Comparison of spinocerebellar ataxia type 3 mouse models identifies early gain-of-function, cell-autonomous transcriptional changes in oligodendrocytes

Abstract

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine-encoding CAG repeat expansion in the ATXN3 gene. This expansion leads to misfolding and aggregation of mutant ataxin-3 (ATXN3) and degeneration of select brain regions. A key unanswered question in SCA3 and other polyglutamine diseases is the extent to which neurodegeneration is mediated through gain-of-function versus loss-of-function. To address this question in SCA3, we performed transcriptional profiling on the brainstem, a highly vulnerable brain region in SCA3, in a series of mouse models with varying degrees of ATXN3 expression and aggregation. We include two SCA3 knock-in mouse models: our previously published model that erroneously harbors a tandem duplicate of the CAG repeat-containing exon, and a corrected model, introduced here. Both models exhibit dose-dependent neuronal accumulation and aggregation of mutant ATXN3, but do not exhibit a behavioral phenotype. We identified a molecular signature that correlates with ATXN3 neuronal aggregation yet is primarily linked to oligodendrocytes, highlighting early white matter dysfunction in SCA3. Two robustly elevated oligodendrocyte transcripts, Acy3 and Tnfrsf13c, were confirmed as elevated at the protein level in SCA3 human disease brainstem. To determine if mutant ATXN3 acts on oligodendrocytes cell-autonomously, we manipulated the repeat expansion in the variant SCA3 knock-in mouse by cell-type specific Cre/LoxP recombination. Changes in oligodendrocyte transcripts are driven cell-autonomously and occur independent of neuronal ATXN3 aggregation. Our findings support a primary toxic gain of function mechanism and highlight a previously unrecognized role for oligodendrocyte dysfunction in SCA3 disease pathogenesis.

Figure 1

Comparison of an atypical SCA3 knock-in mouse model and a corrected knock-in line reveals distinct differences in ataxin-3 (ATXN3) expression and aggregation in the brain. (A) Simplified schematic of the insertion in our previously reported knock-in mice which harbor a duplicate tandem insertion of the targeting vector containing the CAG repeat-expansion in exon 10 (dupKi). FLP-FRT recombination allowed us to excise the duplication and generate a corrected SCA3 knock-in mouse line (Ki). (B) By Western blot, brain lysates of one-year-old heterozygous dupKi (dupKi-het) mice exhibit increased high molecular weight (HMW) aggregated ATXN3 and reduced mutant (mut) ATXN3 monomer (arrowhead), compared to corrected Ki-het mice. (C) Anti-ATXN3 immunohistochemistry demonstrates robust ATXN3 accumulation and puncta in brain regions of 1-year-old dupKi-het mice, including the pons and hippocampus (CA1 and stratum radiatum (StRad)), whereas Ki-het mice show no noticeable ATXN3 accumulation. Homozygous Ki mice (Ki-hom) exhibit modest ATXN3 accumulation in brainstem neuronal nuclei, along with large neuropil inclusions in the StRad, similar to heterozygous dupKi mice.

Figure 2

Ki mice do not exhibit Atxn3 mis-splicing, whereas dupKi mice express different aberrantly-generated Atxn3 transcripts. (A) We examined Atxn3 transcripts with alternative splicing of exon 11, including a shorter 10-exon containing Atxn3 transcript (Atxn3-10e) and a full-length transcript containing all 11 exons (Atxn3-11e), both diagrammed at the 3’ end. RT-PCR demonstrates that Ki mice express wild-type (arrow) and mutant (arrowhead) Atxn3-11e transcript, whereas dupKi-het mice do not express detectable mutant Atxn3-11e transcript. Instead, dupKi-het mice express the previously described Atxn3-10e transcript (red arrowhead) (16), which is not elevated in Ki mice. (B) To test for a predicted Atxn3 transcript containing both mutant exon 10s spliced together in dupKi mice, we performed different PCR reactions on Atxn3 cDNA that was reverse-transcribed with a primer against the Atxn3 3’UTR in homozygous Ki and dupKi mice brain RNA. We provide a diagram of the PCR primers and predicted sizes of amplification products for exon 9 to 11, exon 10 to 11, and exon 10 to exon 10 (3’ to 5’ ends) in Ki and dupKi mice. All three PCR reactions revealed signals in a dupKi-hom mouse consistent with the predicted size for a duplicated exon 10 Atxn3 transcript (diamond). *Non-specific band. (C) Diagram of predicted mutant ATXN3 isoforms in the different SCA3 knock-in lines. Ki mice predominantly express the full-length mutant ATXN3 isoform encoded by the Atxn3-11e transcript. In contrast, dupKi mice predominantly express a mutant ATXN3 isoform slightly truncated at the carboxy-terminus, encoded by the Atxn3-10e transcript, and likely express a predicted mutant ATXN3 encoded from a duplicated exon 10-containing Atxn3 transcript, in which the expanded repeat in the second exon 10 encodes expanded polyserine followed by two arginines and a stop.

