Theme 3 In vitro experimental models

Background: Ongoing disease gene discoveries continue to drive our understanding of the molecular and cellular mechanisms underlying ALS. Causative genes from 60% of ALS families have been identified using modern genetic techniques, but the causal gene defect is yet to be identified in the remaining 40% of families. These remaining families often do not follow true Mendelian inheritance patterns and are challenging to solve using traditional genetic analysis alone. In vitro and in vivo studies have become critical in assessing and validating these ALS candidate genes.Objectives: In this study, we aim to develop and validate the utility of an in vitro functional pipeline for the discovery and validation of novel ALS candidate genes.Methods: A panel of cell based-assays were applied to candidate genes to examine the presence/absence of known ALS pathologies in cell lines as well as human autopsy tissues. These include immunofluorescence, flow cytometry and western blotting to study toxicity, neuronal inclusion formation, interaction with TDP-43, aberrant protein degradation and accumulation in detergent-insoluble cellular fractions. Immunohistochemistry and immunofluorescence were also used to examine if candidates were present in neuronal inclusions from ALS patient spinal cord tissues.Results: The in vitro pipeline was applied to five candidate genes from an ALS family that is negative for known ALS gene mutations. Two candidates were prioritized as top candidates based on their capacity to induce known ALS cellular pathologies. In transfected cells, the variants in these two genes caused a significantly higher toxicity than wild type, formed detergent insoluble inclusions and was able to co-aggregate with TDP-43 in neuronal cells. The variants have also led to protein degradation defects. One of the candidates also co-localised with TDP-43-positive neuronal inclusions in sporadic ALS patient post-mortem tissues, a signature pathology of ALS.Discussion and conclusions: We have demonstrated the utility of a functional prioritization pipeline and successfully prioritized two novel candidate ALS genes. These genes, and its associated pathways, will be further investigated through the development of animal models to establish if there is support for its role in ALS. New ALS genes offer fresh diagnostic and therapeutic targets and tools for the generation of novel animal models to better understand disease biology and offer preclinical testing of candidate treatments for ALS in the future.