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Cryo\u2010EM revealed the existence of disease\u2010specific tau folds, which are challenging to replicate <jats:italic>in vitro<\/jats:italic>. We studied three full\u2010length recombinant 0N3R tau forms (the wild\u2010type and the C322A and C322S variants) using an RT\u2010QuIC assay with brain homogenate seeding. C322A tau formed filaments resembling AD paired helical filaments (PHFs) but with a more open C\u2010shaped core. C322S tau yielded structurally distinct filaments with an ordered C\u2010terminal region. Both mutants seeded further aggregation, whereas the wild\u2010type showed poor reproducibility and mainly unfolded aggregates. These results highlight the importance of optimised conditions to produce disease\u2010relevant tau filaments and aid the development of targeted therapies.<\/jats:p><jats:p><jats:boxed-text content-type=\"box\" position=\"anchor\"><jats:caption><jats:title>Impact statement<\/jats:title><\/jats:caption><jats:p>We investigated the seeded assembly of 0N3R tau and its two mutational variants C322A and C322S, using Alzheimer's disease brain homogenates in a real\u2010time quaking\u2010induced conversion (RT\u2010QuIC) assay. The C322A variant formed filaments partially resembling the AD PHF structure, showing the importance of optimised conditions to produce disease\u2010relevant tau filaments.<\/jats:p><\/jats:boxed-text><\/jats:p>","DOI":"10.1002\/1873-3468.70141","type":"journal-article","created":{"date-parts":[[2025,9,30]],"date-time":"2025-09-30T18:05:44Z","timestamp":1759255544000},"page":"2768-2778","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Serial amplification of tau filaments using Alzheimer's brain homogenates and <scp>C322A<\/scp> or <scp>C322S<\/scp> recombinant tau"],"prefix":"10.1002","volume":"599","author":[{"given":"Alessia","family":"Santambrogio","sequence":"first","affiliation":[{"name":"Centre for Misfolding Diseases, Yusuf Hamied Department of Chemistry, University of Cambridge  UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2152-1476","authenticated-orcid":false,"given":"Sofia","family":"L\u00f6vestam","sequence":"additional","affiliation":[{"name":"MRC Laboratory of Molecular Biology  Cambridge UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9998-4630","authenticated-orcid":false,"suffix":"II","given":"Michael A.","family":"Metrick","sequence":"additional","affiliation":[{"name":"Centre for Misfolding Diseases, Yusuf Hamied Department of Chemistry, University of Cambridge  UK"},{"name":"Laboratory of Neurological Infections and Immunity, Rocky Mountain Laboratories, Division of Intramural Research, National Institute for Allergy and Infectious Diseases  Hamilton MT USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2969-810X","authenticated-orcid":false,"given":"Thomas","family":"L\u00f6hr","sequence":"additional","affiliation":[{"name":"Centre for Misfolding Diseases, Yusuf Hamied Department of Chemistry, University of Cambridge  UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peifeng","family":"Xu","sequence":"additional","affiliation":[{"name":"Centre for Misfolding Diseases, Yusuf Hamied Department of Chemistry, University of Cambridge  UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas C. 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