Functional interaction between FUS and SMN in the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is one of the most common motor neuron disease. ALS affects both upper and lower motor neurons, resulting in death within 3-5 years after symptoms onset. The recent identification of at least three RNA binding proteins as responsible for more than 60% of familiar cases of ALS highlighted the possibility that disturbed RNA metabolism could be a major pathogenic mechanism underlying the disease. Among all, FUS is an RNA binding protein, with a mainly nuclear localization, involved in several functions both in the nucleus, such as DNA transcription and RNA metabolism, and in the cytosol, as mRNA transport along the axons. Mutations of FUS, that account for about 5% of familial cases of ALS, mostly occur in the Nuclear Localization Domain (NLS) of the protein, thus inducing the formation of cytoplasmic aggregates, that are detectable in motor neurons of affected patients. FUS is part of the hnRNP family, a class of proteins able to directly bind intronic or exonic sequences to regulate splicing events. It is likely that FUS regulate alternative splicing also through the cooperation with SMN, a protein crucial for the maturation of the spliceosome. Indeed it has been demonstrated that FUS binds with components of the spliceosomalsnRNPs (small nuclear Ribonucleo Proteins, composed by small nuclear RNAs, snRNAU, and a set of different protein co-factors), such as SMN and proteins of the SMN-complex, thus contributing to the assembly and/or the maturation of the spliceosomal machinery. According to the role of FUS in the control of splicing, is not surprising that in FUS-related ALS impairment in the correct alternative splicing regulation occurs. Thisevidence suggests that alternative splicing defects could participate to the disease, linking ALS to another motor neuron disease where not only a deregulation of the splicing machinery occurs, but also, interestingly, SMN functions are directly involved: Spinal Muscular Atrophy (SMA). In SMA, similarly to what happens in ALS, the lower motor neurons are affected and patients, usually newborns, die for respiratory failure within few years after the diagnosis. SMA is caused by the depletion of Smn1gene, that encodes for SMN protein, the major constituent of the complex that mediates the maturation of spliceosomalsnRNPs. Indeed, in SMA patients and in animal models the correct assembly of snRNPs is affected, thus resulting in a general imbalance of the splicing regulation. Altogether these data suggest that FUS and SMN might cooperate to the same molecular pathway, i.e. alternative splicing regulation, and that disturbances in SMN-regulated functions, either caused by depletion of SMN protein (as in the case of SMA), or by pathogenic interactions between FUS and SMN (as in the case of ALS) might be a common theme in both ALS and SMA. This work was aimed at characterizing in vivo the role of SMN in the pathogenesis of FUS-related ALS. To this aim we used a mouse that overexpresses the human wild-type FUS (hFUS +/+) as ALS model, and we analyzed molecular phenotypes usually induced by SMN depletion. In particular, we observed the alteration of the splicing pattern of some target genes, whose splicing has been demonstrated affected in SMA models, that are particularly relevant for motor neuron viability. Furthermore we observed a decrease number of mature snRNPs from nuclei of motor neurons, that is a typical feature of tissues from SMA patients and models, thus suggesting a possible functional overlapping between FUS and SMN. However, molecular phenotypes induced by SMN lowering in SMA, such as the shortage of the RNAUs and their altered assembly in the core of the spliceosome, has not been observed in FUS mice. These data suggest that ALS and SMA converge in the alteration of the same pathway, but probably through different mechanisms. Yet, the shortage of SMN expression in this ALS model, obtained by crossing FUS mice (hFUS +/+) with SMN heterozygous knock-out mice (Smn +/-), did not modify the disease course nor the molecular phenotypes analyzed, thus reinforcing the existence of a complex interplay between FUS and SMN in the regulation of alternative splicing and gene expression.