Functional characterization of hnRNPA2/B1 splicing isoforms: implications for the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a disease involving motor neuron degeneration, which leads to progressive muscle atrophy and weakness. Even though ALS is believed to have a large genetic component, only a minority of ALS cases, around 10%, have at least one other family member affected and are inherited in an autosomal dependent manner, and thus are classified as familial ALS (fALS) cases (Kirby et al., 2016). Increasing evidence supports a key role of RNA dysmetabolism in ALS pathogenesis; specifically, alternative splicing (AS) dysregulation has been repeatedly reported in the case of FUS mutations, which are associated with fALS (Butti and Patten, 2019). In a previous paper from our laboratory (Mirra et al., 2017), it was shown that mice overexpressing wtFUS, which recapitulate human ALS disease, exhibit a major alteration in the mRNA splicing of hnRNPA2/B1 (A2/B1), an RNA binding protein with key roles in RNA metabolism and in particular in AS regulation, which is mutated in familial ALS (Kim et al., 2013). A2/B1 has four splicing isoforms, which can be distinguished by the presence or absence of exon 2 and/or exon 9. Spinal cords of mice overexpressing wtFUS are characterized by a significant skipping of exon 9 from mature RNA and a subsequent change in the relative expression of the four RNA isoforms (Mirra et al., 2017). More recently, preliminary data showed that this alteration leads to a shift of the four protein isoforms, with decreased expression levels of exon9-containing isoforms (A2 and B1) and concurrently increased levels of isoforms lacking exon9 (A2?9 and B1?9). Interestingly, isoforms lacking exon 9 form aggregates and accumulate in the cytoplasm of degenerating motor neurons of wtFUS mice. Moreover, in the spinal cord of wtFUS mice, we found significant alterations in the mRNA splicing of genes that have common splicing alterations in case of either FUS or A2/B1 downregulation, identified after a comparative analysis of current datasets of mouse models depleted of either FUS or A2/B1. Overall, these data suggest that FUS-mediated alterations in AS regulation of A2/B1 might impair its functions and cause a pathogenic cascade of AS changes, eventually promoting motor neuron degeneration. On these grounds, the overall objective of my thesis work is to provide a characterization of the different A2/B1 isoforms to shed light on their potential pathogenic role.