Trans-Activation Response DNA-binding Protein 43 (TDP-43) is a major constituent of proteinaceous inclusions characteristic of most forms of amyotrophic lateral sclerosis (ALS) and ubiquitin-positive frontotemporal lobar degeneration (FTLD). Normally a nuclear protein, TDP-43 translocates to the cytoplasm and forms pathogenic inclusions in the disease-state, where the protein is often phosphorylated and cleaved and co-localises with stress granules.

This thesis explores the features of TDP-43 pathology in established cell models using microscopy techniques to investigate the relationship of pathological aggregate species and cellular dysfunction. We have characterized stable cell models expressing EGFP-TDP-43 to identify key pathological hallmarks of disease at the molecular level, using confocal microscopy imaging. Using various chemical stress inducers to recapitulate disease pathology, we were able to induce TDP-43 aggregation and to characterise features of the self-assemblies within the cells. We further investigated the properties of these putative pathogenic aggregate species using FLIM (Fluorescence lifetime imaging microscopy) and demonstrate decreased fluorescence lifetimes of EGFP-tagged TDP-43 following MG132 stress induction and we correlated these data with an in vitro model system of liquid droplet formation. Using structured illumination microscopy (SIM) we detail the interactions of TDP-43 aggregate species with subcellular structures to begin investigating the relationship between the aggregate species and the cellular milleu. We demonstrate the use of SIM to enhance the use of mitochondria and lysosomes as markers to screen for TDP-43-induced cellular dysfunction.

Finally, we describe the use of a novel cell model utilising a biarsenical dye system as a promising alternative to commonly used fluorescent proteins for studying TDP-43 dynamics. We demonstrate improved scope for this model by incorporating it into a more physiologically relevant cell line and introducing the A315T and M337V disease-related mutational variants. This model holds promise for studying the mechanisms underlining the cell-to-cell spread of pathology and to identify therapeutic targets.