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Exercise and Genetic Rescue of SCA1 via the Transcriptional Repressor Capicua

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Science  04 Nov 2011:
Vol. 334, Issue 6056, pp. 690-693
DOI: 10.1126/science.1212673

Abstract

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by expansion of a translated CAG repeat in Ataxin-1 (ATXN1). To determine the long-term effects of exercise, we implemented a mild exercise regimen in a mouse model of SCA1 and found a considerable improvement in survival accompanied by up-regulation of epidermal growth factor and consequential down-regulation of Capicua, which is an ATXN1 interactor. Offspring of Capicua mutant mice bred to SCA1 mice showed significant improvement of all disease phenotypes. Although polyglutamine-expanded Atxn1 caused some loss of Capicua function, further reduction of Capicua levels—either genetically or by exercise—mitigated the disease phenotypes by dampening the toxic gain of function. Thus, exercise might have long-term beneficial effects in other ataxias and neurodegenerative diseases.

Spinocerebellar ataxia type 1 (SCA1) is characterized by a progressive loss of motor skills, usually beginning with impaired gait and balance (1). As with other neurodegenerative diseases, the disease protein Ataxin-1 (ATXN1) is abundantly expressed in most neurons, yet some neuronal populations are more vulnerable than others. In SCA1, cerebellar Purkinje cells are first to show dysfunction; eventually, other neuronal populations—including deep cerebellar and brainstem nuclei—are affected, leading to premature death (2). Although exercise has beneficial effects on many brain functions (3), it is not clear whether it would be protective in SCA1 or would accelerate neuronal demise by increasing the activity and metabolic demands on these already vulnerable neuronal populations, as has been suggested for other neurodegenerative diseases (4, 5).

To determine the effects of exercise in SCA1, we implemented a mild exercise regimen in Atxn1154Q knock-in mice, which bear 154 CAG repeats targeted into the endogenous mouse locus so as to create a model that recapitulates all aspects of SCA1 (6). From 4 to 8 weeks of age, wild-type (WT) or Atxn1154Q mice were placed on a fixed-speed rotarod apparatus five times per week, whereas control mice were placed on an immobile rotarod apparatus. At 10 weeks of age, we found no significant improvements in motor performance (fig. S1, A and B), but Atxn1154Q mice that were exercised showed a remarkable and highly significant extension of life span (Fig. 1A).

Fig. 1

Exercise extends life span in Atxn1154Q mice through up-regulation of EGF and consequential down-regulation of Cic. (A) Median survival was 272 days for nonexercised Atxn1154Q mice versus 317 days for exercised Atxn1154Q mice (45-day extension, n = 6 mice per group, P < 0.01 by log-rank test). (B) Exercise caused a 50% increase in brainstem EGF levels in WT mice and a 74% increase in Atxn1154Q mice measured with enzyme-linked immunosorbent assay (mean ± SEM, n = 5 mice per group). (C) Exercise caused a 24 and 22% decrease in brainstem Cic levels in WT and Atxn1154Q mice, respectively, measured with quantitative reverse transcription polymerase chain reaction RT-PCR (mean ± SEM, n = 5 mice per group). (D) Western blotting for Cic demonstrates an exercise-induced decrease in Cic levels in the brainstem, whereas Atxn1 or Atxn1154Q remain unaffected. (E) Primary brainstem neuronal cultures treated with recombinant EGF for 72 hours show a dose-dependent decrease in the level of Cic but not Atxn1.

To determine the molecular mechanism of this rescue, we measured the expression of several growth factors in vulnerable tissues (cerebellum and brainstem) 1 week after the last exercise session. We found a sustained increase in the level of epidermal growth factor (EGF) in the brainstem but not the cerebellum (Fig. 1B and fig. S1). Because Drosophila data indicate that Capicua (Cic) lies downstream of EGF signaling (7), and we previously showed that Cic interacts with Atxn1 (8), we measured Cic levels after exercise and found a significant decrease in the brainstem (Fig. 1, C and D) but not the cerebellum (fig. S1B). Exercise did not affect brainstem Atxn1154Q protein levels (fig. S1F). Additionally, primary brainstem neuronal cultures treated with recombinant EGF for 72 hours showed a dose-dependent decrease in Cic levels (Fig. 1E). Thus, EGF regulates Cic levels in vivo and in vitro, and reduction of Cic might modulate the survival of Atxn1154Q mice.

To formally test the effect of reduced Cic levels on SCA1 phenotypes, we generated a loss-of-function allele of Cic and back-crossed these mice onto a C57BL/6 strain (fig. S2A). Two isoforms of Cic, long (Cic-L) and short (Cic-S), are transcribed from alternative promoters. Cic-L−/− mice completely lack the Cic-L isoform, with ~10% of Cic-S remaining (fig. S2B). Cic-L+/− mice had a ~50% reduction of both Cic-L and Cic-S. Cic protein is reduced in the Atxn1−/− cerebellum (8), and we also found a Cic dose-dependent reduction of Atxn1 and its paralog Ataxin-1–Like (Atxn1L) in WT, Cic-L+/−, and Cic-L−/− cerebella (fig. S2B), confirming the interdependency of Cic and Atxn1 paralog proteins in vivo.

