Ronin overexpression induces cerebellar degeneration in a mouse model of ataxia

Abstract

Spinocerebellar ataxias (SCAs) are a group of genetically heterogeneous inherited neurodegenerative disorders characterized by progressive ataxia and cerebellar degeneration. Here, we used a mouse model to test a possible connection between SCA and Ronin (Thap11), a polyglutamine-containing transcriptional regulator encoded in a region of human chromosome 16q22.1 that has been genetically linked to SCA type 4. We report that transgenic expression of Ronin in mouse cerebellar Purkinje cells leads to detrimental loss of these cells and the development of severe ataxia as early as 10 weeks after birth. Mechanistically, we find that several SCA-causing genes harbor Ronin DNA-binding motifs and are transcriptionally deregulated in transgenic animals. In addition, ectopic expression of Ronin in embryonic stem cells significantly increases the protein level of Ataxin-1, the protein encoded by Atxn1, alterations of which cause SCA type 1. This increase is also seen in the cerebellum of transgenic animals, although the latter was not statistically significant. Hence, our data provide evidence for a link between Ronin and SCAs, and suggest that Ronin may be involved in the development of other neurodegenerative diseases.

Keywords: Ataxia; Copy number variation; Embryonic stem cells; Polyglutamine disease; Purkinje cells.

Fig. 1.

Purkinje cell-specific transgenic expression of Ronin leads to reduced rotarod performance. (A) Schematic of a Ronin monomer, in which the C-terminal Thap domain is separated from the N-terminal coiled coil domain by a 29/30 Q long polyQ tract. (B) RT-PCR analysis of mRNA isolated from cerebella of transgenic and wild-type (wt) animals from three independent mouse lines (C6, C15 and C26). (C) Quantification of the mRNA levels shown in B. Data are shown as box plots; each circle represents the signal from one animal. n=3 per line. The boxes represent the 25-75th percentiles, and the whiskers extend 1.5× the interquartile range from the 25th and 75th percentiles. The median is indicated. The center lines show the median. Actb, Actin B. (D) Rotarod analyses of a representative set of animals from transgenic L7-Ronin (Ronin^tg) animals (red lines) from three different mouse lines (C6, C15 and C26) at 10-12 weeks (left) and 20-22 weeks (right) of age compared to wild-type littermates (black lines). The latency to fall in seconds in four trials (T1-T4) on three consecutive days (D1-D3) is shown. Data are mean±s.e.m. P-values were calculated using mixed hierarchical models and describe the difference between the performance average (four trials per animal) of wild-type control and Ronin^tg animals for each day of the trial (indicated in black) or all days (average of 12 trials per animal) combined (indicated in red). n=13, 24 or 21 for transgenic, and n=14, 13 or 7 for wild-type animals of lines C6, C15 and C26, respectively. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

Fig. 2.

Purkinje cell-specific transgenic expression of Ronin leads to ataxia. (A) Gait pattern at 58 weeks of age. Red, front paws; black, hind paws. (B) Quantification of hindleg step length in transgenic and control animals at the indicated time points. The boxes represents the 25-75th percentiles, and the whiskers extend 1.5× the interquartile range from the 25th and 75th percentiles. The median is indicated. Each circle represents the average step length of each animal tested. n=5, 5, 3, 4, 3 and 3 from left to right. At least 15 steps were measured per animal. The center lines show the median. **P≤0.01; n.s., not significant (unpaired two-tailed t-test comparing the average values per animal between groups). Ctrl, control; tg, transgene. (C) Kyphosis, ledge performance and gait were scored between 0 and 3 according to the scoring scheme described by Guyenet et al. (2015) at the indicated time points. Data are mean±s.d. n=5, 7 and 7 animals per genotype per time point (10, 30 and 60 weeks, respectively). Control (wild type) littermates showed phenotype scores of 0 in all categories at all time points. (D) Typical kyphosis phenotype seen sporadically (left) and clasping behavior during a course of 20 s (right) observed in 1-year-old animals as quantified in C.

Fig. 3.

Purkinje cell-specific transgenic expression of Ronin leads to loss of Purkinje cells and cerebellar degeneration. (A) Quantification of the coronal diameter ratio between cerebellum and cerebrum at the indicated ages. Each animal is represented by a circle. n=4 at 5 weeks, n=3 at 11, 20 and 58 weeks. The center lines show the medians. P=5.37548E-03, 3.30734E-03, 6.34538E-05 and 1.71945E-05 from left to right. (B) Macroscopic pictures of brains isolated from 58-week-old animals as quantified in A. (C) Quantification of the number of Purkinje cells per 100 µm Purkinje cell layer in transgenic and control (wild type) animals after H&E staining of sagittal cerebellar sections. Data are presented as box plots. Each circle represents the average number of Purkinje Cells (PC) per animal; n=3. The boxes represent the 25-75th percentiles, and the whiskers extend 1.5× the interquartile range from the 25th and 75th percentiles. The center lines show the median. Sixty-five measures per animal across several sections of three animals per genotype at each time point were performed. P=0.802551867, 0.000191325, 6.33405E-05 and 2.62519E-0.5 from left to right. (D) Representative images of sagittal cerebellar sections after H&E-staining as quantified in C. (E) Immunofluorescence microscopy of cerebellar sections after staining with a specific anti-calbindin antibody was used to visualize Purkinje cells. **P≤0.01; ***P≤0.001; ****P≤0.0001; n.s., not significant (unpaired two-tailed t-test comparing the average values per animal between groups).

Fig. 4.

Transgenic expression of Ronin induces Atxn1. (A) Representative western blots of protein fractions collected by size exclusion chromatography of cerebellar extracts (left) or mouse ESC (mES) cell lysates (right). The size exclusion standards thyroglobulin (667 kDa) and ADH (150 kDa) are indicated. (B) Western blot analyses of equal protein amounts of cerebellar extracts from 10-week-old animals (left) or ESC lysates (right) of the indicated genotypes. (C) Quantification of transgenic Ronin (left) and Atxn1 (right) protein levels relative to GADPH, as detected by western blotting of cerebellar extracts obtained at 5 and 10 weeks of age. The boxes represent the 25-75th percentiles, and the whiskers extend 1.5× the interquartile range from the 25th and 75th percentiles. All samples are indicated as circles. Center lines show medians. **P≤0.01 (unpaired two-tailed t-test). wt, wild type.

Fig. 5.

RNA-seq analyses of L7-Ronin^tg animals at 5 weeks of age. (A) MA plot of RNA-seq data showing the log-fold change (M-value) against the log-average (A-value). Points are colored in red if the adjusted P-value was less than 0.1. Points that fall outside the window are plotted as open triangles. (B) GSEA of Ronin targets that are differentially expressed in Ronin^tg cerebella compared with wild-type control cerebella. (C) Expression of genes (indicated on the left) that have been identified to cause SCAs (indicated on the right), as well as Grid2, the loss of which causes the Lurcher phenotype in mice. Genes that are Ronin targets in ESCs are marked with an asterisk; genes that are differentially expressed with P≤0.5 are marked in blue; those with an adjusted P-(padj)≤0.1 are highlighted in red. (D) Top categories enriched for genes that were downregulated ≤1.25-fold in Ronin^tg cerebella versus wild-type controls, sorted by their P-values. (E) GSEA of cerebellar markers in Ronin^tg cerebella compared with wild-type control cerebella.