Alterazioni nella regolazione dello splicing alternativo nella Sclerosi Laterale Amiotrofica associata al gene fus

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects both the lower motor neurons in the brainstem and spinal cord, and the upper motor neurons in the motor cortex. This initially leads to muscle atrophy and then to death for respiratory failure within 2-5 years after symptoms onset. Recently, the discovery of mutations in many genes involved in RNA metabolism, such as FUS, C9orf72 and TDP43, focused the attention of ALS research on the possible involvement in ALS pathogenesis of RNA-related dysfunctions. FUS is a nuclear protein supposed to have several functions in DNA and RNA metabolism, and it has been found in cytoplasmic aggregates that characterize tissues from patients. Importantly, FUS binds both SMN, whose mutations are responsible for the motor neuron disease Spinal Muscular Atrophy (SMA) and snRNPs, thus indicating a role of FUS in the regulation of mRNA splicing. Moreover, ALS-associated mutant FUS stalls snRNPs in the cytoplasm and affect alternative splicing, thus suggesting that alterations of splicing regulation might participate in the disease. In order to investigate the nature of the binding between FUS and snRNPs, we transfected NSC-34 cells with plasmids containing FUS and FUS mutants lacking different domains and we performed RNA-immunoprecipitations on these cells. The analysis of the results showed that the first Arginine glycine-glycine cassette (RGG1) and the RNA recognition domain (RRM) are those responsible of the interaction with snRNA U1. To verify if this interaction is required for FUS to regulate splicing, we co-transfected NSC-34 cells with plasmids containing FUS variants lacking RGG1 and RRM domains together with the splicing reporter pSMN2. Compared to control cells, the ratio between the splicing isoforms of the reporter pSMN2 is different in cells transfected with FUS, while those transfected with mutants have patterns similar to the non transfected cells, suggesting that these domains are important for FUS ability to regulate alternative splicing. In light of these results, and in order to extend to an in vivo ALS model the notion that splicing misregulation might be involved in disease pathogenesis, we decided to look for some RNA targets whose splicing pattern was altered in an in vivo model of SMA pathogenesis. To this aim, we performed splicing assays in homozygous transgenic mice overexpressing human FUS that develop ALS like symptoms. We found that hnRNP A2/B1 and Neurexin2 splicing patterns are different between these mice and controls. Notably, alterations in hnRNP A2/B1 splicing pattern parallels the disease progression. We then performed immunohistochemistry analysis on spinal cords from FUS transgenic mice to see if these splicing changes were reflecting an alteration in Gems. We found a significantly reduced number of Gems compared to controls in motor neurons of diseased FUS mice, similarly to what has been observed in mice modeling of SMA. Altogether, these findings support the hypothesis that ALS and SMA share similar pathogenic mechanisms that involve alteration in the regulation of alternative splicing by SMN. To reinforce this notion, heterozygous, non symptomatic transgenic mice overexpressing human FUS (FUS +/-) were crossed with heterozygous mice knock-out for SMN (SMN +/-), generating a new transgenic line, FUS +/-; SMN +/-, that expresses a higher amount of FUS WT in a reduced SMN levels background. Intriguingly, these mice behave normally and do not show any impairment in motor activity, indicating that a reduction in SMN levels is not sufficient to induce the appearance of ALS symptoms in heterozygous FUS transgenic mice. These mice are now being used to generate FUS +/+; SMN +/- mice to assess the effect of SMN depletion in symptomatic FUS mice. In conclusion, this research indicates that FUS requires RGG1 and RRM domains to bind snRNP U1 and to regulate alternative splicing, which is a process that seems to be crucial in motor neurons. The alteration of RNA splicing, therefore, could be a common and primary cause of ALS and other neurodegenerative pathologies.