Maintenance of basal levels of autophagy in Huntington's disease mouse models displaying metabolic dysfunction

Abstract

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expanded polyglutamine repeat in the huntingtin protein. Neuropathology in the basal ganglia and in the cerebral cortex has been linked to the motor and cognitive symptoms whereas recent work has suggested that the hypothalamus might be involved in the metabolic dysfunction. Several mouse models of HD that display metabolic dysfunction have hypothalamic pathology, and expression of mutant huntingtin in the hypothalamus has been causally linked to the development of metabolic dysfunction in mice. Although the pathogenic mechanisms by which mutant huntingtin exerts its toxic functions in the HD brain are not fully known, several studies have implicated a role for the lysososomal degradation pathway of autophagy. Interestingly, changes in autophagy in the hypothalamus have been associated with the development of metabolic dysfunction in wild-type mice. We hypothesized that expression of mutant huntingtin might lead to changes in the autophagy pathway in the hypothalamus in mice with metabolic dysfunction. We therefore investigated whether there were changes in basal levels of autophagy in a mouse model expressing a fragment of 853 amino acids of mutant huntingtin selectively in the hypothalamus using a recombinant adeno-associate viral vector approach as well as in the transgenic BACHD mice. We performed qRT-PCR and Western blot to investigate the mRNA and protein expression levels of selected autophagy markers. Our results show that basal levels of autophagy are maintained in the hypothalamus despite the presence of metabolic dysfunction in both mouse models. Furthermore, although there were no major changes in autophagy in the striatum and cortex of BACHD mice, we detected modest, but significant differences in levels of some markers in mice at 12 months of age. Taken together, our results indicate that overexpression of mutant huntingtin in mice do not significantly perturb basal levels of autophagy.

Figure 1. Body weight changes in mice injected with rAAV-htt853 vectors and validation of htt expression.

A) Mice bilaterally injected with rAAV5-htt853-79Q displayed increased body weight compared to mice injected with rAAV5-htt853-18Q and uninjected animals already 2 weeks post injection. Animals bilaterally injected with rAAV5-htt853-18Q showed increased body weight compared to uninjected controls 6 weeks post injection (n = 14/group; * and ^#p<0.05 after Bonferroni post-hoc test; *statistical significant difference compared to uninjected controls while # expresses statistical significance compared to rAAV5-htt853-18Q). B) qRT-PCR verified the expression of htt853-18Q and htt853-79Q relative to uninjected controls in mice 8 weeks post-injection of rAAV5-htt853 vectors (n = 7/group). C) Representative Western blot detected the expression of endogenous wt htt as well as htt853-18Q and htt853-79Q in mice 8 weeks post injection of rAAV5-htt853 vectors.

Figure 2. Maintenance of basal levels of autophagy in mice overexpressing htt fragments in the hypothalamus.

The expression levels of selected autophagy markers were analyzed by Western blot in mice bilaterally injected with rAAV5-htt853-18Q or rAAV5-htt853-79Q and compared to uninjected controls 8 weeks post injection (n = 6–7/group). The data displayed maintenance of the autophagy flux, despite the presence of a clear metabolic phenotype in the injected mice. The data in the graphs are expressed as mean ± SEM relative to the uninjected controls. The Western blots are representative of one sample for each group.

Figure 3. Basal levels of autophagy in the hypothalamus of BACHD mice.

The expression levels of selected autophagy markers were analyzed by Western blot in hypothalamic tissue from BACHD mice at 2, 6 and 12 months of age and compared with gender- and age-matched controls (n = 3–6/group). The data showed a substantial maintenance of the autophagy flux, despite the presence of a metabolic phenotype in the BACHD mice already at early time points. The expression level of a marker for selective autophagy p62 showed a progressive accumulation in both BACHD and wt mice (Bonferroni post-hoc test: wt p = 0.003, BACHD p = 0.02). The data in the graphs are expressed as mean ± SEM, relative to the 2 months wt controls. The Western blots are representative of one sample for each group.

Figure 4. Basal levels of autophagy in the cerebral cortex of BACHD mice.

The expression levels of selected autophagy markers were analyzed by Western blot in cortical tissue from BACHD mice at 2, 6 and 12 months of age and compared with gender- and age-matched controls (n = 3–6/group). The data show that the basal levels of autophagy were substantially unaltered in the cortex of BACHD mice with the exception of the LC3II/I ratio, which is reduced in 12 months old BACHD when compared with wt controls (Genotype effect in two-factor ANOVA: F_(1,25) = 4.715, p = 0.040; Student’s un-paired T-test as post hoc test: p = 0.006 at 12 months). The data in the graphs are expressed as mean ± SEM, relative to the 2 months wt controls. The Western blots are representative of one sample for each group.

Figure 5. Basal levels of autophagy in the striatum of BACHD mice.

Selected autophagy markers were analyzed by Western blot of striatal tissue from BACHD mice at 2, 6 and 12 months of age and compared with gender- and age-matched controls (n = 3–6/group). The data show that the basal levels of autophagy were unaltered in the striatum of BACHD mice. The data in the graphs are expressed as mean ± SEM, relative to the 2 months wt controls. The Western blots are representative of one sample for each group.