RNA Dysregulation in a Novel Human Model of TDP43 Proteinopathy

Amyotrophic lateral sclerosis (ALS) is a devastating and invariably fatal neurodegenerative disease. In patients, relentless motor neuron (MN) degeneration triggers progressive muscle weakness, paralysis, and death within a few years of diagnosis. Despite the massive clinical and genetic heterogeneity of ALS, over 97% of patients present with TDP43 pathology. This involves the nuclear-to-cytoplasmic mislocalisation and aggregation of TDP43, an important nuclear regulator of mRNA splicing. In this thesis, we describe a novel in vitro model allowing on-demand mislocalisation of endogenous TDP43, without its mutation or overexpression. We engineered TDP43-GFP induced pluripotent stem cells, where GFP was knocked into the C-terminus of endogenous TARDBP. In MNs, expression of an anti-GFP nanobody fused to a strong nuclear export signal (NES) allows for rapid mislocalisation of TDP43 into the cytoplasm. Our model successfully captures key pathological features of ALS, including dendritic defects, apoptosis, microRNA dysregulation, as well as hyperphosphorylation and, likely, aggregation of TDP43. Simultaneously, the nuclear loss of TDP43 results in widespread alternative splicing errors. In a doxycycline-inducible version of this model, we demonstrate that mis-splicing occurs immediately following mislocalisation. This highlights that splicing errors are one of the core early features of TDP43 proteinopathy. Little is currently known about many of the proteins that are depleted due to TDP43 pathology-mediated mis-splicing. This includes the three neuronally enriched proteins ACTL6B, ELAVL3, and CELF5. Utilising shRNAs, we independently knocked down these transcripts in healthy MNs to investigate their impact on neuronal morphology and the transcriptome. Depletion of ACTL6B profoundly reduced dendrite complexity and impacted chromatin regulation, suggesting that ACTL6B may be a novel therapeutic target. The TDP43 mislocalisation model presented here has the potential to be a valuable tool for studying the wide spectrum of TDP43 proteinopathies, and act as a crucial platform for the development of therapeutics for the treatment of ALS.