INDUCED PLURIPOTENT STEM CELLS (IPSC) TO STUDY PATHOMECHANISMS ASSOCIATED TO AMYOTROPHIC LATERAL SCLEROSIS (ALS)

Amyotrophic Lateral Sclerosis (ALS) is a currently incurable and adult-onset neurodegenerative disease, characterized by the progressive and selective loss of upper and/or lower motor neurons, leading to a relentless and severe muscular atrophy with rapid death of patients, usually due to respiratory failure. The majority of ALS patients (90%) manifest a sporadic form with a multifactorial etiology, while 10% of cases are familial with more than 30 causative genes identified so far. The main genetic cause of ALS is represented by the intronic GGGGCC hexanucleotide repeat expansion (HRE) in C9ORF72 gene which is polymorphic in healthy subjects (2-23 units), while in ALS patients it expands from 30 to more than 4000 units. The pathomechanisms associated to C9ORF72 HRE include a loss of function due to C9ORF72 protein haploinsufficiency because of reduced transcription, and a toxic gain of function caused by both the formation of pathological HRE- containing RNA foci and the synthesis of dipeptide repeat proteins (DPR) resulting from RAN translation of the HRE-containing transcripts. C9ORF72-associated defects include impairment of RNA metabolism, genomic stability, DNA damage response (DDR), nuclear pore integrity and axonal transport. The study of ALS pathomechanisms has always been hampered by the lack of suitable experimental models that could properly mimic the pathophysiology of the disease. The quite recent possibility to obtain patient-derived induced pluripotent stem cells (iPSC) represents a great advancement to study neurodegenerative disorders, because iPSC maintain the individual genetic background and can be differentiated into different neuro- glial cell lineages, including motoneurons and cortical neurons. In my PhD project I exploited iPSC, reprogrammed from C9ORF72 ALS patients, to study DNA damage and DDR and their possible link with actin cytoskeleton remodeling, and to test a novel RNA- based therapeutic approach to reduce C9ORF72 pathology. I also established a new patient-derived iPSC line to elucidate the different pathomechanisms associated with KIF5A gene mutations. To study DNA damage and DDR, we differentiated three C9ORF72 and two healthy control iPSC lines, already reprogrammed in the lab, into both neural stem cells (iPSC-NSC) and motor neurons (iPSC-MN) at different maturation time (day 34 and 56). DNA damage was induced with the radiomimetic agent Neocarzinostatin and the phosphorylated histone H2AX (gH2AX) was used as marker of DNA breakage. By measuring the mean number of gH2AX- positive foci, the γH2AX mean fluorescence intensity in the cell nucleus and the percentage of γH2AX foci-positive cells, we found that the presence of the C9ORF72 HRE did not impact on the extent of DNA damage and DDR ability in iPSC-NSC. C9ORF72 iPSC-MN (day 34) displayed a higher extent of DNA damage at physiological level compared to wild-type controls and they were able to recover the induced DNA damage after 6 hours rescue. However, prolonging the maturation time of iPSC-MN in vitro (day 56), we observed both a significant higher extent of DNA damage and an impaired ability of C9ORF72 iPSC-MN to activate DDR compared to control iPSC-MN. During my 3-months internship in the USA, I applied an innovative method to differentiate cortical neurons from one C9ORF72 and one isogenic wild-type i3-iPSC line using an inducible system. I found that modulation of the F-actin cytoskeleton using actin-modifier drugs had an impact on the incidence of DNA damage, DDR and chromatin remodeling. To decrease C9ORF72-associated pathology, I used iPSC to test a novel RNA-based approach consisting in a modified spliceosomal U1 snRNA specifically designed to bind the sense transcript of the C9ORF72 HRE. We first demonstrated U1 snRNA efficacy in reducing RNA foci and DPR formation in HEK293T cells, then we validated our results in C9ORF72 iPSC-MN infected with U1-lentiviral vectors. We found that modified U1 snRNA could significantly decrease both the mean number of pathological RNA foci/cell and the percentage of C9ORF72 iPSC-MN forming RNA foci. I also took advantage of iPSC technology to establish a new in vitro model to study why different mutations in KIF5A gene may cause ALS or hereditary spastic paraplegia (HSP). KIF5A encodes for a microtubule motor protein involved in the anterograde transport in neurons. I reprogrammed fibroblasts from a HSP patient carrying a novel KIF5A mutation (p.R17Q) into iPSC and also generated its isogenic wild-type and a lof iPSC line using CRISPR/Cas9 gene editing. I differentiated these iPSC lines into MN to study and compare their differentiation capacity, mitochondria morphology and distribution along neurites as well as the axonal transport of mitochondria through live-cell imaging assay. The results obtained in my PhD thesis provide evidence that patient-derived iPSC and iPSC- MN represent suitable in vitro platforms both to model C9ORF72 and KIF5A-related pathogenetic mechanisms and to screen new promising therapeutic approaches for the treatment of ALS disease.