The JunAA mutation slows thinning of photoreceptor segments in R7E animals. Immunofluorescence microscopy of retinal cryosections of JunAA-R7E, CT-R7E, JunAA and CT mice. a, Sections were stained with the lectin PNA. b, sections were double stained with anti-rhodopsin antibody (red) and anti-ataxin-7 antibody (green). Nuclei are revealed with DAPI. S, segments; ONL, outer nuclear layer; INL, inner nuclear layer. Thickness of the segments had a tendency to be less reduced in JunAA-R7E than in CT-R7E mice (p=0,08), and no obvious difference was observed between JunAA and CT mice (JunAA-R7E: 21,45 μM +/−4,50, n=3; CT-R7E: 14,97 μM +/−3,66, n=3; CT: 30,63 μM +/−2,12, n=4; JunAA: 32,85 μM +/−5,51, n=4). Statistical analyses were performed using Kruskal-Wallis test followed by Wilcoxon test for post-hoc comparisons

PolyQ-ataxin-7 expression, which is regulated by an Nrl-dependent promoter, is upregulated in JunAA-R7E mice. a, Real-time RT-PCR analysis of total RNA extracted from 3 month JunAA-R7E and CT-R7E animals, using primers specific for recombinant SCA7 and 36B4, as an internal control. JunAA-R7E SCA7 mRNA level was quantified as a percentage of CT-R7E mRNA level. PolyQ-ataxin-7 expression was two-fold increased in JunAA-R7E mice (JunAA-R7E: n=7; CT-R7E: n=5; *, p<0.01, Student’s t-test). b, Western-blotting analysis of insoluble fractions prepared from 3 month CT, JunAA-R7E and CT-R7E retinas, using the 1C2 antibody which detects polyQ. PolyQ-ataxin-7 accumulated as a cleaved insoluble fragment. Accumulation of polyQ-ataxin-7 was more prominent in JunAA-R7E than in CT-R7E animals. c, Real-time RT-PCR analysis of total RNA extracted from 4.5 month JunAA-R7E and CT-R7E animals, using primers specific for Nrl, Crx, Rho, Pdeb and 36B4, as an internal control. JunAA-R7E and CT-R7E mRNA levels were expressed as a percentage of CT level (JunAA-R7E: n=4; CT-R7E: n=5; CT: n=2). Expressions of Nrl, Crx, Rho and Pdeb were not different in JunAA-R7E and CT-R7E animals. d, Analysis of the rod response of JunAA, CT, JunAA-R7E and CT-R7E animals at 4.5 months (JunAA: 175.42 μv +/− 11.11, n=6; CT: 144.52 μv +/− 8,93, n=11, including 7 Junaa/wt and 4 WT, JunAA-R7E: 62.05 μv +/− 3.11, n=7; CT-R7E: 62.91 μv +/− 8.83, n=10, including 7 Junaa/wt-R7E and 3 R7E; *, p<0,05, Kruskal-Wallis followed by Wilcoxon test).

c-Jun phosphorylation modulates rod photoreceptor activity. a,JNK-mediated phosphorylation of c-Jun is abolished in SCA7 transgenic mice expressing mutated c-Jun. Western-blotting analysis of whole cell protein extracts from 3 month old retinas of JunAA transgenic (JunAA-R7E), R7E and control (CT) animals, using anti-phospho-cJun and anti-phospho-JNK antibodies. Antibody against β-tubulin was used as a loading control. b, The JunAA mutation induces an increase in rod photoreceptor activity. Intensity-response curves of the dark-adapted ERG a-wave at 3 months of age for three R7E mice, 5 R7E mice heterozygous for the JunAA mutation (Junaa/wt-R7E), 6 R7E mice homozygous for the JunAA mutation (Junaa/aa-R7E), three wild-type mice (WT), 10 mice heterozygous for the JunAA mutation (Junaa/wt) and 8 mice homozygous for the JunAA mutation (Junaa/aa). c, Comparison of the rod response between JunAA-R7E (Junaa/aa-R7E) and CT-R7E (Junaa/wt-R7E and R7E were pooled to constitute the CT-R7E group), and between JunAA (Junaa/aa) and CT (Junaa/wt and WT were pooled) at 3 months of age. Junaa/wt-R7E and R7E, as well as Junaa/wt and WT animals were not statistically different. Each bar represents the amplitude value calculated by averaging the last 4 dark-adapted ERG amplitudes measured at maximum light intensities and represented on figure 1b (JunAA-R7E: 96.99 μv +/− 3.84 μv, n=6; CT-R7E: 63.59 μv +/− 7.32, n=8; JunAA: 201.00 μv +/− 16.15, n=8; CT: 169.75 μv +/− 8.22, n=13). Statistical analyses were performed using Kruskal-Wallis test followed by Wilcoxon test for post-hoc comparisons; *, p<0,05.

