{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T20:48:19Z","timestamp":1768510099158,"version":"3.49.0"},"posted":{"date-parts":[[2024,1,1]]},"group-title":"Cell Biology","reference-count":103,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2024,1,1]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>The endocytic pathway is both an essential route of molecular uptake in cells and a potential entry point for pathology-inducing cargo. The cell-to-cell spread of cytotoxic aggregates, such as those of \u03b1-synuclein (\u03b1-syn) in Parkinson\u2019s Disease (PD), exemplifies this duality. Here we used a human iPSC-derived induced neuronal model (iNs) prone to death mediated by aggregation in late endosomes and lysosomes of endogenous \u03b1-syn, seeded by internalized pre-formed fibrils of \u03b1-syn (PFFs). This PFF-mediated death was not observed with parental iPSCs or other non-neuronal cells. Using live-cell optical microscopy to visualize the read out of biosensors reporting endo-lysosome wounding, we discovered that up to about 10% of late endosomes and lysosomes in iNs exhibited spontaneous constitutive perforations, regardless of the presence of internalized PFFs. This wounding, absent in parental iPSCs and non-neuronal cells, corresponded to partial damage by nanopores in the limiting membranes of a subset of endolysosomes directly observed by volumetric focused ion beam scanning electron microscopy (FIB-SEM) in iNs and in CA1 pyramidal neurons from mouse brain, and not found in iPSCs or in other non-neuronal cells in culture or in mouse liver and skin.<\/jats:p>\n                <jats:p>We suggest that the compromised limiting membranes in iNs and neurons in general are the primary conduit for cytosolic \u03b1-syn to access PFFs entrapped within endo-lysosomal lumens, initiating PFF-mediated \u03b1-syn aggregation. Significantly, eradicating the intrinsic endolysosomal perforations in iNs by inhibiting the endosomal Phosphatidylinositol-3-Phosphate\/Phosphatidylinositol 5-Kinase (PIKfyve kinase) using Apilimod or Vacuolin-1 markedly reduced PFF-induced \u03b1-syn aggregation, despite PFFs continuing to enter the endolysosomal compartment. Crucially, this intervention also diminished iN death associated with PFF incubation.<\/jats:p>\n                <jats:p>Our results reveal the surprising presence of intrinsically perforated endo-lysosomes in neurons, underscoring their crucial early involvement in the genesis of toxic \u03b1-syn aggregates induced by internalized PFFs. This discovery offers a basis for employing PIKfyve kinase inhibition as a potential therapeutic strategy to counteract synucleinopathies.<\/jats:p>","DOI":"10.1101\/2023.12.30.573738","type":"posted-content","created":{"date-parts":[[2024,1,1]],"date-time":"2024-01-01T23:15:17Z","timestamp":1704150917000},"source":"Crossref","is-referenced-by-count":3,"title":["Constitutive Endolysosomal Perforation in Neurons allows Induction of \u03b1-Synuclein Aggregation by Internalized Pre-Formed Fibrils"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1456-8604","authenticated-orcid":false,"given":"Anwesha","family":"Sanyal","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4001-3389","authenticated-orcid":false,"given":"Gustavo","family":"Scanavachi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7283-7182","authenticated-orcid":false,"given":"Elliott","family":"Somerville","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9225-5558","authenticated-orcid":false,"given":"Anand","family":"Saminathan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1754-6440","authenticated-orcid":false,"given":"Athul","family":"Nair","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Athanasios","family":"Oikonomou","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4202-0328","authenticated-orcid":false,"given":"Nikos S.","family":"Hatzakis","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0559-893X","authenticated-orcid":false,"given":"Tom","family":"Kirchhausen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2024010921165272000_2023.12.30.573738v1.1","doi-asserted-by":"publisher","DOI":"10.15252\/embj.201593411"},{"key":"2024010921165272000_2023.12.30.573738v1.2","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.e16-03-0164"},{"key":"2024010921165272000_2023.12.30.573738v1.3","doi-asserted-by":"publisher","DOI":"10.1080\/15548627.2015.1063871"},{"key":"2024010921165272000_2023.12.30.573738v1.4","doi-asserted-by":"publisher","DOI":"10.3389\/fcomp.2022.777728"},{"key":"2024010921165272000_2023.12.30.573738v1.5","first-page":"879","article-title":"Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson\u2019s disease and dementia with Lewy bodies","volume":"152","year":"1998","journal-title":"Am. 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