King's College London

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease and the most common of the motor neuron diseases. The disease course involves the progressive loss of upper and lower motor neurons in the brain and spinal cord, and the mechanism of this degeneration is not fully understood. Affected individuals usually die within three to five years from symptom onset, often due to respiratory failure. At present, there is no cure, and the drugs which are available are only able to extend life by just a few months and most therapeutic interventions are solely focused on alleviating the symptoms as best as possible. The pathological hallmarks of ALS are hyperphosphorylated and ubiquitinated TDP-43 inclusions in the cytoplasm of spinal cord motor neurons that are present in >95% of cases. TDP-43 binds to DNA and RNA, and is involved in multiple steps in RNA processing. The discovery of mutations in TARDBP, the gene encoding TDP-43, in 1-2 percent of sporadic and familial ALS cases, confirms that TDP-43 dysfunction plays a key role in ALS pathogenesis. The experiments described in this thesis used motor neurons derived from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying TARDBP mutations, in an attempt to replicate pathological features of ALS. Patient-derived motor neurons have endogenous levels of TDP-43 and therefore more likely to be closer to human physiology than transfected cancer cell lines which remain the most common cellular models of ALS. This work successfully demonstrated the presence of TDP-43 proteinopathy in motor neurons derived from patients with three different TARDBP mutations and replicates some aspects seen in post mortem tissues. The mutations studied were M337V and G298S, as well as a novel mutation, K181E which, unusually, is found adjacent to the RRM1 domain rather than the C terminal, where the majority of ALS-related TARDBP mutations are found. TDP-43 proteinopathy is related to a decrease in mitochondrial activity and cellular ATP production in the M337V mutant lines, which are specific to motor neurons. This phenotype was reversed by CRISPR-Cas9 correction of the mutation. Cell cycle regulator p53 is identified as a relevant disease pathway by RNA-seq analysis of mutant M337V iPSCs in comparison to its isogenic control line. It is hoped that insights gained from this work, such as the metabolic defects that are evident even in fairly young neurons, and the implication of p53 as an important disease pathway, that add to our understanding of the disease. This approach, which used highthroughput high-content imaging, can be applied in future work in screening for pathways involved in ALS pathogenesis, and potentially for testing potential new drugs and therapeutics in the future, on TARDBP mutant lines as well as other ALS mutations.