Antisense oligonucleotide-mediated ataxin-1 reduction prolongs survival in SCA1 mice and reveals disease-associated transcriptome profiles

Abstract

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited ataxia caused by expansion of a translated CAG repeat encoding a glutamine tract in the ataxin-1 (ATXN1) protein. Despite advances in understanding the pathogenesis of SCA1, there are still no therapies to alter its progressive fatal course. RNA-targeting approaches have improved disease symptoms in preclinical rodent models of several neurological diseases. Here, we investigated the therapeutic capability of an antisense oligonucleotide (ASO) targeting mouse Atxn1 in Atxn1154Q/2Q-knockin mice that manifest motor deficits and premature lethality. Following a single ASO treatment at 5 weeks of age, mice demonstrated rescue of these disease-associated phenotypes. RNA-sequencing analysis of genes with expression restored to WT levels in ASO-treated Atxn1154Q/2Q mice was used to demonstrate molecular differences between SCA1 pathogenesis in the cerebellum and disease in the medulla. Finally, select neurochemical abnormalities detected by magnetic resonance spectroscopy in vehicle-treated Atxn1154Q/2Q mice were reversed in the cerebellum and brainstem (a region containing the pons and the medulla) of ASO-treated Atxn1154Q/2Q mice. Together, these findings support the efficacy and therapeutic importance of directly targeting ATXN1 RNA expression as a strategy for treating both motor deficits and lethality in SCA1.

Keywords: Drug therapy; Neurodegeneration; Neuroscience.

Figure 1. Reduction of Atxn1 RNA by Atxn1 ASO353 in brain regions of Atxn1^66Q/2Q mice affected by SCA1.

500 μg Atxn1 ASO or vehicle was delivered by bolus i.c.v. injection to 5-week-old Atxn1^66Q/2Q mice. Atxn1 RNA analyzed by qPCR at 2, 12, and 18 weeks after i.c.v. injection. (A) Atxn1 RNA analyzed from the cerebellum; 2-week ASO and vehicle (n = 6), 12-week ASO and vehicle (n = 3), 18-week ASO (n = 5), 18-week vehicle (n = 7). (B) Atxn1 RNA analyzed from the medulla; 2-week ASO and vehicle (n = 6), 12-week ASO and vehicle (n = 3), 18-week ASO (n = 5), 18-week vehicle (n = 7). (C) Atxn1 RNA analyzed from the pons; 2 week and 12-week ASO and vehicle (n = 3), 18-week ASO (n = 5), 18-week vehicle (n = 7). (D) Atxn1 RNA analyzed from the cerebral cortex; 2-week ASO and vehicle (n = 6), 12-week ASO and vehicle (n = 3), 18-week ASO (n = 5), 18-week vehicle (n = 7). Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001, 2-way ANOVA.

Figure 2. Reduction of Atxn1 in the brainstems, cerebella, and Purkinje cells of Atxn1^154Q/2Q mice.

(A) RNA from Atxn1^154Q/2Q mice analyzed by qPCR at 6 and 24 weeks after i.c.v. injection of the ATXN1 ASO353. qPCR data are presented as the average ± SEM; 6-week brainstem and cerebellum (n = 6). (B) Atxn1 RNA brainstems, cerebella, and dissected Purkinje cells (PC layer) from Atxn1^154Q/2Q mice receiving a bolus i.c.v. injection of ASO353 (500 μg) at 16 weeks, with analysis at 18 weeks; brainstem (n = 4), cerebellum (n = 7), Purkinje cell layer (n = 3). qPCR data are presented as mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001, by t test.

Figure 3. Recovery of SCA1-like phenotypes in Atxn1^154Q/2Q mice receiving ASO353 by i.c.v. injection at 5 weeks of age.

Mice were administered ASO353 or vehicle (saline) by bolus i.c.v. injection at 5 weeks. (A) Motor performance on a rotarod between ages 5 weeks and 27 weeks. Data are presented as the mean ± SEM. **P < 0.01, ***P < 0.001, 1-way ANOVA. (B) Motor performance on a balance beam at ages 15 weeks and 26 weeks. Data are presented as the mean ± SEM. **P < 0.01, ***P < 0.001, 1-way ANOVA. (C) Survival, plotted as Kaplan-Meyer curves, for mice receiving a bolus i.c.v. injection of 500 μg ASO353 or vehicle (saline) at 5 weeks. ***P = 0.0001, log-rank Mantel-Cox test; P = 0.0005, Gehan-Breslow-Wilcoxon test. (D) i.c.v. injection of ASO353 fails to improve the failure-to-gain weight phenotype in Atxn1^154Q/2Q mice; WT C57BL/6 uninjected (n = 23), WT C57BL/6 vehicle (n = 16), Atxn1^154Q/2Q uninjected (n = 19), Atxn1^154Q/2Q vehicle (n = 15), and Atxn1^154Q/2Q ASO353 (n = 12).

