Selective histone deacetylase (HDAC) inhibition imparts beneficial effects in Huntington's disease mice: implications for the ubiquitin-proteasomal and autophagy systems

Abstract

We previously demonstrated that the histone deacetylase (HDAC) inhibitor, 4b, which preferentially targets HDAC1 and HDAC3, ameliorates Huntington's disease (HD)-related phenotypes in different HD model systems. In the current study, we investigated extensive behavioral and biological effects of 4b in N171-82Q transgenic mice and further explored potential molecular mechanisms of 4b action. We found that 4b significantly prevented body weight loss, improved several parameters of motor function and ameliorated Huntingtin (Htt)-elicited cognitive decline in N171-82Q transgenic mice. Pathways analysis of microarray data from the mouse brain revealed gene networks involving post-translational modification, including protein phosphorylation and ubiquitination pathways, associated with 4b drug treatment. Using real-time qPCR analysis, we validated differential regulation of several genes in these pathways by 4b, including Ube2K, Ubqln, Ube2e3, Usp28 and Sumo2, as well as several other related genes. Additionally, 4b elicited increases in the expression of genes encoding components of the inhibitor of kappaB kinase (IKK) complex. IKK activation has been linked to phosphorylation, acetylation and clearance of the Htt protein by the proteasome and the lysosome, and accordingly, we found elevated levels of phosphorylated endogenous wild-type (wt) Htt protein at serine 16 and threonine 3, and increased AcK9/pS13/pS16 immunoreactivity in cortical samples from 4b-treated mice. We further show that HDAC inhibitors prevent the formation of nuclear Htt aggregates in the brains of N171-82Q mice. Our findings suggest that one mechanism of 4b action is associated with the modulation of the ubiquitin-proteasomal and autophagy pathways, which could affect accumulation, stability and/or clearance of important disease-related proteins, such as Htt.

Figure 1.

The effects of 4b on the relative body weights of female (A) and male (C) wt and N171-82Q transgenic mice. The relative body weights were based on the weight at 8 weeks of age. Differences in drug- versus vehicle-treated mice were determined by two-way ANOVA (F(1,63) = 11.8; **P = 0.0011). (B) Rotarod performance of vehicle- and HDACi 4b-treated female and male N171-82Q transgenic mice from 8 to 16 weeks of age. Two-way ANOVA revealed significant differences between vehicle- and HDACi 4b-treated female transgenic mice (F(1,135) = 4.2; P = 0.040), as well as a significant effect of drug treatment in male N171-82Q transgenic mice (F(1,126) = 23.5; P < 0.0001). (D) Number of correct choice of first 10 trials in the alternating T-maze test. One-way ANOVA revealed significant differences between vehicle-treated wt and N171-82Q transgenic mice (**P < 0.001), and also a significant difference between vehicle-treated and HDACi 4b-treated N171-82Q mice (*P < 0.05). Bars represent mean score ± SEM (n = 6–7 per group). (E) Effects of HDACi 4b treatment on open-field activity of wt and N171-82Q transgenic mice (n = 15–20 mice per group) are shown over a 5 min test period. Two-way ANOVA revealed significant differences between HDACi 4b-treated and vehicle-treated N171-82Q transgenic mice in ambulatory distance (P = 0.004), vertical time (P = 0.0005), mean velocity (P = 0.0002), vertical counts (P = 0.004) and ambulatory time (P = 0.01). The summary of total effects of HDACi 4b treatment at different doses is shown in Table 1.

Figure 2.

Real-time qPCR analysis showing the effects of 4b on the expression of the indicated ubiquitination-related genes in the striatum of N171-82Q transgenic mice. Values shown are the mean ± SEM expression values (n = 5–6 mice per group) compared with a wt expression level of 1, which is indicated by the dotted line. Significant differences were determined by Student's t-tests (unpaired; one-tailed) at the indicated treatment time points. *P < 0.05; **P < 0.01; ⁺P < 0.08.

Figure 3.

Real-time qPCR analysis showing the effects of 4b on the expression of the indicated ubiquitination-related genes in the cortex of N171-82Q transgenic mice. Values shown are the mean ± SEM expression values (n = 5–6 mice per group) compared with a wt expression level of 1, which is indicated by the dotted line. Significant differences were determined by Student's t-tests (unpaired; one-tailed) at the indicated treatment time points. *P < 0.05; **P < 0.01; ***P < 0.001; ⁺P < 0.08.

Figure 4.

Real-time qPCR analysis showing the effects of 4b treatment on the IKK-related genes in the cortex (A) and striatum (B) of wt and N171-82Q transgenic mice. Values shown are the mean ± SEM expression values (n = 5–6 mice per group). Significant differences were determined by Student's t-tests (unpaired; one-tailed) at the indicated treatment time points. *P < 0.05; **P < 0.01; ⁺P < 0.08.

Figure 5.

Modifications of Htt protein by 4b treatment in the cortex of wt (control) and N171-82Q transgenic mice. (A) Cytosolic and nuclear protein fractions from cortex of vehicle- and 4b-treated mice were probed with antibodies recognizing different phosphorylation and acetylation states of Htt protein. Homogenates were immunoprecipitated (IP) using PW0595 anti-Htt antibody as described in Materials and Methods. Immunoblots (IB) were run using the indicated specific antibodies. (B) Quantitation of band intensity was done for the high molecular weight modified Htt species designated by the arrow, which runs larger than the standard full-length 350 kDa Htt species (*), shown in (A). Bar graphs represent data from n = 6–7 samples for pT3, pS16 and AcK9/pS13/pS16 in the cytoplasmic fractions and n = 2–3 samples for all nuclear fractions and the pS13 cytoplasmic fraction. Statistical significance was determined using Student's t-tests comparing 4b- and vehicle-treated samples. *P < 0.05; **P < 0.01.

Figure 6.

The effect of 4b and 874 on Htt aggregates in different brain regions from N171-82Q transgenic mice. Immunohistochemistry was performed on free-floating sections (25 µm) from vehicle-, 4b- and 874-treated mice (6 weeks of treatment), using the anti-Htt antibody EM48 (1:500 dilution). Aggregates were quantified by counting the number of EM48-positive aggregates per 150 μm × 150 μm field at 64× magnification. Micrographs from caudate putamen are not shown due to very low numbers of aggregates. Bar graphs depict quantification of Htt aggregates from 874-treated N171-82Q transgenic mice (n = 6 per group). PIR, piriform cortex; INS, insular cortex; SS, somatosensory cortex; CPu, caudate putamen. Asterisks denote statistically significant values using Student's t-test, unpaired, two-tailed: *P < 0.05; **P < 0.01.