Studio della localizzazione nucleo-citosol degli RNA messaggeri in modelli cellulari di Sclerosi Laterale Amiotrofica associata a mutazione nel gene C9ORF72

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease. It is characterized by the neurodegeneration of motor neurons in the brain and spinal cord. The result for patients is the inability to coordinate the voluntary muscular movements and this culminates in the death due to respiratory insufficiency within 2-5 years from the diagnosis (Taylor et al., 2016).Approximately 10% of cases of the disease are classified as familial, while 90% are sporadic, although the two forms are clinically indistinguishable. Many genetic factors have been associated with ALS, but their impact on the pathology has been better characterized for the genes C9ORF72, TARDBP (TDP-43), SOD1 and FUS which, together, represent the cause of about 70% of familial cases. The main molecular consequences linked to these predisposing factors are many; they vary from protein aggregation to alterations of RNA metabolism. In particular, the discovery of a GGGGCC hexanucleotide repeat expansion within chromosome 9 open reading frame 72 (C9ORF72) - the most common genetic mutation responsible for the development of ALS - represents a major advance in ALS disease research because it provided strong support to the concept that RNA metabolism defects are critical and causative factors in the development of the disease (Ling et al., 2013). In normal individuals, the GGGGCC sequence is repeated mostly two-five times (range 0-20), while in patients with C9ORF72-linked ALS, the numbers of repeats exceed 30, and it has been estimated that it can reach several hundred or even thousands of repeats (Rohrer et al., 2015). Multiple pathogenic mechanisms are thought to underlie the pathology associated with the G4C2 expansion: haploinsufficiency due to the loss of protein expression from the mutated allele; RNA toxicity caused by the accumulation of repeat-containing transcripts; and generation of toxic protein species (poly-dipeptides, DPRs) by an unconventional mechanism of protein translation, which is known as repeat-associated non-ATG (RAN) translation (Heutink et al., 2014). Recently, in our lab we observed that the expression of an expanded GGGGCC repeat in cultured cells causes translational repression and a marked nuclear accumulation of poly-adenylated mRNA (Rossi et al., 2015). This suggests that defective trafficking of mRNAs - as a consequence of impaired nuclear mRNA export - might contribute to the pathogenesis of C9ORF72-linked ALS. On this basis, the general aim of this study was to evaluate whether the altered mRNA export characterizing C9ORF72 cells might impact on specific mRNAs with ALS-relevant functions by systematically identify, by RNA sequencing (RNAseq), those mRNAs that are specifically altered in their nucleus/cytosol distribution in cellular models expressing a C9ORF72 mutation. To this aim, we extracted nuclear and cytoplasmic RNAs from cultured cells which express an 31 GGGGCC repeats (G4C2)31 ad from control cells. After checking fraction purity and RNA integrity, RNAs prepared from samples in triplicate were analyzed through mRNA sequencing. After assessing the nucleus/cytosol ratio (N/C) of each identified RNA target for each sample, we compared the N/C ratios of cells expressing expanded repeats to those of controls cells, as a measure of the shift in nuclear vs cytosolic localization of mRNAs. Overall, this analysis led to the identification of 60 mRNAs targets that are significantly accumulated in the nucleus of cells expressing (G4C2)31 repeats, which further supports the concept that mRNA export defects might be major feature of C9ORF72 cells. Moreover, although preliminarily, these results confirms the idea that defects in a general mechanism, such as the nuclear export of mRNAs, might have selective effects on a distinct set of target genes, and that the alteration of these genes might explain the specific degeneration of motor neurons, that is the most typical feature of ALS. However, these data do not clarify whether the observed phenotypes depend upon the toxic action of the accumulated expanded RNA, or if the toxic RNA is assisted by a second pathogenic mechanism linked to its translation into poly-dipeptides (DPRs) within the cell. To clarify this point, the second part of our research focused on the characterization of a human cell model, namely HeLa cells, that transiently express the five individual DPRs, poly-glycine/arginine (GR), poly-proline/arginine (PR), poly-glycine/alanine (GA), poly-glycine/proline (GP), poly-proline/alanine (PA), from synthetic cDNAs encoding 100 repeats of DPR without a GGGGCC repeat. Immunofluorescence analysis showed that all five DPRs, even if in different proportions, are able to induce translational repression associated with the formation of stress granules and, especially in cells expressing poly-GR and poly-PR, this is accompanied by a significant accumulation of polyadenylated mRNAs and associated proteins such as PABPc in cells nuclei. The obtained data indicate that the expression of DPRs is sufficient to generate the molecular phenotypes characterizing G4C2 repeat-expressing cells, suggesting that the presence and accumulation of expanded RNAs in cells might be dispensable for C9ORF72 toxicity, or at least that DPRs co-operate with G4C2 RNA in impairing the proper nucleus/cytosol trafficking of mRNAs, thus contributing to the pathogenicity C9ORF72 mutation. In conclusion, we have collected further evidence that alterations in nucleus/cytosol transport play a crucial role in the pathogenesis of SLA, and further studies will be needed to understand the details closely associated to this mechanism.