Figure 4. Differentially expressed genes in the cerebella, pontes, and medullae of Atxn1^154Q/2Q mice receiving vehicle or ASO353 by i.c.v. (A) Genes differentially expressed in vehicle-treated WT versus vehicle-treated Atxn1^154Q/2Q mice from the cerebellum at 18 weeks, the pons at 18 and 28 weeks, and the medulla at 18 and 28 weeks.

The black portion of the bars depicts the number of genes downregulated and the gray portion of the bars depicts the number of genes upregulated in vehicle-treated Atxn1^154Q/2Q mice. (B) Genes differentially expressed in vehicle-treated Atxn1^154Q/2Q versus ASO353-treated Atxn1^154Q/2Q mice from the cerebellum at 18 weeks, the pons at 18 and 28 weeks, and the medulla at 18 and 28 weeks. The black portion of the bars depicts the number of genes downregulated and the gray portion of the bars depicts the number of genes upregulated in ASO353-treated Atxn1^154Q/2Q mice. In A and B, numbers above bars indicate the number genes with differential expression. (C and D) GSEA of the Magenta module compared with differentially expressed gene lists from the cerebella of 18-week-old and medullae of 28-week-old Atxn1^154Q/2Q mice, respectively. Significance was determined by the observed enrichment score compared to a set of null scores generated by permuting the gene ranked order 10,000 times.

Figure 5. Medulla Brown WGCNA module is associated with disease in Atxn1^154Q/2Q mice.

(A) Transcriptional heatmap of Brown module genes for the medulla of vehicle- and ASO353-treated Atxn1^154Q/2Q mice and vehicle-treated WT mice. (B) Transcriptional heatmap of Brown module genes for the cerebella of vehicle and ASO353-treated Atxn1^154Q/2Q mice and vehicle-treated WT mice; ASO-treated Atxn1^154Q/2Q mice (n = 7), vehicle-treated Atxn1^154Q/2Q mice (n = 7), and vehicle-treated WT mice (n = 8).

Figure 6. Gene overlap between cerebellar Magenta and medulla Brown WGCNA modules.

Venn diagram depicting gene overlap between the cerebellar Magenta and medulla Brown WGCNA modules for genes in common between both analyses. The overlap of 65 genes is significant at P < 4.61E-05. The top IPA canonical pathways for each module and the overlapping genes are listed. While no canonical pathways were significant for the overlapping gene set with Benjamini-Hochberg correction for multiple tests, the top canonical pathway in the uncorrected analysis is shown.

Figure 7. ASO treatment effects on neurochemicals in the cerebella and brainstems in Atxn1^154Q/2Q mice.

(A and D) Voxel placement and spectral quality in the cerebellum and brainstem. T₂-weighted images and localized proton MR spectra (LASER, TE = 15 ms, TR = 5 s) are shown for an ASO353-treated Atxn1^154Q/2Q mouse. (B, C, E, and F) Myo-inositol (Ins) and total choline (tCho) concentrations in the cerebella (B and C) and brainstems (E and F) of ASO353-treated Atxn1^154Q/2Q mice, vehicle-treated Atxn1^154Q/2Q mice, and vehicle-treated WT mice at 18 and 28 weeks of age. Sample sizes in B and E are as follows: ASO-treated Atxn1^154Q/2Q mice (n = 11), vehicle-treated Atxn1^154Q/2Q mice (n = 12), and WT mice (n = 11). Sample sizes in C and F are as follows: ASO-treated Atxn1^154Q/2Q mice (n = 16), vehicle-treated Atxn1^154Q/2Q mice (n = 15), and WT mice (n = 15). In the box-and-whisker plots, the bounds of the boxes represent 25th to 75th percentiles, the line in the box is the median, and the whiskers extend to the minimum and maximum values. Group means for neurochemical concentrations were compared with 1-way ANOVA, and P values were corrected for multiple comparisons using the Tukey Method. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.