ASOs are an effective treatment for disease-associated oligodendrocyte signatures in premanifest and symptomatic SCA3 mice

Spinocerebellar ataxia type 3 (SCA3) is the most common dominantly inherited ataxia. Currently, no preventative or disease-modifying treatments exist for this progressive neurodegenerative disorder, although efforts using gene silencing approaches are under clinical trial investigation. The disease is caused by a CAG repeat expansion in the mutant gene, ATXN3, producing an enlarged polyglutamine tract in the mutant protein. Similar to other paradigmatic neurodegenerative diseases, studies evaluating the pathogenic mechanism focus primarily on neuronal implications. Consequently, therapeutic interventions often overlook non-neuronal contributions to disease. Our lab recently reported that oligodendrocytes display some of the earliest and most progressive dysfunction in SCA3 mice. Evidence of disease-associated oligodendrocyte signatures has also been reported in other neurodegenerative diseases, including Alzheimer's disease, Amyotrophic Lateral Sclerosis, Parkinson's disease, and Huntington's disease. Here, we assess the effects of anti-ATXN3 antisense oligonucleotide (ASO) treatment on oligodendrocyte dysfunction in premanifest and symptomatic SCA3 mice. We report a severe, but modifiable, deficit in oligodendrocyte maturation caused by the toxic gain-of-function of mutant ATXN3 early in SCA3 disease that is transcriptionally, biochemically, and functionally rescued with anti-ATXN3 ASO. Our results highlight the promising use of an ASO therapy across neurodegenerative diseases that requires glial targeting in addition to affected neuronal populations. Overall design: RNA was extracted from PBS-perfused, flash-frozen BS/Dien, spinal cord, and cerebellum samples from the left hemisphere. Using the Next Advance Bullet Blender, samples were homogenized in radioimmunoprecipitation assay (RIPA, by Sigma, St. Louis, MO) buffer (750 µL for BS/Dien and 500 µL for cerebellum and spinal cord samples) with added proteinase inhibitor (BioRad, Hercules, CA). A 250 µL aliquot of this lysate was reserved for Western blot Journal Pre-proof19 analysis and the remaining sample was used for RNA extraction. RNA extraction was conducted using the QIAshredder (Qiagen Hilden, Germany), RNeasy Mini Kit (Qiagen, Hilden, Germany), and DNase I kit (Qiagen, Hilden, Germany), then eluted in RNase-free water, according to manufacturer's instructions. RNA samples were assessed by nanodrop to determine concentration and sample purity. For library preparation, 1 µg of total BS/Dien RNA was submitted to the University of Michigan Sequencing Core for Illumina HiSeq library preparation, quality control, and RNA sequencing. WT and Q84 RNA samples were chosen for RNA sequencing based on RIN number (>7 RIN) and, if applicable, CAG repeat expansion size (>average 72Q) as determined by Laragen, Inc. (Culver City, CA).