To determine whether reducing the level of Cic would affect disease course in SCA1, we bred Atxn1154Q male mice to Cic-L+/− female mice in order to generate WT, Cic-L+/−, Atxn1154Q, and Atxn1154Q;Cic-L+/− mice, all on a pure C57BL/6 background. Because of their motor impairments, Atxn1154Q mice showed less total locomotor activity in the open-field assay than did WT mice, but Atxn1154Q;Cic-L+/− mice performed significantly better than did their Atxn1154Q littermates (Fig. 2A). Reduced Cic levels also significantly improved the motor coordination defects normally seen in Atxn1154Q mice (Fig. 2, B and C). Reduced Cic levels also improved the learning and memory deficits that Atxn1154Q mice normally exhibit in the conditioned fear assay (Fig. 2D). These improved phenotypes were accompanied by reduction in neuropathology: Atxn1154Q;Cic-L+/− mice had significantly more Purkinje cells than did Atxn1154Q mice at 40 weeks of age (Fig. 3, A and B). Thus, a 50% reduction of Cic levels is enough to mitigate the behavioral defects and Purkinje cell loss in Atxn1154Q mice.

Fig. 2

Reduction of Cic rescues multiple behaviors in SCA1 mice. Reduction of Cic by 50% in Atxn1154Q mice resulted in motor improvements in the (A) open-field assay, (B) dowel test, (C) rotarod, and (D) conditioned fear assay of learning and memory. Legend for each panel is indicated in (B). Values represent mean ± SEM with n = 9 to 10 mice per genotype. Analysis of variance (ANOVA) with post-hoc t tests were performed. #P < 0.05, ##P < 0.01 between WT and Atxn1154Q mice; *P < 0.05, **P < 0.01 between Atxn1154Q and Atxn1154Q;Cic-L+/− mice.

Fig. 3

Reduction of Cic rescues multiple phenotypes in SCA1 mice. A 50% reduction of Cic in Atxn1154Q mice rescued (A) and (B) Purkinje cell integrity (n = 4 mice per genotype), (C) body weight, and (D) premature lethality (P < 0.05 by log-rank test). The median life span of Atxn1154Q mice in this cohort is in close agreement with the median life span from the unexercised Atxn1154Q cohort (Fig. 1), with some increased variability due to greater numbers of litters being used in this larger cohort. For body weight and lethality, n = 11 WT mice, n = 12 Cic-L+/− mice, n = 14 Atxn1154Q mice, and n = 16 Atxn1154Q;Cic-L+/− mice.

Reduction of Cic levels also improved phenotypes that could be attributed to other brain regions. After weaning, Atxn1154Q mice lost weight progressively, but loss of one Cic allele was enough to significantly rescue this weight loss (Fig. 3C). As with the exercised Atxn1154Q mice, the median age of survival of the Atxn1154Q;Cic-L+/− mice was significantly older than that of Atxn1154Q mice (Fig. 3D). These data probably explain why exercise improved the longevity of Atxn1154Q mice but not the impaired coordination: Exercise reduced Cic levels only in the brainstem and not in the cerebellum.

To determine the mechanism by which constitutive reduction of Cic rescues SCA1, we focused on the cerebellum, the primary site of dysfunction. We examined how the Atxn1154Q protein influenced the transcriptional repressor function of Cic by comparing microarrays of Cic-L−/− cerebella (table S1) with previous microarrays from Atxn1154Q cerebellum (9). We identified many “hyper-repressed” genes (up-regulated in Cic-L−/− but down-regulated in Atxn1154Q), with >50% containing a Cic motif (table S2) (10). We selected several of these hyper-repressed target genes and found that genes that were down-regulated in Atxn1154Q cerebellum were in fact restored to near WT levels in Atxn1154Q;Cic-L+/− cerebellum, with significantly more Cic bound to their promoters in Atxn1154Q versus WT mice (Fig. 4A). This suggests that the mechanism of disease rescue is relief of Cic hyper-repression conferred by polyglutamine-expanded Atxn1.

Fig. 4

Atxn1154Q causes concomitant gain and loss of Cic transcriptional function. (A) Atxn1154Q hyper-repressed cerebellar transcripts containing a Cic motif, but these were normalized to near WT levels in Atxn1154Q;Cic-L+/− mice as measured with quantitative RT-PCR (mean ± SEM, n = 6 mice per genotype), with more Cic bound to their promoters in Atxn1154Q versus WT littermates as measured with chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR, mean ± SEM, n = 4 mice per genotype). (B) Atxn1154Q also derepressed cerebellar transcripts containing a Cic motif, but these transcripts were further up-regulated in Atxn1154Q;Cic-L+/− mice through quantitative RT-PCR (mean ± SEM, n = 6 mice per genotype), with less Cic bound to their promoters in Atxn1154Q versus WT littermates as measured with ChIP-qPCR (mean ± SEM, n = 4 mice per genotype). Data analyzed by means of ANOVA with post-hoc t test. *P < 0.05 between WT and Atxn1154Q; #P < 0.05 between Atxn1154Q and Atxn1154Q;Cic-L+/− mice.

