INVESTIGATION OF C9ORF72 MOLECULAR HALLMARKS AND DEVELOPMENT OF THERAPEUTIC STRATEGIES

The majority of genetic Amyotrophic Lateral Sclerosis (ALS) cases are due to the expansion in the number of an hexanucleotidic repeat in a non-coding site of the locus C9orf72. Many mechanisms for the C9orf72-ALS are suggested, both loss of function and gain of function due to RNA foci and Dipeptide Repeats (DPR) toxicity. Research on C9orf72-ALS is hindered by the lack of satisfactory models and by the difficult access to the target cell type. Animal models fail to recreate all the pathological features and the situation is further complicated by the presence of many disease modifying genes. To overcome theseissues, this study takes advantages of the induced Pluripotent Stem Cells (iPSCs) technology to investigate canonical as well as more recent C9orf72 molecular hallmarks. The iPSCs carry the same genetic background of patients and can be further differentiated in motoneurons (MNs) to study neuronal specific alteration and comparing C9orf72-expanded cell lines with their isogenic corrected counterpart, the influence of disease-modifying genes can be eliminated. We analysed canonical RNA foci and DPR accumulation finding them increased in C9orf72 samples in both models. DNA damage accumulation, a recently discovered C9orf72-ALS feature, was also increased. Moreover, in iPSC-derived MNs expression of SEPT7, STMN1 and STMN2 genes, cytoskeletal regulators with important function in neuronal cells, has been found altered in C9orf72 background. Taking advantage of these established models we could also evaluate the efficacy of a morpholine (MO)-based antisense oligonucleotide (ASO) therapy as a proof of principle for the feasibility of drug screening in these models. We foundthat both our MO oligomers were able to rescue DPR accumulation and DNA damage induction. Global gene expression analysis has also been performed. From this investigation a subtle alteration in genes related with neuronal function in iPSC was detected, while C9orf72 MNs showed deregulation in pathways related to inflammation and cell-to-cell communication suggesting a non-cell-autonomous mechanism for the disease. Interestingly, MOs treatment could rescue these alterations. Moving forward we started the characterization of a 3D organoidbased model of C9orf72-ALS that can reproduce the complexity of central nervous system. We found in organoids DPR accumulation and other disease hallmarks which could not be detected in 2D models, supporting the promising role of this 3D model.