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Investigating the molecular environments and interactions of pathological TDP-43 filaments https://doi.org/10.17863/CAM.113785 Chen, Renren Ryskeldi-Falcon, Benjamin Filamentous transactive response DNA-binding protein 43 (TDP-43) pathology characterises many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementias (FTD). Pathogenic mutations in TARDBP, the gene encoding TDP-43, can cause ALS and FTLD by increasing TDP-43 filament formation, demonstrating a central role of TDP-43 pathology in disease. TDP-43 pathology starts in focal sites in the central nervous system (CNS) and spreads to connected regions over time, which correlates with progressive neurodegeneration and clinical symptoms. There is growing evidence that TDP-43 filaments themselves may spread between neurons, potentially through axonal trafficking and synaptic transfer, and seed the assembly of native TDP-43, a phenomenon known as prion-like propagation. However, the underlying molecular mechanisms are largely unknown. An understanding of these mechanisms may yield therapeutic strategies to intervene with pathological TDP-43 filament formation to slow or prevent disease progression. In this thesis, I aimed to shine a light on these mechanisms by investigating the molecular environments and potential interactions of pathological TDP-43 filaments. First, I established novel human and murine neuronal model systems of stages of pathological TDP-43 propagation by incubation with exogenous TDP-43 filaments extracted from human brain. Second, I developed a time-resolved, antibody-targeted proximity labelling approach to map the molecular environments and potential interactions of pathological TDP-43 in these models at different stages of propagation in an unbiased manner. During the uptake of exogenous filaments, I observed an enrichment of distinct cell-surface receptors. At later stages, I detected an enrichment of specific chaperones and proteins involved in the endolysosomal system, intracellular trafficking, release and cellular degradation systems. Unexpectedly, I found that pathological TDP-43 filaments were most enriched at synapses, especially at the pre-synaptic active zone complex. I validated the presynaptic localisation of TDP-43 filaments in these models and in human tissue using immunofluorescence light microscopy, co-immunoprecipitation and synaptosome preparations. These findings suggest an important role for the pre-synaptic active zone complex in prion-like propagation of TDP-43 filaments and, possibly, resulting toxicity. During my PhD, I have mapped and validated novel molecular environments and potential interactions of pathological TDP-43 filaments at different stages of their prion-like propagation in novel neuronal models. These insights will guide future studies into the molecular mechanisms of propagation and may inform therapeutic strategies to intervene in this process. University of Cambridge 2024-09-01 Thesis Doctoral Doctor of Philosophy (PhD) eng Cambridge Trust https://www.repository.cam.ac.uk/handle/1810/376619 embargo 2025-11-26 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/c55626f5-33a6-4c91-af0b-07cf8f1b3a8f/download a6e460f9465e00244290a5e227129a58 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/bf64f700-a91a-4616-a0f1-594b59d39d0a/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ TDP-43 FTLD ALS Neurodegeneration Synapse Mass spectrometry