Alterazioni nel meccanismo di esporto nucleare degli mRNA nella patogenesi della Sclerosi Laterale Amiotrofica associata a mutazioni nel gene C9orf72

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that affects both lower motor neurons i n the brainstem and spinal cord, and the upper motor neurons i n the motor cortex. Loss of these neurons leads to muscle atrophy and weakness, fasciculations and spasticity and death from respiratory failure, typically within 2 to 3 years from symptom onset. About 10% of ALS is classified as familial (fALS), whereas the remaini ng 90% of cases are considered sporadic (sALS), as they appear to occur randomly throughout the community (Renton et al., 2014). fALS is associated with a wide range of genes that are autosomal dominant or recessive mutations.The recent discovery of mutations in TARDBPand FUSgenes, responsible for about 10% of casesof fSLA, focused the attention on a possible pathogenic role of RNA metabolism, which was observedto bealtered also i n other neurodegenerati ve diseases. Moreover, a non-coding G4C2 repeat expansion in the C9orf72 gene has been identified in 2011 as the major genetic determi nant of ALS. In normal individuals the G4C2 sequence is repeated mostly two-to-fi ve time (range 0-20), whi le in patients with ALS associated to the gene C9orf72the number of repeats exceeds 30, and it has been estimated that it can reach several hundred or even thousands of repeats (Robberecht and Philips, 2013). The understanding of the disease mechanisms caused by the repeat expansion is still i n an early stage. Three main hypotheses are currently being considered to explai n the disease process, including: haploinsufficiency due to the loss of protei n expression from the mutated allele, RNA toxicity caused by the accumulation of repeat containing transcripts,and toxic protei n species generated by repeat-associated non-ATG (RAN) translation (Heuti nk et al., 2014). RNAs containi ng the repeat, which accumulate as RNA foci in the nucleus and/or cytoplasm of affected cells, are able to bind to various RNA-binding proteins, possibly impairing their function. Recently, in our lab we observed that the expression of pathological repeats, which induces the formation of nuclear RNA foci, causes stress granules formation, reduction of protein translation and a marked nuclear accumulation of poly-adenylated mRNAs in cultured cells.So the aim of this research was to i nvestigate how the expansion of the hexanucleotide G4C2 repeat might induce neurodegeneration and, in particular, the functional consequences of (G4C2)31expression on nuclear mRNA export pathway and protei n translation in cultured cells.6As a cellular model we choseHeLa cells transfected with plasmids contai ning pathological (G4C2)31or non-pathological (G4C2)10repeat.The G4C2 sequence is located within an intron,so in this study wedecided tointroduce the intronic sequences of ?-actin gene upstream (i ntron 5') and downstream (i ntron 3') of G4C2 repetitions to make our study model as close as possible to the physiologicalcondition. Fluorescence in situ hybridization (FISH) analysis showed that the expression of the pathological expanded repeatwithi n an i ntronic sequenceleads to the formation of nuclear RNA foci similar to those found i n ALS-C9orf72 patients and that are able to sequester some RNA binding protei ns (RBP). Furthermore, as previously observed, the expression of the intronic(G4C2)31iis able to induce a translational repression i n HeLa cells associated with the formation of stress granules, and accompanied by a considerable accumulation of polyadenylated mRNA in the cell nuclei. We then analyzed in vitro, using pull down assays, andin cells, using fluorescent in situhybridization and immunofluorescence analysis, the ability of the expanded repeat to interact with some key proteins involved in the mRNA nuclear export pathway, specifically NXF1 and ALY/REF. This analysis shows that the (G4C2)31irepeat is able to bind both these protei ns and sequester them into RNA foci,thus possibly i nterfering with their functions and leading to the nuclear retention of poly-adenylated mRNAs.To test this possibility we analyzed the effects of the expanded repeat on the mRNA export mechanism and the role of NXF1 through the use of plasmids coding for a dominant-negati ve mutant form NXF1 (?NXF1) and NXF1 wild type. Interestingly, the overexpression of?NXF1, which causes nuclearmRNA accumulation, reproduces key phenotypes characteri zing C9orf72 cells, including stress granules formation and translational repression. Moreover, the overexpression of wild-type NXF1 is able to reduce G4C2-induced stress granules.In conclusion, this research i ndicates that the expanded repeat G4C2 interferes withthe NXF1-mediated mRNA export pathway, and the resulti ng nuclear mRNA retention might affect translation efficiency and contribute to the pathogenesis of C9orf72-ALS.