SPLICEALS - DISSECTING THE FUNCTIONAL INTERACTION BETWEEN FUS AND HNRNP A2/B1 IN THE PATHOGENESIS OF ALS

Several genetic and experimental findings point to a crucial role of RNA dysfunction in the pathogenesis of ALS. In particular, evidence suggests that mutations in FUS, that are associated with genetic ALS, affect the regulation of alternative splicing (AS) of a selected number of target genes. Recently we have demonstrated that a major target of FUS activity is hnRNP A2/B1, an RNA binding protein with key roles in RNA metabolism, including AS regulation, which is mutated in familial ALS. Further, we have obtained compelling evidence in Drosophila showing that A2/B1 is a potent modifier of FUS toxicity. Overall, these data suggest that A2/B1 might represent a relevant target of FUS in vivo, and that functional alterations of A2/B1 induced by FUS might cause a pathogenic cascade of AS changes, eventually promoting motor neuron degeneration. The general aim of the SPLICEALS project is to validate this hypothesis. In particular, we will use mice, Drosophila and cultured cells to: i. characterize how AS deregulation promoted by FUS affects A2/B1 expression and function; ii. validate these alterations in tissues from mouse models of FUS-ALS; iii. define how modulation of A2/B1 expression impacts on ALS-relevant phenotypes in flies modelling FUS-ALS. Using isoform-specific antibodies, we have observed that the expression of different splice variants of A2/B1 is modified in the lumbar spinal cord of FUS transgenic mice, as well as in fibroblasts from FUS-ALS patients. Further, by using isoform-specific A2/B1 expression constructs, we have pinpointed distinct abilities of A2/B1 variants to localise into stress granules. Finally, we have identified by bionformatics analysis a restricted number of common splicing targets of FUS and A2/B1 and verified that they are affected in diseased mice. Overall, these data support the existence of a pathological connection between FUS and A2/B1 in ALS.