A knockin mouse model of spinocerebellar ataxia type 3 exhibits prominent aggregate pathology and aberrant splicing of the disease gene transcript

Abstract

Polyglutamine diseases, including spinocerebellar ataxia type 3 (SCA3), are caused by CAG repeat expansions that encode abnormally long glutamine repeats in the respective disease proteins. While the mechanisms underlying neurodegeneration remain uncertain, evidence supports a proteotoxic role for the mutant protein dictated in part by the specific genetic and protein context. To further define pathogenic mechanisms in SCA3, we generated a mouse model in which a CAG expansion of 82 repeats was inserted into the murine locus by homologous recombination. SCA3 knockin mice exhibit region-specific aggregate pathology marked by intranuclear accumulation of the mutant Atxn3 protein, abundant nuclear inclusions and, in select brain regions, extranuclear aggregates localized to neuritic processes. Knockin mice also display altered splicing of the disease gene, promoting expression of an alternative isoform in which the intron immediately downstream of the CAG repeat is retained. In an independent mouse model expressing the full human ATXN3 disease gene, expression of this alternatively spliced transcript is also enhanced. These results, together with recent findings in other polyglutamine diseases, suggest that CAG repeat expansions can promote aberrant splicing to produce potentially more aggregate-prone isoforms of the disease proteins. This report of a SCA3 knockin mouse expands the repertoire of existing models of SCA3, and underscores the potential contribution of alternative splicing to disease pathogenesis in SCA3 and other polyglutamine disorders.

Figure 1.

Generation of a SCA3 knockin mouse expressing mutant Atxn3 (Q82). (A) Schematic of the generation of the SCA3 knockin mouse in which the endogenous murine (CAA)(CAG)₅ was replaced with a human CAG-expanded sequence, (CAG)₂(CAAAAG)(CAG)₈₂, by homologous recombination. The neomycin (neo) selection cassette, flanked by FRT sites, was removed by FLPe recombination. (B) PCR across the CAG repeat shows the expanded repeat in heterozygous (Atxn3Q82/Q6) and homozygous (Atxn3Q82/Q82) SCA3 knockin mice. (C) SCA3 knockin mice show modest intergenerational repeat length instability with a tendency for CAG repeat contraction upon maternal transmission. (D) Western blotting shows expression of mutant Atxn3 accompanied by increased aggregates in the stacking gel in 1-year-old Atxn3Q82/Q6 hindbrain lysates. (E) Electrophoresis of lysates from (D) on 3% SDS–PAGE further illustrates high-molecular-weight aggregates. (F) About 30-week-old homozygous Atxn3Q82/Q82 mice express only mutant Atxn3 with aggregates in the stack. (D and F) Arrow indicates wild-type (WT) Atxn3 and arrowhead indicates mutant Atxn3.

Figure 2.

Mutant Atxn3 accumulation in the SCA3 knockin mouse brain. (A) Immunohistochemical staining (IHC) for Atxn3 shows nuclear accumulation in the DCN of a 10-week-old Atxn3Q82/Q6 mouse. Scale bar = 20 μm, inset scale bar = 2 μm. (B) IHC of 1-year-old Atxn3Q82/Q6 mice shows increased diffuse nuclear staining and intranuclear puncta/inclusions in several brain regions, including the brain stem (medulla shown), DCN and the hippocampus (CA1 region is shown). Higher magnification insets show puncta and inclusions in nuclei. (C) Intranuclear accumulation of Atxn3 (DCN shown) is accelerated in an ∼7-month-old homozygous Atxn3Q82/Q82 mouse compared with a heterozygous littermate. Scale bar = 20 μm (D) Intranuclear puncta and inclusions in brainstem neurons from SCA3 knockin mice frequently co-stain with p62, whereas p62 is predominantly cytoplasmic in wild-type neurons. The nuclear border is outlined by a dashed line. Scale bar = 10 μm. (E) Performance of 1-year-old Atxn3Q82/Q6 (Q82/Q6) mice (n = 9) did not differ from age-matched WT mice (n = 10) on motor behavior tasks, including from left to right, 5 mm balance beam, accelerating rotarod and open field exploration. Graphs represent mean + SE.

