Bryce-Smith, Samuel; (2025) Characterising the dysregulation of TDP-43 mediated alternative polyadenylation in ALS. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Nuclear loss and cytoplasmic aggregation of TAR DNA-binding protein 43 (TDP-43) occurs in up to 97% of amyotrophic lateral sclerosis (ALS) and 45% of frontotemporal dementia (FTD) patients. TDP-43 point mutations also cause ALS and FTD, indicating a central role of TDP-43 dysfunction in these disorders. TDP-43 nuclear depletion impairs cryptic splicing repression, disrupting key neuronal gene expression. Disease-causative TDP-43 mutations present a splicing gain-of-function, implicating altered RNA processing in ALS-FTD pathogenesis. This thesis analyses RNA-sequencing data from cellular and in vivo models to investigate how TDP-43 dysfunction affects RNA alternative polyadenylation (APA) regulation and its relevance in disease. Firstly, I present results from a community-driven benchmarking study, in which I was a core contributor, to evaluate algorithms for APA detection and quantification using bulk RNA sequencing data. We developed a framework for reproducible, extensible benchmarking, highlighting the poor performance of de-novo inference methods relative to reference-based methods. Using these results, I analysed bulk RNA-sequencing data from TDP-43 mutant mouse models to contrast the effects of loss and gain-of-function mutations on APA regulation within 3’UTRs, uncovering a gain-of-function mimicking that of alternative splicing. Lastly, I developed a computational pipeline to identify novel APA events extending 3’UTRs and occurring within introns. I systematically analysed neuronal TDP-43 depletion bulk RNA-seq datasets to identify cryptic APA events. I integrated post-mortem RNA-seq data from the New York Genome Consortium ALS/FTD dataset to identify events specifically expressed in patients and brain regions affected by TDP-43 proteinopathy, highlighting putative biomarkers and disease contributors. Finally, I integrated orthogonal omics data profiling translation and RNA stability to highlight transcription factors with cryptic 3’UTR extensions that increase RNA stability and translation. Altogether, this thesis expands knowledge of TDP-43 dysfunction impacts on RNA regulation, raising novel disease effectors and markers of TDP-43 proteinopathy for further study

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