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.

Oligonucleotide therapy mitigates disease in spinocerebellar ataxia type 3 mice

“…Disease-specific oligodendrocyte-enriched genes 6 were selected for transcriptional analysis in the diencephalon of vehicle-treated versus ASO-5 treated mice at 22 weeks of age (Fig 4B). Of the eight transcripts analyzed, six were confirmed to exhibit significant gene expression changes in vehicle-treated Q84/Q84 mice compared to WT mice.…”

“…Though neuronal loss must be a major contributor to the progressive symptoms of SCA3, studies have also begun to implicate non-neuronal cell dysfunction in SCA3 6,43 . For example, in our recent study exploring differentially expressed genes in mouse models of SCA3, the most abundantly altered transcripts reside in oligodendrocytes 6 . The reasons for this preferential effect on oligodendrocytic transcription are unclear, but our demonstration here that ATXN3 accumulates in the nuclei of non-neuronal cells in the diencephalon of Q84/Q84 mice is consistent with a cell-autonomous effect.…”

“…iQ SYBR green quantitative PCR was performed on the diluted cDNA following the manufacturer’s protocol (Bio-Rad, Hercules, CA). Average adjusted relative quantification analysis was performed using previously described primers 6,19 .…”

“…Average-adjusted relative quantification analysis was performed using previously described primers. 6,19 Immunoblotting Protein lysates from macrodissected diencephalon and cerebellar tissue were processed as previously described 19 and stored at −80 C. Forty micrograms of total mouse brain protein lysate were resolved in 4 to 20% gradient sodium dodecyl sulfate-polyacrylamide electrophoresis gels and transferred to 0.45 μm nitrocellulose membranes. Membranes were incubated overnight at 4 C with various antibodies: mouse anti-ATXN3 (1H9; 1:1,000, MAB5360; Millipore, Billerica, MA), mouse anti-GFAP (1:20,000, 3670S; Cell Signaling Technology, Danvers, MA), rabbit anti-IBA1 (1:500, 016-20001; Wako, Osaka, Japan), rabbit anti-BAX (1:500, 2772S; Cell Signaling Technology, Danvers, MA), mouse anti-BCL-2 (1:500, 610538; BD Biosciences, San Jose, CA), mouse anti-p62 (1:1,000, ADI-SPA-902-D; Enzo Life Sciences, New York, NY), rabbit anti-BECLIN1 (1:1,000, ab207612; Abcam, Cambridge, UK), mouse anti-GAPDH (1:5,000, MAB374; Millipore, Billerica, MA), and rabbit anti-α-Tubulin (1:5,000, #S2144, Cell Signaling Technology).…”

“…3,4 SCA3 is 1 of 9 neurodegenerative diseases caused by an expanded, polyglutamine-coding repeat in the disease gene. 5 The mutant SCA3 disease protein, ATXN3, acts through a dominant toxic mechanism that is still poorly understood, 1,6 and mice lacking ATXN3 are phenotypically normal. 7 Thus, suppression of the disease gene, ATXN3, represents a promising approach to slow or block the neurodegenerative cascade in SCA3.…”