Clioquinol down-regulates mutant huntingtin expression in vitro and mitigates pathology in a Huntington's disease mouse model

Abstract

In investigating the role of metal ions in the pathogenesis of Huntington's disease, we examined the effects of clioquinol, a metal-binding compound currently in clinical trials for Alzheimer's disease treatment, on mutant huntingtin-expressing cells. We found that PC12 cells expressing polyglutamine-expanded huntingtin exon 1 accumulated less mutant protein and showed decreased cell death when treated with clioquinol. This effect was polyglutamine-length-specific and did not alter mRNA levels or protein degradation rates. Clioquinol treatment of transgenic Huntington's mice (R6/2) improved behavioral and pathologic phenotypes, including decreased huntingtin aggregate accumulation, decreased striatal atrophy, improved rotarod performance, reduction of weight loss, normalization of blood glucose and insulin levels, and extension of lifespan. Our results suggest that clioquinol is a candidate therapy for Huntington's disease and other polyglutamine-expansion diseases.

Fig. 1.

CQ down-regulates expanded polyQ-egfp fluorescence in vitro. Bars and error bars represent mean + SEM. n ≥ 3 for each data set. Fluorescence microscopy and quantitation of EGFP-, Htt^exon1-Q²⁵-egfp-, and Htt^exon1-Q¹⁰³-egfp-expressing cells. (Bar, 100 μm.) **, P < 0.03.

Fig. 2.

CQ down-regulates polyQ protein expression and decreases cell death in vitro. Bars and error bars represent mean + SEM. n ≥ 3 for each data set. Western data are normalized to the GAPDH signal. (A) Western blotting and quantitation of Htt^exon1-Q¹⁰³-egfp (polyQ Ab) in HEK293 cell lysates; times posttransfection as indicated. (B) Viability (propidium iodide staining) of Htt^exon1-Q¹⁰³-egfp cells treated with CQ. *, P ≤ 0.03; **, P < 0.002 vs. vehicle control. (C) Western blotting and quantitation of Htt^exon1-Q¹⁰³-egfp (polyQ Ab) and EGFP (GFP Ab) in cell lysates. ***, P = 0.02.

Fig. 3.

CQ does not affect polyQ mRNA levels or protein degradation. Bars and error bars represent mean + SEM. n ≥ 3 for each data set. (A) Northern blotting and quantitation of Htt^exon1-Q¹⁰³-egfp transcripts. (B) Fluorescence microscopy of mutant protein accumulation in Htt^exon1-Q¹⁰³-egfp-expressing cells in presence of MG132 ± CQ. (Bar, 100 μm.) (C) Western analysis of cells treated with MG132 and CQ. (D) Pulse–chase analysis of Htt^exon1-Q¹⁰³-egfp turnover in the presence and absence of CQ.

Fig. 4.

CQ inhibits mutant Htt aggregate accumulation in vivo. Bars and error bars represent mean + SEM. (A) Representative Western blotting and quantitation of high molecular weight mutant Htt in whole-brain homogenates from 11-week-old R6/2 transgenic mice. Wt, wild-type; Tg, vehicle-treated R6/2; CQ, CQ-treated R6/2. n = 3. *, P = 0.01. (B) Inhibition of neuronal intranuclear inclusion formation by treatment with CQ. Representative images of striatum and cortex from 11-week-old mice; wild-type, R6/2-vehicle treated, or R6/2-CQ treated, as indicated. Htt aggregates were visualized by using EM48 Ab. Results were similar for six or more animals in each treatment group. (Bar, 50 μm.)

Fig. 5.

CQ decreases polyQ-mediated pathology in vivo. Bars and error bars represent mean + SEM. Representative images of the cerebrum of 11-week-old mice and quantitation of lateral ventricle areas, showing decreased striatal atrophy in 11-week-old CQ-treated R6/2 mice. n ≥ 6 for each treatment group. *, P < 0.001 vs. vehicle-treated mice.

Fig. 6.

CQ positively effects behavior, weight, and survival in vivo. Bars and error bars represent mean + SEM. (A) Illustration of clasping behavior. (Left) Wild-type mouse. (Right) Ten-week-old R6/2 mouse exhibiting clasping. (B) Clasping score at 10 weeks of age in CQ and vehicle-treated R6/2. n = 8. *, P < 0.0004 vs. vehicle. (C) Rotarod testing of CQ and vehicle-treated R6/2. Bars and error bars represent mean + SEM. Black bars, vehicle-treated; gray bars, CQ-treated. n ≥ 6 for each group. *, P < 0.03; **, P < 0.001 vs. vehicle-treated animals. (D) Body weight over time of wild-type (filled circles), vehicle-treated R6/2 (open circles), and CQ-treated R6/2 (filled triangles); symbols and bars represent mean ± SEM. n ≥ 6 for each group, *, P < 0.01 vs. vehicle-treated animals by Student's t test; also, P < 0.0001 overall, for CQ-treated vs. vehicle-treated animals by ANOVA with post hoc Bonferroni/Dunn testing. (E) Kaplan–Meier analysis of R6/2 lifespan, vehicle treated (closed circles) vs. CQ treated (open circles). n = 5 per group. P = 0.0018 by log-rank Mantel–Cox test.