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Studies of hyperphosphorylated tau aggregation and cytotoxicity 10.17863/CAM.93908 Meng, Jonathan Klenerman, David Diffusible aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by protein kinase A in conjugation with either glycogen synthase kinase 3b or stress-activated protein kinase 4 enabled recombinant wild-type tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites, highresolution native mass spectrometry to measure the degree of phosphorylation, and superresolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, compared with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4-dependent responses in human macrophages. We also found that non-phosphorylated tau could polymerize into amorphous, cytotoxic aggregates when induced by hyperphosphorylated tau in situ, having similar structural and functional properties to the self-assembled hyperphosphorylated tau aggregates. These results together offer two alternative mechanisms of how hyperphosphorylation may lead to aggregates that are potentially damaging to cells and drive neuroinflammation in tauopathies. University of Cambridge 2022-12-29 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/346489 controlled.access https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3d76ba75-d850-4b59-98be-128078d68e6c/download 6a1e9f49c404cd14ce907508db970634 https://www.rioxx.net/licenses/all-rights-reserved/ tau Alzheimer's disease hyperphosphorylation