c-Jun phosphorylation contributes to the regulation of Nrl expression. a, Real-time RT-PCR analysis of total RNA extracted from adult (4.5 months) JunAA and CT retinas, using primers specific for Nrl, Crx, Rho, Gnat1, Pdeb and RhoK. Primers amplifying 36B4 or cyclophilin were used as internal controls. JunAA mRNA levels are quantified as a percentage of CT mRNA, after normalization to internal control level. Each bar represents the mean value +/− SEM (JunAA: n= 3; CT: n=5). Nrl, Rho, Gnat1 and Pdeb expressions were significantly increased in JunAA mice. Stastistical analysis was performed using a Student’s t-test. *, p<0.05. Similar results were obtained at 3 months (not shown). b, Real-time RT-PCR analysis of total RNA extracted from 3 month JunAA-R7E and CT-R7E retinas. JunAA-R7E and CT-R7E mRNA levels were expressed as a percentage of CT mRNA, after normalization to internal control level (JunAA-R7E: n=7; CT-R7E: n=5, CT: n=2). Nrl and Pdeb expressions were significantly increased in JunAA-R7E mice, compared to CT-R7E. **, p<0.01, Student’s t-test. c and d, Western-blotting analysis of whole cell protein extracts from 3 month JunAA-R7E, CT-R7E, JunAA and CT retinas, using anti-Nrl antibody (c) and anti-rhodopsin antibody (d). Anti-β-tubulin antibody was used as control. The anti-Nrl antibody detected multiple bands, the upper bands corresponding to phosphorylated forms of Nrl. Fold-change of Nrl and Rho levels were calculated from 3 to 6 retinas for each mouse genotype. Nrl and Rho are increased in JunAA, when compared to CT mice (p<0.001 and p<0.05, respectively). Nrl is also increased in JunAA-R7E, when compared to CT-R7E mice (p<0.05), while Rho is not statistically different between JunAA-R7E and CT-R7E mice (p=0.16). Statistical analyses were performed using Kruskal-Wallis test followed by Wilcoxon test for post-hoc comparisons

DNase footprinting and reporter assays of the Nrl promoter. a, DNase I footprinting of the proximal mouse Nrl promoter using bovine retinal nuclear extract. Footprints are marked by lines. and numbered I-1 through I-7. (−), no RNE; (+) with RNE. The footprints corresponding to the EboxAP1/CRE site and the Ret1/PCEI AP-1 site are I-5 and I-6 respectively. b, DNA sequences of the proximal mouse Nrl promoter. Boxed regions indicate DNase footprints and brackets show presumed binding sites for transcription factors. The location of the putative AP1 sites TGATCTCA and TCATCTTATTGGA are denoted by the letters ‘A’ and ‘B’, respectively. c, Luciferase experiments testing the effect of Jun or JunAA on wildtype (Nrl-l) and mutant (Nrl-l-mAP1B) promoter. Nrl-l-mAP1B lacks the putative AP1 site TCATCTTATTGGA. Addition of Jun (Jun Nrl-l) or JunAA (JunAA Nrl-l) slightly decreased luciferase activity of the Nrl promoter. Mutation of the putative AP1 site eliminated the repressive effect of Jun (Jun Nrl-l mutB) and JunAA (JunAA Nrl-l mutB). Activities that are statistically different from controls (Student’s t-test, p<0.001) are marked with an asterisk. d, Western-blotting analysis of whole cell protein extracts from 3 month JunAA-R7E, CT-R7E and CT retinas, using anti-c-Jun antibody. The anti-β-tubulin antibody was used as loading control. C-Jun protein is increased in SCA7 transgenics, compared to wild-type mice, and the JunAA mutation results in decreased levels of c-Jun protein in retinas of SCA7 animals. Fold-change of c-Jun levels were calculated from 2 to 3 retinas for each mouse genotype.