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Investigating the effects of tau mutations in induced pluripotent stem cell-derived neurons 10.17863/CAM.42277 Ostick, Janine Louise Spillantini, Maria Grazia Microtubule-associated protein tau (MAPT) is a neuronal protein which promotes microtubule assembly and stabilisation. The MAPT gene is alternatively spliced to give six tau isoforms: three with 3 microtubule-binding repeats (3R, excluding exon 10) and three with 4 microtubule-binding repeats (4R, including exon 10). MAPT mutations cause the progressive, degenerative disorder fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17T), in which tau becomes abnormally phosphorylated and aggregates. FTDP-17T related MAPT mutations can either alter exon 10 splicing and the 3R:4R tau isoform ratio (such as N279K), or affect tau’s biochemical properties (such as P301L and V337M). In this project, I differentiated induced pluripotent stem cells (iPSCs) derived from control subjects and people with the N279K or V337M MAPT mutations to monolayer neurons. There was an increase in the proportion of neurons containing tau phosphorylated at the AT8 epitope in N279K cultures after 50 days, and more 4R tau was detected earlier in the N279K cultures than in controls, confirming the action of the N279K mutation. All monolayer neural cultures matured at similar rates in terms of synapse formation, neuronal marker expression and astrocyte production, and I did not detect other FTDP-17T-relevant phenotypes in MAPT-mutant cultures, perhaps due to the time points (50 and 80-100 days) at which I examined them. To investigate the effects of the N279K mutation independent of iPSC line genetic background, I attempted to create isogenic iPSC lines using CRISPR. This was not successful, but I obtained isogenic control and P301L iPSCs from Axol Bioscience, from which, along with the patient-derived P301L MAPT-mutant iPSCs, I made cerebral organoids (COs). Similar amounts of neuronal, astrocyte, and synaptic proteins were detected in the control and MAPT-mutant COs, as were cortical layer markers indicating a forebrain-like fate. Phosphorylation at the AT8 epitope was detected in the control and MAPT-mutant COs, as was tau misfolding, detected using the MC-1 antibody. In conclusion, some alterations similar to those observed in FTDP-17T brains can be observed in monolayer iPSC-derived neurons and perhaps in iPSC-derived COs. However, some phenotypes may be masked by experimental variability which needs to be addressed in the future. University of Cambridge 2019-08-01 Thesis Doctoral Doctor of Philosophy (PhD) en Funded by the MRC-DTP and the Sackler Foundation https://www.repository.cam.ac.uk/handle/1810/295215 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3b96b25d-ce98-4481-a0c4-9e427fad5b86/download f48ca546548757c5f7b150c939952164 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/9e040c72-e6a4-4bf4-8919-27b6514c9dd5/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ tau mapt ipsc