Chronic treatment with 17-DMAG improves balance and coordination in a new mouse model of Machado-Joseph disease - PubMed (original) (raw)

Chronic treatment with 17-DMAG improves balance and coordination in a new mouse model of Machado-Joseph disease

Anabela Silva-Fernandes et al. Neurotherapeutics. 2014 Apr.

Abstract

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disease currently with no treatment. We describe a novel mouse model of MJD which expresses mutant human ataxin-3 at near endogenous levels and manifests MJD-like motor symptoms that appear gradually and progress over time. CMVMJD135 mice show ataxin-3 intranuclear inclusions in the CNS and neurodegenerative changes in key disease regions, such as the pontine and dentate nuclei. Hsp90 inhibition has shown promising outcomes in some neurodegenerative diseases, but nothing is known about its effects in MJD. Chronic treatment of CMVMJD mice with Hsp90 inhibitor 17-DMAG resulted in a delay in the progression of their motor coordination deficits and, at 22 and 24 weeks of age, was able to rescue the uncoordination phenotype to wild-type levels; in parallel, a reduction in neuropathology was observed in treated animals. We observed limited induction of heat-shock proteins with treatment, but found evidence that 17-DMAG may be acting through autophagy, as LC3-II (both at mRNA and protein levels) and beclin-1 were induced in the brain of treated animals. This resulted in decreased levels of the mutant ataxin-3 and reduced intranuclear aggregation of this protein. Our data validate this novel mouse model as a relevant tool for the study of MJD pathogenesis and for pre-clinical studies, and show that Hsp90 inhibition is a promising therapeutic strategy for MJD.

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Figures

Fig. 1

Fig. 1

Generation of a transgenic mouse model of MJD carrying the expanded ATXN3 with 135 CAGs. (A) Schematic diagram of the plasmid CMVMJDAT3Q135_1.5 used for the generation of cDNA MJD transgenic mice. (B) Western blot anti-ATXN3 in different CNS regions (cerebellum, forebrain and brainstem) of CMVMJD135 mice with 14–16 weeks of age. In all lanes, the endogenous mouse ataxin-3 (Atxn3) is detected at about 42 kDa. An approximately 80-kDa protein corresponding to expanded ATXN3 is detected in transgenic animal lysates. (C) qRT-PCR analysis of human ATXN3 and murine Atxn3 mRNA expression levels. Values are presented as mean ± SEM (n = 4). (D) CMVMJD135 mice at 40 weeks of age presenting body posture deficit with lower pelvic elevation and hunchback (2), limb clasping (4) and reduced hindlimb tonus (6) in comparison with wt animals (1,3,5). (E) Assessment of body weight with age (n = 11–12 for each group). Asterisks indicate significant differences between wt control and CMVMJD135 transgenic mice. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

Fig. 2

Body balance impairment in CMVMJD135 mice. Balance beam test performance of wt and transgenic animals using square (A,B) and round beams (C–E) is depicted at 10 and 22 weeks of age. Values are presented as mean ± SEM (n = 8–13 for each genotype and gender). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Fig. 3

Abnormal footprint pattern in transgenic mice. (A) Representative walking footprint patterns of 14-, 28- and 41-week-old CMVMJD135 mice. Qualitatively, the patterns generated clearly differ, showing that transgenic mice display foot-dragging. At late stages CMVMJD135 mice also demonstrate shorter strides and an irregular left–right step pattern as compared with the wt control mice. (B) Quantitative analysis of foot-dragging. Quantitative analysis of the footprint patterns of wt and transgenic mice, based on measurements of (C) distance between front and hind footprint overlap and (D) stride length through age. Values are presented as mean ± SEM (n = 7 and 10 for wt and transgenic, respectively). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Fig. 4

Motor incoordination on the rotarod and in the swimming tank in CMVMJD135 mice. (A) 22–25- and 40-week-old wt and CMVMJD135 male mice (n = 7–15) were tested for motor coordination in the swimming tank, by assessing the latency to swim over a 60-cm distance to a visible escape platform. Transgenic mice displayed swimming impairments given by a significant increase in the time spent to cross the 60-cm distance. Rotarod analysis of CMVMJD135 and wt animals were tested in (B) an accelerating rod (4–40 rpm) and (C) at constant speeds. The mean ± SEM of the latency to fall at each speed level was recorded. Rotarod deficit was present at 20 weeks of age and older (shown for 40 weeks); CMVMJD135 transgenic mice displayed a progressive decline in performance on the rotarod with increasing rotation speed. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Fig. 5

