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Loss of Hsp70 exacerbates pathogenesis but not levels of fibrillar aggregates in a mouse model of Huntington's disease - PubMed

  • ️Thu Jan 01 2009

Loss of Hsp70 exacerbates pathogenesis but not levels of fibrillar aggregates in a mouse model of Huntington's disease

Jennifer L Wacker et al. J Neurosci. 2009.

Abstract

Endogenous protein quality control machinery has long been suspected of influencing the onset and progression of neurodegenerative diseases characterized by accumulation of misfolded proteins. Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) tract in the protein huntingtin (htt), which leads to its aggregation and accumulation in inclusion bodies. Here, we demonstrate in a mouse model of HD that deletion of the molecular chaperones Hsp70.1 and Hsp70.3 significantly exacerbated numerous physical, behavioral and neuropathological outcome measures, including survival, body weight, tremor, limb clasping and open field activities. Deletion of Hsp70.1 and Hsp70.3 significantly increased the size of inclusion bodies formed by mutant htt exon 1, but surprisingly did not affect the levels of fibrillar aggregates. Moreover, the lack of Hsp70s significantly decreased levels of the calcium regulated protein c-Fos, a marker for neuronal activity. In contrast, deletion of Hsp70s did not accelerate disease in a mouse model of infectious prion-mediated neurodegeneration, ruling out the possibility that the Hsp70.1/70.3 mice are nonspecifically sensitized to all protein misfolding disorders. Thus, endogenous Hsp70s are a critical component of the cellular defense against the toxic effects of misfolded htt protein in neurons, but buffer toxicity by mechanisms independent of the deposition of fibrillar aggregates.

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Figures

Figure 1.
Figure 1.

Deletion of Hsp70.1 and Hsp70.3 decreases survival in the R6/2 mouse, but not in mice infected with prions. A, Kaplan–Meier survival curve for the indicated genotypes [R6/2−/−;Hsp70+/+ (n = 21), R6/2tg/−;Hsp70+/+ (n = 18), R6/2−/−;Hsp70−/+ (n = 27), R6/2tg/−;Hsp70−/+ (n = 22), R6/2−/−;Hsp70−/−(n = 18), and R6/2tg/−;Hsp70−/− (n = 18)] demonstrates that the absence of Hsp70.1/3 significantly decreased survival of R6/2 mice (log rank: p = 0.033). No nontransgenic, Hsp70 heterozygous knock-out, or Hsp70 homozygous knock-out mice died during the 14 week time course. B, C, Kaplan–Meier survival curves for Hsp70+/+ (n = 11, and 12, respectively) and Hsp70−/− (n = 19) mice inoculated intracranially with 3.5 LC50 22L prion or 3.5 LC50 RML prion indicate that deletion of Hsp70.1/3 did not affect survival (log rank: p = 0.207 and 0.495, respectively).

Figure 2.
Figure 2.

Deletion of Hsp70.1/3 worsens motor deficits in R6/2 mice. A–D, Deletion of Hsp70.1/3 decreases the latency to fall of R6/2 mice (two-way ANOVA: p < 0.05) (A), and increases severity of clasping (two-way ANOVA: p < 0.001) (B), tremor (two-way ANOVA: p < 0.001) (C), and grooming (two-way ANOVA: p < 0.03) (D). E, F, Deletion of Hsp70.1/3 decreases R6/2 spontaneous activity (two-way ANOVA: p < 0.001) but has only a moderate effect on locomotor activity. Error bars indicate SEM. Note that in the absence of the R6/2 transgene, the loss of one or both copies of Hsp70.1/3 does not influence any of the presented outcome measures.

Figure 3.
Figure 3.

Deletion of Hsp70.1/3 exacerbates the physical phenotypes of R6/2 mice. The absence of Hsp70.1/3 significantly exacerbates the weight loss phenotype (two-way ANOVA: p < 0.05) (A), and worsens the coat appearance (two-way ANOVA: p < 0.001) (B), body position (two-way ANOVA: p < 0.02) (C), and tail position (two-way ANOVA p < 0.001) (D) of R6/2 mice. Error bars indicate SEM. Note that in the absence of the R6/2 transgene, the loss of one or both copies of Hsp70.1/3 does not influence any of the presented outcome measures.

Figure 4.
Figure 4.

Deletion of Hsp70.1/3 increases the size of inclusion bodies in R6/2 mice. A, Representative images (600×) of inclusion bodies in the neocortex of R6/2 mice as detected with the anti-htt antibody EM48. B, Quantification of inclusion body number in the neocortex shows that R6/2tg/−;Hsp70−/− mice have an increase in the density of inclusion bodies compared with R6/2tg/−;Hsp70+/+ mice, although this difference only showed a trend toward statistical significance (p = 0.086). C, Representative images (1000×) illustrating the size of inclusion bodies in the neocortex of R6/2 mice as detected with the anti-htt antibody EM48. D, Quantification of inclusion body size shows that the average size of the inclusion bodies was significantly larger (p < 0.001) in R6/2tg/−;Hsp70−/− mice compared with R6/2tg/−;Hsp70+/+ mice. Statistical comparisons were performed by one-way ANOVA (n = 6–11 mice per group).

Figure 5.
Figure 5.

Deletion of Hsp70.1/3 does not modulate levels of SDS-insoluble fibrillar protein aggregates formed by mutant htt exon 1 in R6/2 mice. A, B, Deletion of Hsp70.1/3 does not alter the levels of EM48 reactive SDS-insoluble aggregates (normalized to GAPDH reactivity) measured with Western immunoblots in 14-week-old R6/2 brain homogenates (Student's t test p = 0.96). C, D, Treatment of brain homogenates with formic acid liberates an SDS-resistant monomeric/oligomeric mutant huntingtin exon 1 species. The levels of formic acid-sensitive monomers/oligomers (normalized to GAPDH reactivity) appeared to increase in the absence of Hsp70.1/3, but did not reach statistical significance (Student's t test p = 0.15). E, F, The levels of SDS-insoluble EM48-positive fibrillar aggregates in brain measured by a filter-trap assay do not change in the absence of Hsp70.1/3 (Student's t test p = 0.89). G, H, Formic acid-treated brain homogenates were subjected to the filter-trap assay, which showed no change in EM48 immunoreactivity in the absence of Hsp70.1/3 (Student's t test p = 0.90). I, A native agarose gel used to examine EM48 immunoreactive oligomeric species in R6/2 brain homogenates shows no apparent change in the absence of Hsp70.1/3.

Figure 6.
Figure 6.

Deletion of Hsp70.1/3 exacerbates the loss of c-Fos immunoreactivity and other neuropathological deficits in R6/2 mice. A, B, Quantification of c-Fos immunohistochemistry in the neocortex from 14-week-old mice shows that R6/2tg/−;Hsp70−/− mice have a significant decrease (p < 0.05) in c-Fos levels compared with R6/2tg/−;Hsp70+/+ mice. C, D, Quantification of synaptophysin immunohistochemistry in the neocortex from 14-week-old mice shows that R6/2tg/−;Hsp70+/− and −/− mice have a significant decrease (p < 0.05) in synaptophysin levels compared with R6/2−/−;Hsp70−/− mice. E, F, Quantification of Iba1 and GFAP immunohistochemistry in the neocortex from 14-week-old mice shows that R6/2tg/−;Hsp70+/− and −/− mice have a significant increase (p = 0.0297) in Iba1 levels, and a trend toward increased GFAP levels (p = 0.1048), respectively, compared with R6/2−/−;Hsp70−/− mice.

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