Cell targeted phenotyping: understanding molecular genetic mechanisms of C9ORF72 ALS in motor neurons

Motor neuron disease affects 2 in every 100,000 people each year. Amyotrophic lateral sclerosis (ALS), the most prevalent form of motor neuron disease, presents in adulthood with the loss of both upper and lower motor neurons. Approximately 10% of ALS cases are familial with dominant inheritance, while the remaining 90% are sporadic in nature. A large hexanucleotide repeat expansion in the C9ORF72 gene causes 7% of sporadic and 40% of familial cases, making the C9ORF72 gene expansion the most prevalent cause of ALS known to date. At present there is no cure for ALS and only treatments such as Riluzole to prolong life for a few months are available. There is a need to better understand disease progression and develop therapeutic approaches. The differentiation of motor neurons from induced pluripotent stem cells (iPSC), derived from patients with the C9ORF72 gene expansion, provides a model allowing exploration of the downstream effects of the mutation whilst maintaining the patientâs genetic background. However, these neurons are typically immature and may not fully recapitulate disease phenotypes as presented by patients. Therefore, generating mature iPSC-derived motor neurons is critical for the study of adult onset diseases including ALS, as they allow for improved functional assays and form a more physiological model for drug testing. In Chapter 3, two motor neuron differentiation protocols were initially trialled before selection of the final protocol and further optimisation trials to create mature motor neurons. In Chapter 4, patient and control iPSC-derived motor neurons were analysed for the expression of motor neuron specific markers, excitability and internal calcium phenotypes. Motor neuron marker (SMI-32) analysis detected a significant difference in expression at the early time point, day 40 during MN differentiation, between patient and control cells. A phenotypic difference was also observed at day 40 showing increased excitability and increased internal calcium release in patient versus control cells. In Chapter 5, iPSC-derived motor neurons were further engineered to express optogenetic tools in a cell specific or sub-compartment specific manner to allow for improved selective functional studies including synaptic vesicle release and intrinsic membrane potential studies. Combining optogenetic and reporter systems particularly for electrophysiological studies as used here, not only allows for selective functional analysis but also tracking and visualisation of live motor neurons in culture. This study generated a dual optogenetic and motor neuron reporter lentiviral vector based on the Cre/Lox system, able to both track and activate live motor neurons in culture. Analysing the downstream effects of the gene mutation in neurons, as above, is crucial. However, analysing how the gene itself leads to dysfunction is equally important. It is still unknown by which mechanism the C9ORF72 gene expansion causes disease, three mechanisms including haploinsufficiency, toxic RNA and toxic protein are hypothesised. Fluorescent in situ hybridisation aimed to further explore and add a novel approach to the question of haploinsufficiency. In the final results Chapter 6, the expression of the C9ORF72 gene itself was probed using fluorescent in situ hybridisation in patient cells to infer information on the expression of the expanded vs non-expanded alleles. One patient showed a mislocalisation of the expanded allele towards the nuclear periphery compared to the non-expanded allele suggesting reduced C9ORF72 gene expression. This project overall aims to further elucidate disease pathophysiology to allow future development of therapeutic approaches.