Phenotype of CMVMJD135 mice detected in the SHIRPA protocol. (A) Strength and fine motor coordination evaluation in the hanging wire grip test, measured by the latency to fall off the grid. Values are presented as mean ± SEM (n = 11–12 for each group). (B) At 19 weeks of age animals demonstrated hindlimb tonus alterations, (C) a decrease in forelimb grip strength and (D) a higher percentage of animals displaying clasping of the hindlimbs when compared to controls. Values are presented as percentage of animals with different scores in the SHIRPA protocol (n = 11–12 for each group). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Fig. 6

Neuropathology of CMVMJD135 mice. (A) Brain weight analysis at 42–43 weeks of age of wt (n = 5) and CMVMJD135 male mice (n = 4). (B) Volume and total cell number quantification in different brain regions of transgenic (n = 9) and control (n = 5) male animals using stereological methods. (C) Anti-ataxin-3 immunohistochemistry (rabbit anti-MJD1.1) of wt (a-c) and CMVMJD135 mice (d-f) at approximately 20–35 weeks of age. CMVMJD135 mice exhibited nuclear and cytoplasmic inclusions positive for ataxin-3 in different regions of the CNS, namely, pontine nuclei (d), reticulotegmetal nucleus of pons (e) and spinal cord neurons (f); Cresyl violet brain sections in the pontine nuclei of wt (g) and in CMVMJD135 (i) mice. Higher magnification of neuronal cells in the wt (h) and transgenic mice (j). Images are representative of the results obtained for six animals per genotype, three each female and male. Scale bar: 20 μm

Fig. 7

Fig. 7

17-DMAG improves motor coordination of CMVMJD135 mice. (A) Motor swimming test was performed at 16, 22, 24 and 30 weeks of age (n = 10–12 animals for each tested group) and the mean latency of the third day test was analyzed. Transgenic mice displayed swimming impairments, given by a significant increase in the time spent to swim across 60 cm distance, and 17-DMAG treatment improved their performance. (B) Rotarod test was performed at 22 and 30 weeks of age (n = 10–12 for each tested group) to evaluate motor coordination at constant speed in the rod (8, 15, 20, 24 and 31 rpm). 17-DMAG treatment was able to rescue this phenotype at 22 weeks of age. (C) Balance beam test was performed at 14, 16, 22, 24 and 30 weeks of age (n = 10–12 for each tested group. 17-DMAG treatment significantly improved balance deficits at 22 and 24 weeks of age. Values are presented as mean ± SEM. (D) Quantitative analysis of the percentage of animals showing foot-dragging. 17-DMAG treatment delays the onset and progression of this symptom. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 8

Fig. 8

17-DMAG treatment reduced the levels of human ataxin-3 and the aggregate load in CMVMJD135 mice brain. (A) Neuronal inclusions were counted in the pontine nuclei of 30-week-old animals treated with vehicle or 17-DMAG (n = 3 for each condition). Four slides of each animal were used for the analysis. (B) qRT-PCR analysis of human ATXN3 mRNA expression levels (n = 4). (C) Anti-ataxin-3 western-blot (rabbit anti-MJD1.1) of 30-week-old CMVMJD135 mice, either vehicle or treated with 17-DMAG (n = 4 for each condition), were performed in the brainstem. Mutant human ataxin-3 has a molecular weight of approximately 80 kDa. Values are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 9

Fig. 9

17-DMAG is able to induce autophagy in CMVMJD135 animals. ELISA assays, western blots and qRT-PCR analysis of HSR and autophagy markers were performed in the brainstem or forebrain of 16-, 20- or 30-week-old wt and CMVMJD135 mice (n = 4–5) treated with vehicle or 17-DMAG (25 mg/kg), 12 hours after the last treatment. (A) Hsp70, Hsp40 mRNA expression levels were normalized for the housekeeping gene HPRT. (B) ELISA assay for Hsp70 quantification. Anti- Beclin-1 (C) and anti-LC3 (D) western-blots. LC3-II was normalized both for tubulin and LC3-I; Beclin-1 was normalized for tubulin. Values are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001

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