Disease progression despite early loss of polyglutamine protein expression in SCA7 mouse model

Abstract

Nine neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxia type 7 (SCA7) are caused by an expansion of a polyglutamine (polyQ) stretch in the respective proteins. Aggregation of expanded polyQ-containing proteins into the nucleus is a hallmark of these diseases. Recent evidence indicates that transcriptional dysregulation may contribute to the molecular pathogenesis of these diseases. Using SCA7 and HD mouse models in which we recently described a retinal phenotype, we investigated whether altered gene expression underlies photoreceptor dysfunction. In both models, rhodopsin promoter activity was early and dramatically repressed, suggesting that downregulation of photoreceptor-specific genes plays a major role in polyQ-induced retinal dysfunction. Because the rhodopsin promoter drives mutant ataxin-7 expression in our SCA7 mice, we also assessed whether downregulation of mutant SCA7 transgene would reverse retinopathy progression and aggregate formation. Although residual expression of mutant ataxin-7 was found negligible from 9 weeks of age, SCA7 transgenic mice showed a progressive decline of photoreceptor activity leading to a complete loss of electroretinographic responses from 1 year of age. At this age, aggregates were cleared in only half of the photoreceptors, indicating that their formation is not fully reversible in this model. We demonstrate here that abolishing full-length mutant ataxin-7 expression did not reverse retinopathy progression in SCA7 mice, raising the possibility that some polyQ-induced pathological events might be irreversible.

Figure 1.

PolyQ expansions downregulate normal recombinant ataxin-7 in R7E and R6 retinas. A-E, Immunofluorescence analysis of 10 μm retinal cryosections from 10-week-old R7N (A), R7E (B), R7N-R7E (C), R6/2 (D), and R7N-R6/2 (E) mice using anti-ataxin-7 (2A10 in red; A-C, E) and anti-Huntingtin (1259 in green; D, E) antibodies. Nuclei were counterstained with DAPI (blue), and merged images are shown. Normal recombinant ataxin-7 displayed a homogenous nuclear ring-like staining (A), whereas mutant ataxin-7 (B) or mutant Huntingtin (D) formed aggregates. Rod photoreceptors from double transgenic animals contained only NIs (C, E). A small number of rods contained Huntingtin aggregates together with ataxin-7 [Q₁₀] (E, arrowhead). Scale bar, (A-F) 10 μm. F, Western blot analysis of retinal extracts from R7N, R7N-R7E, and R7N-R6/2 animals at 5 and 10 weeks of age probed with anti-ataxin-7 (1C1) antibody. Anti-actin antibody was used as an internal loading control. PolyQ expansion in ataxin-7 or Huntingtin induced an early decrease of normal recombinant ataxin-7 that progressively led to undetectable levels in retinal homogenates from 10-week-old R7N-R7E and R7N-R6/2 mice.

Figure 2.

PolyQ expansions downregulate recombinant ataxin-7 but not endogenous ataxin-7 expression. A, Northern blots of total RNA isolated from R7N, R7E, and R7N-R7E retinas at 3, 5, and 9 weeks of age were hybridized with SCA7 and Gapd probes. At 3 weeks of age, high levels of transgene mRNA were detected in single and double transgenic animals. Later, SCA7 transgene expression was sustained in R7N mice but severely reduced in R7N-R7E mice. B, Real-time RT-PCR analysis of total RNA extracted from 32 week R7N and R7N-R6/1 retinas, using primers specific for recombinant human SCA7 and Hprt as an internal control. Accordingly, we observed a reduced expression of normal SCA7 transgene in R7N-R6/1 mice. SCA7 mRNA level is quantified as a percentage of R7N mice, after normalization to Hprt levels. Each bar represents the mean value ± SEM (n = 2). C, Quantification of endogenous mouse Sca7 expression in wild-type and R7E mice at 9 weeks of age by real-time RT-PCR using primers specific for endogenous Sca7 and Arbp as an internal control. No difference could be detected between wild-type and R7E animals at 10 weeks of age. Sca7 levels are represented as a percentage of the mean of wild-type littermate mice after normalization to Arbp levels. Each bar represents the mean value ± SEM (n = 2). Each panel is representative of at least two independent experiments performed on different mice. WT, Wild type.

Figure 3.