Figure 3.

Extranuclear inclusions in the hippocampus of SCA3 knockin mice. (A) Large extranuclear inclusions are concentrated in the stratum radiatum (SR) of the hippocampus 1-year-old Atxn3Q82/Q82 mice. Right, immunofluorescence of a different Atxn3Q82/Q82 mouse showing that inclusions do not colocalize with nuclear DAPI. Bottom panel shows magnified view of inclusions. (B) Hippocampal aggregates often stain ubiquitin positive, including both intranuclear inclusions in CA1 pyramidal neurons (left) and extranuclear inclusions in the stratum radiatum (right). (C) Extranuclear inclusions show overlap with dendritic markers MAP2 and SMI32. (D) Large Atxn3 extranuclear inclusions in the stratum radiatum of a 2-year-old Atxn3Q82/Q6 mouse stain for RTN3, a marker for dystrophic neurites. (E) 1-year-old Atxn3Q82/Q6 mice (n = 9) did not differ from wild-type mice (n = 9) in tests of fear conditioning, including freezing to context (left) and tone (right). Scale bars in (A) top and bottom panels are 50 and 10 μm, respectively. Scale bars in (B)–(D) are 10 μm.

Figure 4.

Alternative splicing of the mutant Atxn3 transcript is enhanced in SCA3 knockin mice. (A) Diagram of 3′ alternative splicing of the human ATXN3 transcript, showing the 10 exon-containing ATXN3 transcript (right) generated from retention of intron 10, which encodes a hydrophobic segment that may accelerate mutant ATXN3 aggregation. (B) RNA-sequencing on the pons of Atxn3Q82/Q82 mice shows elevated reads in intron 10 following the CAG repeat-containing exon 10. Genomic sequence near the end of the reads in intron 10 (boxed) contains a putative polyadenylation (polyA) site ATTAAA (underlined). (C) 3′ RACE in an Atxn3Q82/Q6 mouse amplified a 300 bp band containing this putative polyA site (underlined) followed by a polyA tail (black, bold). (D) Wild-type and homozygous Atxn3Q82/Q82 mice showed similar frequency of sequencing reads in exon 11 and the 3′ UTR of the SCA3 knockin mice. (E) Levels of predicted full-length Atxn3 transcript from the RNA-sequencing (FPKMs) do not significantly differ between wild-type (n = 7) and Atxn3Q82/Q82 mice (n = 7). Graph represents mean + SD.

Figure 5.

Expression of 10 exon Atxn3/ATXN3 transcript in SCA3 mouse models and SCA3 human fibroblasts. (A) Diagram of 10 exon ATXN3/Atxn3 (ATXN3/Atxn3-10e) transcript and arrows indicating location of primer pairs used for qPCR and non-qPCR on reversed-transcribed RNA. (B) Atxn3-10e transcript is highly upregulated (∼8-fold) while total Atxn3 transcript is only modestly upregulated (∼1.5-fold) in heterozygous SCA3 knockin mice (n = 3). (C) ATXN3-10e transcript is also upregulated (∼2-fold) in YAC mice expressing the full human ATXN3 gene with 84 CAG repeats (YAC84Q, n = 4) compared with 15 CAG repeats (YAC15Q, n = 4). (D) ATXN3-10e transcript levels in SCA3 fibroblasts (n = 6) did not significantly differ from non-disease control fibroblasts (n = 4). qPCR in (B)–(D) was normalized to Gapdh, TRIP11 and ACTB, respectively. (E) PCR reveals the presence of mutant Atxn3-10e cDNA in SCA3 knockin mice only. (F and G) PCR shows expanded and non-expanded ATXN3-10e in both YAC mouse lines, all SCA3 fibroblast lines, and three of four non-disease control fibroblast lines. Amplification of ATXN3 exon 7 to exon 11 indicates the presence of ATXN3-11e in all fibroblast lines. Graphs represent the mean + SD. *P < 0.02 by an unpaired t-test.