Exploring the role of non-coding RNAs in ALS: from disease onset to possible applications to counteract muscle degeneration

Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease whose causes remain largely unknown and whose therapeutic options are ineffective in the long term. Several studies suggest that non-coding RNAs and transcription factors, important regulators of gene expression, are critical for ALS onset and progression. Increasing evidence suggests that molecular alterations in muscle fibers in the presymptomatic phase can trigger ALS pathology. However, there is still a huge gap in the knowledge about the molecular mechanisms that compromise the muscle synapses and the consequences during the regeneration process. In the present study, we investigated the role of microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and transcription factors (TFs) in the regulation of muscle functioning and plasticity. First, we analyzed the non-coding and coding differentially expressed genes of skeletal muscle from SOD1*G93A mice, an established model for ALS studies. We identified 3 miRNAs (miR-152, miR-193a, and miR-193b) that were predicted to target genes that encode proteins with key functions in the maintenance of neuromuscular junction (NMJ). Our analysis revealed an aberrant expression of genes that encodes for acetylcholine receptors (AChRs) subunits and the results strongly suggest that the 3 miRNAs are involved in the adult-to-fetal (ε to γ) switch of AChRs isoforms in ALS. In vitro assays confirmed that the miRNAs indeed bind the 3’UTR of target genes and effectively induce their degradation. In vivo functional assays corroborate the involvement of miR-152, miR-193a, and miR-193b in the onset of myopathies. The overexpression of the pooled miRNAs in healthy mice induced loss of muscle mass and weight reduction. The mechanisms responsible for muscle atrophy induction were also investigated. High levels of miR-152, miR-193a, and miR-193b induce the impairment of synaptic transmissions and disrupt NMJ functioning. Interestingly, we also observed the fast-to-slow myofiber shift phenomenon as a consequence of the treatment. Altogether, these results suggest that miR-152, miR-193a, and miR-193b could possibly trigger ALS, since their aberrant expression occurs in the early-stage disease. Next, we evaluated the crosstalk between miRNAs-lncRNAs-TFs in muscle functions to reveal novel networks disturbed in myopathies and in muscle development. The transcriptomic analysis associated with specific bioinformatic tools for the predictions of intermolecular interactions allowed us to identify 139 networks that may be involved in the modulation of muscle functions. To select the targets of interest for further characterization, we investigated the expression of a list of those regulatory genes in other muscle atrophy models (denervation and starvation) and during in vitro differentiation. The dynamic expression of the lncRNA Gas5 and the TF Runx1 (possible targets of miR-30) lead us to select them for the next analyses. The in vitro overexpression of three miRNAs from the miR-30 family corroborated our predictions, specifically the upregulation of miR-30 caused the downregulation of Runx1 and the isoforms 5 and 6 of Gas5. Our gain-of-function assays showed that miR-30e promoted myotube fusion, producing thicker myotubes. The transcriptomic analysis of myotubes overexpressing miR-30e revealed its involvement in muscle functions and its ability to negatively regulate the cell cycle. Differently, myoblasts overexpressing Gas5 overexpress genes that promote apoptosis. We showed that miR-30 and Gas5 behave diametrically opposite with regard to regulation of 6 mitochondrial structure. miR-30 promotes mitochondria network fusion while Gas5 induces mitochondria fragmentation and disruption of mitochondria cristae. These results highlight the importance of non-coding RNAs in the regulation of muscle cell activity also guiding them toward disease development and pave the way for new treatments for ALS and other myopathies.