Rhodopsin mRNA (Rho) and protein levels are severely reduced in SCA7 and HD mice. A, Northern blot of total RNA extracted from R7N and R7E mice at 3, 6, and 9 weeks of age was hybridized with Rho and Gapd probes. Constant expression of Rho in aging R7N mice contrasted with a progressive diminution of Rho mRNA in R7E mice from 3 to 9 weeks of age. B, Quantification of Rho expression in wild-type, R7E (at 3.5, 6, and 9 weeks of age), homozygous R7E (at 9 weeks of age), and R6/2 mice (at 10 weeks of age) (n = 3 for each group) by real-time RT-PCR using primers specific for Rho and Ppia as an internal control. This time-course analysis revealed that Rho downregulation was progressive, leading to a fivefold reduction of Rho mRNA levels from 6 weeks of age. Downregulation of Rho mRNA in homozygous 9-week-old R7E mice is approximately twice that in age-matched heterozygous R7E mice. A similar downregulation of Rho expression was detected in 10-week-old R6/2 mice. Rho levels are represented as a percentage of the mean of wild-type littermate mice (n = 12) after normalization to Ppia levels. Each bar represents the mean value ± SEM (n = 3). C, Western blot analysis of retinal homogenates from R7N, R7E, and R6/2 animals at 10 weeks of age, using anti-rhodopsin and anti-actin antibodies, confirmed this dramatic reduction of Rho expression at the protein level. Each panel is representative of at least two independent experiments performed on different mice. WT, Wild type.

Figure 4.

Early loss of mutant SCA 7 expression contrast with continuous worsening of retinal dysfunction. A, Real-time RT-PCR analysis of total RNA extracted from R7E retinas at 3, 6, 9, 20, 45, and 100 weeks of age using primers specific for recombinant human SCA7 and Arbp as an internal control. Maximal levels of mutant transgene expression were detected at 3 weeks of age when photoreceptor differentiation was complete. Early and dramatic downregulation of mutant SCA7 mRNA was detected from 6 weeks of age and led to negligible levels of expression. SCA7 mRNA levels are quantified as a percentage of 3-week-old R7E mice after normalization to Arbp. Each bar represents the mean value ± SEM (n = 4-6). B, Scotopic ERG response of wild-type and R7E mice at 3.5, 6, 9, 14, 29, 53, 79, and 100 weeks of age. A complete ERG time-course analysis of R7E mice revealed a progressive and continuous worsening of rod photoreceptor function. Rod activity is quantified as the mean amplitude of the a-wave recorded at the four maximal light stimulus intensities (from 0 to 1.4 log cd sec m-2). Each bar represents the mean value ± SEM (n = 4-7).

Figure 5.

Long-term rod photoreceptor dysfunction eventually leads to a late onset and limited neurodegeneration. A, Time-course analysis of scotopic ERG response from wild-type and R7E mice at 5, 9, 26, 52, and 100 weeks of age. Each recording corresponds to the average response of one animal at maximal light intensities (from 0 to 1.4 log cd sec m-2). Single recordings revealed a progressive decrease of rod electrophysiological activity up to flat responses. B, C, Progression of retinopathy in aging R7E mice assessed by histological (B) and clinical (C) examination. Decrease of ERG response was concomitant to progressive thinning of the segment layer. Histologically, the photoreceptor layer showed first abnormalities at 5 weeks of age and was then completely disrupted by numerous whorls. Later, flattening of the ONL was concordant with the disappearance of white spots by funduscopy performed in 100-week-old R7E mice that showed areas of abnormal pigmentation. Each panel is representative of observations performed on at least three other mice. WT, Wild type; RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; INL, inner nuclear layer. Scale bar, (A-C) 40 μm.

Figure 6.

Partial disappearance of mutant ataxin-7 NIs in the photoreceptor nuclear layer of 52-week-old R7E mice. DAB immunostaining was performed on 5 μm paraffin-embedded retinal sections from 26-week-old (top panel) and 52-week-old (bottom panel) R7E mice using anti-ataxin-7 (1261) antibody. Although aggregates were present in most rods at 26 weeks of age, the proportion of rods bearing NIs was reduced to approximately one-half in 52-week-old R7E mice. Counterstaining with methyl green revealed a significant number of rod nuclei without any ataxin-7 immunoreactivity. Thus, partial aggregate clearance is not attributable to photoreceptor cell loss. Scale bar, 20 μm.

Figure 7.

Time-course assessment of phenotypic and molecular events occurring during R7E mice lifespan. High expression of mutant ataxin-7 in rod photoreceptors is restricted to a narrow time window of ∼3-4 weeks. Photoreceptor differentiation is normal in R7E mice, because we did not detect any abnormalities until 3 weeks of age. Altered rhodopsin expression, concomitant with decreased rod ERG response, is the earliest anomaly and occurred at 3-4 weeks of age. At 8 weeks of age, most rods contain NIs, and the photoreceptor layer is disrupted by numerous whorls. From this time point, drastic reduction of mutant SCA7 expression leads to partial aggregate reversion. Despite negligible influx of mutant protein, we observed a progressive and complete loss of rod normal function that strikingly does not immediately lead to their degeneration.