miR760 regulates ATXN1 levels via interaction with its 5' untranslated region.

Identifying modifiers of dosage-sensitive genes involved in neurodegenerative disorders is imperative to discover novel genetic risk factors and potential therapeutic entry points. In this study, we focus on Ataxin-1 (ATXN1), a dosage-sensitive gene involved in the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1). While the precise maintenance of ATXN1 levels is essential to prevent disease, the mechanisms that regulate ATXN1 expression remain largely unknown. We demonstrate that ATXN1's unusually long 5' untranslated region (5' UTR) negatively regulates its expression via posttranscriptional mechanisms. Based on recent reports that microRNAs (miRNAs) can interact with both 3' and 5' UTRs to regulate their target genes, we identify miR760 as a negative regulator that binds to a conserved site in ATXN1's 5' UTR to induce RNA degradation and translational inhibition. We found that delivery of Adeno-associated virus (AAV)-expressing miR760 in the cerebellum reduces ATXN1 levels in vivo and mitigates motor coordination deficits in a mouse model of SCA1. These findings provide new insights into the regulation of ATXN1 levels, present additional evidence for miRNA-mediated gene regulation via 5' UTR binding, and raise the possibility that noncoding mutations in the ATXN1 locus may act as risk factors for yet to be discovered progressive ataxias.

Cis-acting single nucleotide polymorphisms alter MicroRNA-mediated regulation of human brain-expressed transcripts.

Substantial variability exists in the presentation of complex neurological disorders, and the study of single nucleotide polymorphisms (SNPs) has shed light on disease mechanisms and pathophysiological variability in some cases. However, the vast majority of disease-linked SNPs have unidentified pathophysiological relevance. Here, we tested the hypothesis that SNPs within the miRNA recognition element (MRE; the region of the target transcript to which the miRNA binds) can impart changes in the expression of those genes, either by enhancing or reducing transcript and protein levels. To test this, we cross-referenced 7,153 miRNA-MRE brain interactions with the SNP database (dbSNP) to identify candidates, and functionally assessed 24 SNPs located in the 3'UTR or the coding sequence (CDS) of targets. For over half of the candidates tested, SNPs either enhanced (4 genes) or disrupted (10 genes) miRNA binding and target regulation. Additionally, SNPs causing a shift from a common to rare codon within the CDS facilitated miRNA binding downstream of the SNP, dramatically repressing target gene expression. The biological activity of the SNPs on miRNA regulation was also confirmed in induced pluripotent stem cell (iPSC) lines. These studies strongly support the notion that SNPs in the 3'UTR or the coding sequence of disease-relevant genes may be important in disease pathogenesis and should be reconsidered as candidate modifiers.

VWA3A-derived ependyma promoter drives increased therapeutic protein secretion into the CSF.

Recombinant adeno-associated viral vectors (rAAVs) are a promising strategy to treat neurodegenerative diseases because of their ability to infect non-dividing cells and confer long-term transgene expression. Despite an ever-growing library of capsid variants, widespread delivery of AAVs in the adult central nervous system remains a challenge. We have previously demonstrated successful distribution of secreted proteins by infection of the ependyma, a layer of post-mitotic epithelial cells lining the ventricles of the brain and central column of the spinal cord, and subsequent protein delivery via the cerebrospinal fluid (CSF). Here we define a functional ependyma promoter to enhance expression from this cell type. Using RNA sequencing on human autopsy samples, we identified disease- and age-independent ependyma gene signatures. Associated promoters were cloned and screened as libraries in mouse and rhesus macaque to reveal cross-species function of a human DNA-derived von Willebrand factor domain containing 3A (VWA3A) promoter. When tested in mice, our VWA3A promoter drove strong, ependyma-localized expression of eGFP and increased secreted ApoE protein levels in the CSF by 2-12× over the ubiquitous iCAG promoter.

Disruption of the ATXN1-CIC complex reveals the role of additional nuclear ATXN1 interactors in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative disease in that it is caused by a mutation in a broadly expressed protein, ATXN1; however, only select populations of cells degenerate. The interaction of polyglutamine-expanded ATXN1 with the transcriptional repressor CIC drives cerebellar Purkinje cell pathogenesis; however, the importance of this interaction in other vulnerable cells remains unknown. Here, we mutated the 154Q knockin allele of Atxn1154Q/2Q mice to prevent the ATXN1-CIC interaction globally. This normalized genome-wide CIC binding; however, it only partially corrected transcriptional and behavioral phenotypes, suggesting the involvement of additional factors in disease pathogenesis. Using unbiased proteomics, we identified three ATXN1-interacting transcription factors: RFX1, ZBTB5, and ZKSCAN1. We observed altered expression of RFX1 and ZKSCAN1 target genes in SCA1 mice and patient-derived iNeurons, highlighting their potential contributions to disease. Together, these data underscore the complexity of mechanisms driving cellular vulnerability in SCA1.

Decreasing mutant ATXN1 nuclear localization improves a spectrum of SCA1-like phenotypes and brain region transcriptomic profiles.

Spinocerebellar ataxia type 1 (SCA1) is a dominant trinucleotide repeat neurodegenerative disease characterized by motor dysfunction, cognitive impairment, and premature death. Degeneration of cerebellar Purkinje cells is a frequent and prominent pathological feature of SCA1. We previously showed that transport of ATXN1 to Purkinje cell nuclei is required for pathology, where mutant ATXN1 alters transcription. To examine the role of ATXN1 nuclear localization broadly in SCA1-like disease pathogenesis, CRISPR-Cas9 was used to develop a mouse with an amino acid alteration (K772T) in the nuclear localization sequence of the expanded ATXN1 protein. Characterization of these mice indicates that proper nuclear localization of mutant ATXN1 contributes to many disease-like phenotypes including motor dysfunction, cognitive deficits, and premature lethality. RNA sequencing analysis of genes with expression corrected to WT levels in Atxn1175QK772T/2Q mice indicates that transcriptomic aspects of SCA1 pathogenesis differ between the cerebellum, brainstem, cerebral cortex, hippocampus, and striatum.

Modulation of ATXN1 S776 phosphorylation reveals the importance of allele-specific targeting in SCA1.

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disorder characterized by motor incoordination, mild cognitive decline, respiratory dysfunction, and early lethality. It is caused by the expansion of the polyglutamine (polyQ) tract in Ataxin-1 (ATXN1), which stabilizes the protein, leading to its toxic accumulation in neurons. Previously, we showed that serine 776 (S776) phosphorylation is critical for ATXN1 stability and contributes to its toxicity in cerebellar Purkinje cells. Still, the therapeutic potential of disrupting S776 phosphorylation on noncerebellar SCA1 phenotypes remains unstudied. Here, we report that abolishing S776 phosphorylation specifically on the polyQ-expanded ATXN1 of SCA1-knockin mice reduces ATXN1 throughout the brain and not only rescues the cerebellar motor incoordination but also improves respiratory function and extends survival while not affecting the hippocampal learning and memory deficits. As therapeutic approaches are likely to decrease S776 phosphorylation on polyQ-expanded and WT ATXN1, we further disrupted S776 phosphorylation on both alleles and observed an attenuated rescue, demonstrating a potential protective role of WT allele. This study not only highlights the role of S776 phosphorylation to regulate ATXN1 levels throughout the brain but also suggests distinct brain region-specific disease mechanisms and demonstrates the importance of developing allele-specific therapies for maximal benefits in SCA1.