Studio del ruolo di SMN nella patogenesi della sclerosi laterale amiotrofica associata al gene FUS

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig disease, is a neurodegenerative disease that affects both upper and lower motor neurons, resulting in progressive weakness, muscles atrophy and, finally, death usually in 3-5 years after symptom onset. ALS is classified in familiar ALS (fALS), 10% of the cases, and sporadic ALS (sALS), the remaning 90%, based on the presence of gene mutation in the familiar lineage. There are many genetic causes for ALS, pointing to diverse disease mechanisms as possibly involved; yet, a number of mutations has recently been identified in ALS affecting proteins involved in RNA metabolism such as TDP43, C9orf72 and FUS, and this last is the object of this dissertation. FUS is a nuclear protein having an important role in both DNA and RNA metabolism. Although its location is mainly nuclear, FUS is also present in the cytoplasm, where it forms cytoplasmic inclusion in ALS patients and mice model tissues. It is supposed that FUS has an important role in alternative splicing: indeed, it binds snRNPs and SMN, which are both very important players in spliceosome assembly and function. Moreover, several works showed that ALS-associated mutant FUS holds snRNPs in the cytoplasm causing splicing alteration. These evidence suggest that alternative splicing defects could participate in the disease, similarly to what occurs in another motor neuron disease: spinal muscular atrophy (SMA). The purpose of this work was to investigate the functional overlapping between FUS and SMN. To this aim, mice characterised by wild type FUS over-expression (FUS +/+), which develop a severe ALS-like pathology, were used as an in vivo model to investigate whether SMN dysfunction has a role in ALS pathogenesis.First, the alternative splicing of selected genes that change in SMA mice, as well as in tissues from SMA patients has been analysed in FUS +/+ mice. The results show that the splicing of Adarb1, Ataxn2, Dusp22, Mphosph9, Agrin, Gria, hnRNP A2/B1, Mapk8, Vps16, C19orf54, Parp1 is affected in FUS +/+ mice compared to control FUS -/- mice, similarly to what is observed in SMA. Interestingly, the majority of these changes are specific for FUS-induced motor neuron degeneration, as they are not present in mice mimicking the degeneration occurring in SOD1-G93A ALS, nor in spinal and bulbar muscular atrophy (SBMA) model mice. These results suggest a clear overlapping between FUS and SMN. To verify this hypothesis, the ALS phenotypes, as well as and the alternative splicing changes, that characterise FUS +/+ mice have been studied in the same mice where the levels of SMN were reduced by genetic depletion of the Smn1 gene. Surprisingly, symptoms onset, motor capability (analysed by motor test both in neonatal and adult age) and age of death are unaffected. Further, the alternative splicing of the above mentioned genes does not show any difference between the two genotypes. In conclusion, the results of this work indicate that the pathogenesis of FUS-associated ALS recapitulates, in vivo, crucial molecular features of the degenerative process that characterise mouse models of SMA, and in particular the alteration in the alternative splicing pattern of genes that have important roles in motor neuron function. Since these phenotypes are not modified by SMN shortage, however, it is plausible that this occurs through independent, yet converging, pathways that still need to be identified.