Dantrolene is neuroprotective in Huntington's disease transgenic mouse model

Abstract

Background: Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine expansion in the Huntingtin protein which results in the selective degeneration of striatal medium spiny neurons (MSNs). Our group has previously demonstrated that calcium (Ca2+) signaling is abnormal in MSNs from the yeast artificial chromosome transgenic mouse model of HD (YAC128). Moreover, we demonstrated that deranged intracellular Ca2+ signaling sensitizes YAC128 MSNs to glutamate-induced excitotoxicity when compared to wild type (WT) MSNs. In previous studies we also observed abnormal neuronal Ca2+ signaling in neurons from spinocerebellar ataxia 2 (SCA2) and spinocerebellar ataxia 3 (SCA3) mouse models and demonstrated that treatment with dantrolene, a ryanodine receptor antagonist and clinically relevant Ca2+ signaling stabilizer, was neuroprotective in experiments with these mouse models. The aim of the current study was to evaluate potential beneficial effects of dantrolene in experiments with YAC128 HD mouse model.

Results: The application of caffeine and glutamate resulted in increased Ca2+ release from intracellular stores in YAC128 MSN cultures when compared to WT MSN cultures. Pre-treatment with dantrolene protected YAC128 MSNs from glutamate excitotoxicty, with an effective concentration of 100 nM and above. Feeding dantrolene (5 mg/kg) twice a week to YAC128 mice between 2 months and 11.5 months of age resulted in significantly improved performance in the beam-walking and gait-walking assays. Neuropathological analysis revealed that long-term dantrolene feeding to YAC128 mice significantly reduced the loss of NeuN-positive striatal neurons and reduced formation of Httexp nuclear aggregates.

Conclusions: Our results support the hypothesis that deranged Ca2+ signaling plays an important role in HD pathology. Our data also implicate the RyanRs as a potential therapeutic target for the treatment of HD and demonstrate that RyanR inhibitors and Ca2+ signaling stabilizers such as dantrolene should be considered as potential therapeutics for the treatment of HD and other polyQ-expansion disorders.

Figure 1

Caffeine potentiates glutamate-induced Ca²⁺ signals in YAC128 and WT MSN cultures. Representative 340/380 nm Fura-2 ratio traces are shown for YAC128 (red) and WT (green) after application of 2.5 μM glutamate (A), 2.5 mM caffeine (B), and 2.5 μM glutamate + 2.5 mM caffeine simultaneously (C). The average differences between the peak ratio and base line ratio for each treatment was calculated and shown as mean ± SE, n = number of cells (D). *p < 0.05 compared to corresponding genotype treated with 2.5 μM glutamate only, ^#p < 0.05 comparing YAC128 to WT, and ns = not significant using one-way ANOVA with Tukey's post test. WT, wild type; YAC, YAC128; glu, glutamate.

Figure 2

Dantrolene protects YAC128 MSNs from glutamate-induced apoptosis. Quantification of glutamate-induced apoptotic cell death of cultured 14DIV YAC128 (closed) and WT (open) MSNs, with (red) or without (black) 1 h dantrolene (Dan) pre-treatment. Shown are the mean ± SE of % TUNEL positive cells = (number of TUNEL positive MSNs/number of MSNs) × 100%. n = number of fields counted, Stats. WT, wild type; Q128, YAC128.

Figure 3

Dantrolene feeding improves performance of YAC128 mice in the beam-walk test. Motor coordination performance of WT and YAC128 mice in dantrolene trial. A-F. Beam walk assay. The average time to cross the beam (A, C, E) and the average number of foot slips on the beam (B, D, F) are shown for beam-walking experiments performed with 17 mm round plastic beam (A, B), 11 mm round plastic beam (C, D), and 5 mm square wood beam (E, F). The data for WT control mice (open black circles), YAC128 control mice (open red circles), WT mice fed with dantrolene (filled black circles), and YAC128 mice fed with dantrolene (filled red circles) are shown as mean ± SE (for the numberof mice (10) in each experimental group, see Table 2) 2, 4, 7, 9.5, 11.5, and 13.5 (washout) months of age. While counting the foot slips of the mice with "crawling behavior, " we considered every step as one foot slip. *p < 0.05, significant differences between control WT group and control SCA3 group. "xF" on C and E mouse fell off the beam and failed the test where x = number of mice. *p < 0.05 using one-way ANOVA with Tukey's post test. WT, wild type; YAC, YAC128; Ctrl, control; Dan, dantrolene.

Figure 4

Dantrolene feeding improves gait abnormalities in YAC128 mice. Gait analysis of WT and YAC128 mice in dantrolene trial. (A). The footprint patterns of 13.5-month-old WT and YAC128 for control and dantrolene groups are shown. (B) Stride lengths (cm) and (C) front/hind footprint overlaps (cm) of WT and YAC128 mice in dantrolene trial are shown as mean ± SE (n = number of mice (10) in each groupsee Table 2), *p < 0.05 using one-way ANOVA with Tukey's post test. WT, wild type; YAC, YAC128; Ctrl, control; Dan, dantrolene.

Figure 5

Dantrolene feeding reduces NeuN-positive cell loss in YAC128 striata. (A) The brain weight of control (PBS-fed) and dantrolene-fed WT and YAC128 mice was measured at 14 months of age after 4% paraformaldehyde perfusion. The brain weight is shown as mean ± SE (g) (n = number of mice (10) in each groupsee Table 2). (B) The striatal slices from 14 months old control (PBS-fed) and dantrolene-fed WT and YAC128 mice were stained by the neuronal nuclear marker NeuN. Representative images are shown. (C) Striatal cell counts obtained as a result of stereological analysis of NeuN-stained striatal slices from 14 months old control (PBS-fed) and dantrolene-fed WT and YAC128 mice. The MSN numbers are shown as mean ± SE (n = number of mice, see Table 2). ***p < 0.001, *p < 0.05 using one-way ANOVA with Tukey's post test. WT, wild type; YAC, YAC128; Ctrl, control; Dan, dantrolene.

Figure 6

Dantrolene feeding inhibits Htt^exp aggregation in YAC128 striatal cells. The striatal slices from 14 months old control (PBS-fed) and dantrolene-fed WT and YAC128 mice were stained with an anti-Htt monoclonal antibody (dark grey). Nuclei were counter-stained with cresyl violet (blue). Representative images of 3 mice are shown. WT, wild type; YAC, YAC128; Ctrl, control; Dan, dantrolene.