFP-Q80 aggregates (arrows) but remain low in cells expressing GFP or GFP-Q19. Depicted are paired images in which the top row shows Hsp70 immunofluorescence (red) merged with nuclear staining (blue), and the bottom row shows Hsp70 immunofluorescence merged with GFP fluorescence (green). B, Time course of GFP-Q80 aggregate formation and Hsp70 induction. The number of transfected PC12 neurons containing aggregates increases over time, reaching ∼80% 5 d after transfection. A subset of transfected cells shows marked Hsp70 induction, peaking 3 d after transfection. Markedly induced Hsp70 only occurred in aggregate-containing cells and never in cells in which the mutant polygln protein remained diffusely distributed.C, Aggregate formation and Hsp70 induction both occur in a glutamine repeat length-dependent manner but with different thresholds. Shown are the percentages of transfected cells with aggregates and the percentages with induced Hsp70, 3 d after transfection. Although GFP-Q35 and Q56 form aggregates, Hsp70 induction was never observed with GFP-Q35 aggregates and only rarely with GFP-Q56 aggregates. Results in B and C represent the means ± SD for two independent experiments.

Fig. 5.

Polygln-mediated cell death and relationship to aggregate formation. Polygln toxicity in PC12 neurons occurs in a glutamine repeat length-dependent manner and is correlated with the presence of aggregates. A, Shown are fluorescence images of representative transfected PC12 neurons expressing GFP-Q19 (which remains diffuse in cells) and GFP-Q80 (which readily forms aggregates). Aggregate-containing cells (arrows) become rounded, nonrefractile, and nonadherent and frequently display morphological features of cell death, such as membrane blebbing and condensed nuclei, illustrated here by DAPI staining. B, Time course of inclusion formation by GFP-Q80 versus GFP-Q19 in PC12 neurons.C, Time course of cell death in PC12 cells expressing GFP-Q19 or Q80. The rate of death in GFP-Q80-expressing cells is significantly increased over that in GFP-Q19-expressing cells, which have a low rate of death similar to that in mock-transfected cells analyzed in parallel. D, Cell death is directly correlated with the presence of inclusions. Three days after transfection, cells were identified by fluorescence as diffusely stained or having inclusions. Shown are the percentage of cells in each of these two categories that were then scored as dead. In GFP-Q80-expressing cells, nearly half of all inclusion-containing cells were scored as dead. In contrast, diffusely staining GFP-Q80 cells were scored as dead at a rate similar to the background rate in GFP-Q19 cells. Results in B–D represent the means ± SD of two independent experiments.

Fig. 6.

Overexpression of Hsp40 chaperones reduces aggregation of truncated and full-length ataxin-3. COS7 cells were cotransfected with plasmids encoding HA-Q78 (A,C, E) or NLS-ataxin-3 (Q78) (B, D, F), together with empty control plasmid or plasmids encoding HDJ-1 (A, B), HDJ-2 (C,D), or Hsp70 (E,F). Suppression of aggregate formation occurred with HDJ-1 and HDJ-2 but not Hsp70. A partially deleted form of HDJ-2 lacking the DnaJ domain (del9–107) also suppressed aggregation of mutant ataxin-3. Shown are the mean values of three independent experiments. Statistically significant differences, determined by paired t test, are indicated by asterisks(*p < 0.05; **p < 0.01).

Fig. 7.

Hsp40 chaperone HDJ-1 suppresses aggregation and toxicity of polygln protein in PC12 neural cells. PC12 neurons transfected with plasmids encoding GFP-Q56 (A,B) or GFP-Q80 (C, D), together with HDJ-1 or empty control plasmid, were scored for aggregates and cell death under blinded conditions. HDJ-1 partially suppressed aggregate formation and cell death with both GFP-polygln proteins. Shown are the means of three independent experiments. Statistically significant differences, determined by pairedt test, are indicated by asterisks(*p < 0.05; **p < 0.01).