The genetic landscape of ALS/MND in Turkey and alsin protein-related motor neuron pathology in ipsc-derived cortical neurons

After Alzheimer's and Parkinson's diseases, Amyotrophic Lateral Sclerosis (ALS) is the third most common adult-onset neurodegenerative disease, characterized by progressive muscle weakness and atrophy due to the degeneration of both upper and lower motor neurons. ALS exhibits a broad spectrum of phenotypic variability and involves complex genetic factors. Approximately 10-15% of patients inherit the disease from their parents while the remainder appear sporadic. Monogenic mutations in various genes, particularly C9ORF72, SOD1, TARDBP and FUS, account for disease development in 70% of familial and 5-8% of sporadic cases worldwide, pointing to the strong genetic component in ALS pathophysiology. In the first part of this PhD thesis, the genetically heterogeneous, yet inbred Turkish population was investigated to explore and revisit the basis of ALS genetics. A cohort of 1200 index patients of Turkish origin were included in the study, 15% (176/1200) having familial ALS. Parental consanguinity was present in 14.1% (170/1200) of cases and in 26.5% (319/1200) of patients, the parents were from the same region. All patients were tested for C9ORF72 hexanucleotide repeat expansion. The HRE mutation was detected in 27% (47/176) of familial and 2% (23/1024) of sporadic cases. Further genetic analysis was conducted on 577 patients, using Next Generation Sequencing techniques. The most frequently implicated genes in the cohort were SOD1 (n=33), OPTN (n=18), TARDBP (n=10) and FUS (n=5), which, together with C9ORF72, explained 47% of familial and 4.7% sporadic cases. Additionally, 10% of the familial and 3% of sporadic cases were solved with rare ALS-associated genes, pointing to a strong locus heterogeneity among the Turkish cohort under study Genetic analysis in some families uncovered unique and previously unrecognized situations that diverge from the established inheritance patterns and phenotypic associations reported in other studies. These findings emphasize the significance of our research in revealing novel mechanisms and underscore the critical role of genetics in the pathophysiology of ALS. Homozygous loss of function (lof) mutations in the alsin Rho guanine nucleotide exchange factor ALS2 (ALS2) gene are responsible for three distinct juvenile motor neuron diseases: jALS, IAHSP and jPLS. The alsin protein, encoded by the ALS2 gene, contains three domains with putative guanine exchange factor activity for Ras superfamily GTPases. Disruptions in these domains have been shown to cause neuronal dysfunction, ultimately leading to neurodegeneration. In the second part of this thesis, a patient with a homozygous termination mutation in ALS2 gene was studied and the consequences of alsin lof in patient-derived iPSCs and induced cortical neurons were investigated. Our study revealed that alsin-deficient cell lines exhibit defects in endovesicular trafficking, along with significant impairments and delays in receptor-mediated endocytosis. Additionally, decreased activation of Rac1-GTPase and prominent morphological abnormalities were observed in these cells. This research is significant in presenting the alsin pathophysiology in patient-derived induced cortical cells for the first time.