Fig 2: Transgenic mice with conditional overexpression of SREK1 in forebrain neurons display global alteration of Huntington’s disease-related transcripts. (A) Double transgenic mouse line TgSREK1 was generated by crossing tTA-expressing mice under the control of CamKII promoter (CamKII-tTA mice) with mice carrying a tTA-responsive SREK1 construct (β-Gal-BiTetO-SREK1 mice). (B) Immunohistochemistry with anti-β-Gal antibody in sagittal sections from a 1.5-month-old TgSREK1 mouse and a single-transgenic β-Gal-BiTetO-SREK1 control mouse showing transgenic expression in cortex (Cx), striatum (St) and hippocampus (Hipp) of TgSREK1 mice, but not in non-forebrain structures like thalamus (Thal). Scale bar = 500 μm. (C) Immunoblot of SREK1 protein levels in striatum and cortex of 1.5-month-old TgSREK1 and wild-type (WT) mice, and its quantification with respect to β-actin (Student’s t-test; **P < 0.01). (D) GSEA analysis showing landscape of enrichment score (green line) for the KEGG molecular signature corresponding to ‘Huntington’s disease’. Black vertical bars above the red and blue regions correspond to upregulated and downregulated transcripts, respectively in TgSREK1 mice with respect to wild-type. (E) The different types of alternative splicing events and the abundance of those with an absolute dPSI > 10 between wild-type and TgSREK1 mice. (F) Diagram of the short (e5short) and long (e5long) versions of exon 5 (E5) of TAF1 generated by an alternative 3′ splice site (Alt3′ ss). The e5long version carries 63 additional nucleotides (+63 nt) on its 5′ site. (G) Histogram shows PSI of the e5long and e5short events observed by RNA-seq in 3.5-month-old wild-type (n = 3) and TgSREK1 (n = 3) mice. (H) Ratios in 3.5-month-old wild-type (n = 7) and TgSREK1 (n = 5) mice of the PSI values of e5long over e5short obtained by RNA-seq and of e5long over e5short transcript isoforms by qRT-PCR. Data represent mean + SEM. AS = alternative splicing.

Fig 3: Knockdown of SRSF6 in SH-SY5Y cells leads to decreased SREK1 levels with a subsequent decrease in TAF1. (A) Representative western blots of SRSF6, SREK1 and TAF1 proteins in SH-SY5Y cells upon transduction with scramble RNA (n = 6), shRNA targeting SRSF6 (shSRSF6) (n = 6), or shRNA targeting SREK1 (shSREK1) (n = 6). (B) Bar charts show quantification of SRSF6, SREK1 and TAF1 protein levels (normalized to β-actin) after interference of SRSF6 or SREK1 versus the scramble condition. ANOVA followed by Games-Howell, *P < 0.05; **P < 0.01; ***P < 0.001. Data represent mean + SEM.

Fig 4: SREK1 overexpression rescues TAF1 levels and improves striatal atrophy and motor coordination of Huntington’s disease mice. (A and B) Representative western blots of TAF1 protein levels in striatum (A) and cortex (B) of 3.5-month-old Control (n = 6), TgSREK1 (n = 5), R6/1 (n = 7) and combined R6/1:TgSREK1 (n = 5) mice. Histograms show quantification of TAF1 normalized to VINCULIN and represented in % respect to Control levels. (C) Quantification of genes analysed by RT-PCR in 3.5-month-old R6/1 (n = 5) and R6/1:TgSREK1 (n = 4) normalized to Actb. (D) Quantification of e5long/e5short transcript isoform ratio in Control (n = 5), R6/1 (n = 6) and R6/1:TgSREK1 (n = 5) mice. (E) Rotarod test. Evolution of the mean latency to fall from the rod in the four accelerating testing trials of Control (eight males and seven females); R6/1 (10 males and seven females); and R6/1:TgSREK1 (12 males and 10 females) mice at 2, 3.5 and 5 months of age. Analysis of variance (ANOVA), followed by Games-Howell, **P < 0.01. (F) Representative images of DARPP-32-stained sagittal sections from Control, R6/1 and R6/1:TgSREK1 mice at 3.5 months of age. (G) Quantification of the striatal area measured in sagittal sections at two different lateral coordinates of Control (n = 3), R6/1 (n = 4) and R6/1:TgSREK1 (n = 4) mice. ANOVA, followed by Games-Howell, *P < 0.05. (E) Representative fields showing immunostaining of cleaved caspase-3 (left) and quantification apoptotic (cleaved caspase-3-positive) cells in Control (n = 3), R6/1 (n = 4) and R6/1:TgSREK1 (n = 4) mice. ANOVA followed by Games-Howell. Data represent mean ± SEM.

Fig 5: Decreased SREK1 and TAF1 levels in striatum and cortex of Huntington’s disease mouse model. Representative immunoblots of SREK1 (A) and TAF1 (C) protein levels in striatum and cortex of 3.5-month-age R6/1 mice (n = 8) and wild-type (WT) mice (n = 7) and their quantification normalized to β-actin or vinculin (Student’s t-test; *P < 0.05; ***P < 0.001). Data represent mean + SEM. (B) SREK1- and (D) TAF1-immunohistochemistry staining in striatum (St) and cortex (Cx) of R6/1 and wild-type mice.