Figure 3

RNA-seq of the pons in mouse lines differing in ATXN3 expression and aggregation identifies transcriptional correlates to ATXN3 aggregation that are also altered in SCA3 human brainstem. (A) Total number of differentially expressed (DE) transcripts in the different mouse models correlates with the level of ATXN3 aggregation in a gene dose-dependent manner. Left, a table demonstrating ATXN3 expression level and ATXN3 aggregation for each genotype, with aggregation color-coded by red intensity corresponding to the amount of ATXN3 aggregation in each genotype. (B) Venn diagrams of the overlap in DE transcripts across mouse lines show a greater degree of shared transcripts between mice with higher ATXN3 aggregation, with the greatest overlap occurring between dupKi and YAC84Q mice, both of which have robust aggregation. In contrast, ATXN3-KO mice shared relatively few DE transcripts with any SCA3 mouse model, including Ki-hom and dupKi-hom mice, which do not express any wild-type ATXN3. (C) Visualization of the relative fold-change for 163 DE transcripts of YAC84Q mice as a heat map shows a concordant transcriptional signature in genotypes exhibiting increased ATXN3 aggregation. ATXN3-KO and dupKi-hom mice were profiled in an independent experiment and are shown separately. (D) Quantitative RT-PCR (qRT-PCR) confirmed key upregulated and downregulated candidate genes in the pons of homozygous YAC84Q mouse (n = 3) relative to YAC15Q mice (n = 3). *P < 0.05 by Student’s t-test. (E), Left, Western blot showing increased TNFRSF13C and ACY3 signal in SCA3 human brainstem lysates compared to Alzheimer disease brainstem controls (AD). Right, graphs plotting the relative signal intensity from the blots on the left, with Student’s t-test P-values shown above.

Figure 4

Bioinformatic analysis of the DE transcripts identifies robust oligodendrocyte alterations associated with mutant ATXN3 aggregation. (A) Gene-set enrichment analysis of the 38 shared DE transcripts between YAC84Q, Ki-hom, and dupKi-het mice revealed 5 genes significantly enriched in five ‘Biological Process’ categories associated with myelination. (B) for each genotype, visualization of the activation z-scores for the top altered Diseases and Functions categories through Ingenuity® Pathway Analysis software. This analysis highlighted several categories pointing to demyelination in mouse lines exhibiting increased aggregation. (C) 34 of the 38 DE transcripts commonly altered across all three aggregation-prone lines could be binned, based on the Brain RNA-Seq website (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html)(26), according to the cell-type in which each is maximally expressed. The majority of these transcripts are most highly expressed in oligodendrocytes. (D) Brain RNA-Seq showed that Atxn3 is widely expressed throughout the brain, including in oligodendrocytes. (E) Co-immunofluorescence for ATXN3 and OLIG2 in the brainstem of homozygous YAC84Q mice confirmed that ATXN3 signal is present in oligodendrocyte nuclei and is increased in comparison to wild-type oligodendrocytes. Right three images represent region within white box of leftmost image.

Figure 5

Oligodendrocyte-specific manipulation of the CAG repeat expansion in mice suggests mutant ATXN3 acts cell-autonomously to elicit transcriptional changes in oligodendrocytes. (A) The dupKi mutant Atxn3 locus harbors two LoxP sites that allowed us to excise the duplicate exon 10 by crossing dupKi mice with mice expressing Cre under either a Nestin promoter (dupKi-het + N-Cre) to delete the duplication throughout the brain, or an Olig2 promotor (dupKi-het + O-Cre) to delete the duplication only in oligodendrocytes. (B,C), RT-PCR of Atxn3 transcripts in the brain showed that dupKi-het + N-Cre mice exhibit corrected splicing of Atxn3, with increased mutant Atxn3-11e expression (black arrowhead) and reduced Atxn3-10e expression (red arrowhead), whereas Atxn3 mis-splicing was not rescued in dupKi-het + O-Cre mice. Wild-type Atxn3-11e (black arrow) is identified in all mice. (D) qRT-PCR verified that the Atxn3-10e transcript is significantly reduced in dupKi-het + N-Cre mice, but not in dupKi-het + O-Cre mice. (E,F) dupKi-het + N-Cre mice demonstrate increased soluble mutant ATXN3 (E) and markedly reduced ATXN3 aggregation in the brain (F) relative to dupKi-het mice, whereas dupKi-het + O-Cre mice show no differences in monomeric mutant ATXN3 level or HMW ATXN3 aggregation compared to dupKi-het littermates. Arrow: wild-type (WT) ATXN3, arrowhead: mutant (mut) ATXN3. (G), Anti-ATXN3 immunofluorescence showed that ATXN3 inclusions in the hippocampal pyramidal layer of dupKi-het mice are no longer present in dupKi-het + N-Cre mice, whereas dupKI-het + O-Cre mice continue to exhibit inclusions similar to dupKi-het littermates. (H) qRT-PCR on the pons showed that the robust elevation of Acy3 and Tnfrsf13c transcript levels in dupKi-het mice is rescued in both dupKi-het + N-Cre and dupKi-het + O-Cre mice. *P < 0.05 by t-test or one-way ANOVA.