In Drosophila, overexpression of Atxn182Q is rescued by overexpression of Cic and is exacerbated by Cic reduction (8, 11). Although this is likely due to a titration of the endogenous Drosophila Cic away from its normal transcriptional targets, it suggests that polyglutamine-expanded Atxn1 could cause a loss of Cic transcriptional function (derepression). We identified many “de-repressed” genes [up-regulated in both Cic-L−/− and Atxn1154Q cerebella (table S3)] that were in fact even further up-regulated in Atxn1154Q;Cic-L+/− cerebellum, with significantly less Cic bound to their promoters in Atxn1154Q versus WT mice (Fig. 4B). This suggests that polyglutamine-expanded Atxn1 does indeed cause Cic to lose some transcriptional repressor activity, but this could not explain the genetic rescue; if partial loss of Cic activity was the driving factor in pathogenesis, the disease phenotype would be exacerbated in Atxn1154Q;Cic-L+/− mice. We propose that the polyglutamine-expanded Atxn1 causes Cic to bind more tightly to certain transcriptional targets (hyper-repressing them) while concomitantly causing Cic to bind less to—and thus up-regulate (derepress)—other transcriptional targets. Genetic or exercise-induced reduction of Cic relieves the toxic hyper-repressive activity despite the concomitant loss of normal repressive function (fig. S3), which is consistent with the fact that the gain-of-function mechanism is the driving mechanism for toxicity in SCA1. Whereas other polyglutamine diseases are caused by a gain-of-function mechanism despite partial loss of function of the involved protein mediated through different protein partners (9, 1113), here we have demonstrated that polyglutamine-expanded protein can cause concomitant gain- and loss-of-function effects on the same native protein partner. The level of polyglutamine-expanded Atxn1 protein was reduced by ~9% in Atxn1154Q;Cic-L+/− mice as compared with Atxn1154Q mice (fig. S2, C and D), and although this could possibly contribute to the phenotypic rescue, we suggest that the rescue is more likely caused by relief of Cic-dependent hyper-repression. Therapeutics aimed at lowering Cic levels or disrupting the Cic-Atxn1154Q protein interaction could potentially ameliorate the disease.

The effect of exercise on life span was long lasting, well after its discontinuation, underscoring the potential value of exercise beyond motor improvements. Thus, SCA1 individuals might benefit from an exercise program early in disease course (14, 15). Genetic reduction of Cic also increased the life span of Atxn1154Q mice (by 3.5 weeks), but the magnitude of the survival effect was greater (~6 weeks) in exercised Atxn1154Q mice. Thus, other pathways such as enhanced growth factor signaling are likely to contribute to the effect. We cannot rule out the possibility that more intense or longer-duration exercise could cause a sustained EGF increase and Cic decrease in the cerebellum that could lead to motor improvements. The exercise regimen we chose was quite gentle; a more rigorous or sustained exercise paradigm that engages the cerebellum more intensely might reduce cerebellar Cic levels and improve cerebellar phenotypes. It is encouraging that exercise and the accompanying increase in neuronal activity and metabolic demands do not seem to exacerbate the disease process in vulnerable neuronal populations, which may be important in a variety of neurodegenerative disorders.

Supporting Online Material

www.sciencemag.org/cgi/content/full/334/6056/690/DC1

Materials and Methods

Figs. S1 to S3

Tables S1 to S4

References (16–18)

References and Notes

  1. Acknowledgments: J.D.F. and H.Y.Z. conceived of the study and designed experiments. J.D.F., C.M.B., A.N.C., and Y.G. performed behavioral assays and provided input on analysis. J.D.F. and J.C.-B. performed molecular work and analysis. P.Y., H.K., and C.S. analyzed microarray data. J.D.F. and H.Y.Z. wrote the manuscript with input from J.C.-.B, A.F., and H.T.O. We are grateful to G. Schuster for the generation of mutant mice and the members of the H.Y.Z. laboratory for comments and discussions on the manuscript. This research was supported by NIH grants NS27699, NS27699-20S1–ARRA, and HD24064 (Baylor College of Medicine–Intellectual and Developmental Disabilities Research Center) to H.Y.Z.; 1F32NS055545 to J.D.F.; and NS022920 and NS045667 to H.T.O. H.Y.Z. is an investigator with the Howard Hughes Medical Institute, holds a patent on SCA1 diagnostic testing, and is on the scientific advisory board of Pfizer Neuroscience Program.
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