RNA Metabolism alteration in amyotrophic lateral sclerosis models

Project1: Unraveling the impact of microRNA on Amyotrophic Lateral Sclerosis pathogenesis. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that specifically affects upper and lower motor neurons leading to progressive paralysis and death. There is currently no effective treatment. Thus, identification of the signaling pathways and cellular mediators of ALS remains a major challenge in the search for novel therapeutics. Recent studies have shown that microRNA have a significant impact on normal CNS development and onset and progression of neurological disorders. Based on this evidence, in this study we test the hypothesis that misregulation of miRNA expression play a role in the pathogenesis of ALS. Hence, we exploited human neuroblastoma cell lines expressing SOD(G93A) mutation as tools to investigate the role of miRNAs in familiar ALS. To this end, we initially checked the key molecules involved in miRNAs biogenesis and processing on these cells and we found a different protein expression pattern. Subsequently, we performed a genome-wide scale miRNA expression, using whole-genome small RNA deep-sequencing followed by quantitative real time validation (qPCR). This strategy allowed us to find a small group of up and down regulated miRNA, which are predicted to play a role in the motorneurons physiology and pathology. We measured this group of misregulated miRNA by qPCR on cDNA derived from (G93A) mice at different stage of disease and furthermore on cDNA derived from lymphocytes from a group of sporadic ALS patients. We found that mir-129-5p was up-regulated in cells, mice and in patients and we validated that HuD as mir129-5p target. It has been reported that ELAVL4/HuD plays a role in neuronal plasticity, in recovery from axonal injury and multiple neurological diseases. Furthermore, we generated stable cell line overespressing mir129-5p and we found a reduction in neurite outgrowth and in the expression of differentiation markers in compare to control cells. Taken together these data strongly suggest that microRNAs play a role in ALS pathogenesis and in particular that mir129-5p can affect neuronal plasticity by modulating ELAVL4/HuD level. Project 2: FUS/TLS depletion leads an impairment of cell proliferation and DNA Damage Response. FUS/TLS (fused in sarcoma/translocated in liposarcoma) protein, a ubiquitously expressed RNA-binding protein, has been linked to a variety of cellular processes, such as RNA metabolism, microRNA biogenesis and DNA repair. However, the precise role of FUS protein remains unclear. Recently, FUS has been linked to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disorder characterized by the dysfunction and death of motor neurons. Based on the observation that some mutations in the FUS gene induce cytoplasmic accumulation of FUS aggregates, we decided to explore a loss-of-function hypothesis (i.e. inhibition of FUS’ nuclear function) to unravel the role of this protein. To this purpose, we generated a SH-SY5Y human neuroblastoma cell line which expresses a doxycycline induced shRNA targeting FUS and that specifically depletes the protein. In order to characterize this cell line we performed growth proliferation and survival assays. From these experiments emerged that FUS-depleted cells display alterations in cell proliferation. In order to explain this observation, we tested different hypothesis (e.g. apoptosis, senescence or slow-down growth). We observed that FUS-depleted cells growth slower than control cells. Based on the notion that FUS interacts with the miRNA processing proteins (Morlando et al. 2012), to explain this phenotype, we looked at miRNAs expression and we found an up-regulation of mir-7. Interestingly, this up-regulation is also observed in cells that express the ALS-linked FUS R521C mutation. Finally, since an increasing number of work correlated FUS with DNA damage and repair we explored the effects of DNA damage in FUS-depleted cells by monitoring important components of DNA Damage Response (DDR). We found that FUS depletion had an effect on the initial level of DNA damage by inducing the phosphorylation of H2AX in basal condition and that it delayed DSB repair when acute DNA damage occurs. Interestingly, genotoxic treatment resulted in changes in the subcellular localization of FUS in normal cells. We are currently exploring on one hand the mechanism by which FUS depletion leads to DNA damage, and on the other the functional significance of FUS relocalization after genotoxic stress. Taken together, these studies may contribute to the knowledge of the role of FUS in these cellular processes and will allow us to draw a clearer picture of mechanisms of neurodegenerative diseases.