{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:40:35Z","timestamp":1785404435249,"version":"3.56.0"},"posted":{"date-parts":[[2025,8,6]]},"group-title":"Neuroscience","reference-count":42,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,8,6]],"date-time":"2025-08-06T00:00:00Z","timestamp":1754438400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["2021FIB-41"],"award-info":[{"award-number":["2021FIB-41"]}],"id":[{"id":"https:\/\/ror.org\/05avmtm72","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R35GM151999"],"award-info":[{"award-number":["R35GM151999"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000065","name":"National Institute of Neurological Disorders and Stroke","doi-asserted-by":"crossref","award":["F31NS132517"],"award-info":[{"award-number":["F31NS132517"]}],"id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000065","name":"National Institute of Neurological Disorders and Stroke","doi-asserted-by":"crossref","award":["NS062829"],"award-info":[{"award-number":["NS062829"]}],"id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000065","name":"National Institute of Neurological Disorders and Stroke","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000065","name":"National Institute of Neurological Disorders and Stroke","doi-asserted-by":"crossref","award":["R01NS101686"],"award-info":[{"award-number":["R01NS101686"]}],"id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,9,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Calcium-phosphate (CaP) is a ubiquitous inorganic compound that plays an important structural role in healthy bone and teeth formation, but its pathologic buildup can occur in dyshomeostatic calcium disorders like Alzheimer\u2019s disease and Leigh syndrome. The nexus of pathologic extracellular CaP in the nervous system is not well understood, but prior evidence suggests mitochondria could be a source. We have observed mitochondria-sized sheet-like CaP aggregates within functional wild type cortical neuron cultures at 1 and 20 DIV. Neurons were extracted from embryonic day 18 (E18) rat embryos following standard protocols to study neuronal structure and function. We have used a combination of cryo-ET, cryo-CLEM, and LDSAED to demonstrate that these aggregates are octacalcium phosphate-like, are associated with mitochondria, and that at least a portion are extruded via migrasomes. Visually similar aggregates were previously observed in Huntington\u2019s disease model neurons, but in that study they were not observed in WT controls. These findings show that this CaP aggregation process occurs routinely in WT neurons and may reveal an important link for how mitochondria may participate in calcification, highlighting them as potential therapeutic targets in neurological disorders characterized by pathological calcification, such as Alzheimer\u2019s disease.<\/jats:p>","DOI":"10.1101\/2025.08.04.668590","type":"posted-content","created":{"date-parts":[[2025,8,6]],"date-time":"2025-08-06T20:40:15Z","timestamp":1754512815000},"source":"Crossref","is-referenced-by-count":0,"title":["Primary cortical neurons precipitate and extrude large mitochondria-associated calcium-phosphate sheets with a bone-precursor-like ultrastructure"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5329-4629","authenticated-orcid":false,"given":"Erik D.","family":"Anderson","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3276-9338","authenticated-orcid":false,"given":"Christopher A.","family":"Cronkite","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4202-4129","authenticated-orcid":false,"given":"Philip R.","family":"Baldwin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-4362-4339","authenticated-orcid":false,"given":"Carlota P.","family":"Abella","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6711-0656","authenticated-orcid":false,"given":"Joseph G.","family":"Duman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-0169-0640","authenticated-orcid":false,"given":"Ashleigh N.","family":"Simmonds","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4801-1190","authenticated-orcid":false,"given":"M. Neal","family":"Waxham","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2092-920X","authenticated-orcid":false,"given":"Kimberley F.","family":"Tolias","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1903-1574","authenticated-orcid":false,"given":"Steven J.","family":"Ludtke","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025090914550958000_2025.08.04.668590v2.1","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1016\/j.actbio.2022.03.003","article-title":"Nuclear and cellular, micro and nano calcification in Alzheimer\u2019s disease patients and correlation to phosphorylated Tau","volume":"143","year":"2022","journal-title":"Acta Biomater"},{"key":"2025090914550958000_2025.08.04.668590v2.2","doi-asserted-by":"crossref","unstructured":"Monfrini E , Arienti F , Rinchetti P , Lotti F , Riboldi GM . Brain Calcifications: Genetic, Molecular, and Clinical Aspects. Int J Mol Sci. NLM (Medline); 2023.","DOI":"10.3390\/ijms24108995"},{"key":"2025090914550958000_2025.08.04.668590v2.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.jns.2005.11.033"},{"key":"2025090914550958000_2025.08.04.668590v2.4","unstructured":"Termine JD , Posner AS . Calcium Phosphate Formation in vitro 1. Factors Affecting Initial Phase Separation\u2019. Arch Biochem Biophys. 1970."},{"key":"2025090914550958000_2025.08.04.668590v2.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2011.03.014"},{"key":"2025090914550958000_2025.08.04.668590v2.6","doi-asserted-by":"crossref","unstructured":"Mahamid J , Sharir A , Addadi L , Weiner S. Amorphous calcium phosphate is a major component of the forming fin bones of zebrafish: Indications for an amorphous precursor phase [Internet]. 2008. Available from: www.pnas.org\/cgi\/content\/full\/","DOI":"10.1073\/pnas.0803354105"},{"key":"2025090914550958000_2025.08.04.668590v2.7","doi-asserted-by":"publisher","DOI":"10.1016\/j.bone.2006.02.059"},{"key":"2025090914550958000_2025.08.04.668590v2.8","doi-asserted-by":"crossref","unstructured":"Hasegawa T , Yamamoto T , Tsuchiya E , Hongo H , Tsuboi K , Kudo A , et al. Ultrastructural and biochemical aspects of matrix vesicle-mediated mineralization. Japanese Dental Science Review. Elsevier Ltd; 2017. p. 34\u201345.","DOI":"10.1016\/j.jdsr.2016.09.002"},{"key":"2025090914550958000_2025.08.04.668590v2.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.jprot.2011.04.005"},{"key":"2025090914550958000_2025.08.04.668590v2.10","doi-asserted-by":"publisher","DOI":"10.1016\/s0021-9258(19)39619-x"},{"key":"2025090914550958000_2025.08.04.668590v2.11","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2019.05.038"},{"key":"2025090914550958000_2025.08.04.668590v2.12","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.23.1.21"},{"key":"2025090914550958000_2025.08.04.668590v2.13","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1208916109"},{"key":"2025090914550958000_2025.08.04.668590v2.14","doi-asserted-by":"crossref","unstructured":"Pei D dan , Sun J long , Zhu C hui , Tian F cong , Jiao K , Anderson MR , et al. Contribution of Mitophagy to Cell-Mediated Mineralization: Revisiting a 50-Year-Old Conundrum. Advanced Science. 2018;5.","DOI":"10.1002\/advs.201800873"},{"key":"2025090914550958000_2025.08.04.668590v2.15","doi-asserted-by":"crossref","unstructured":"Wuthier RE , Rice GS , Wallace JEB , Weaver RL , Legeros RZ , David Eanes E . Calcified Tissue International Precipitation of Calcium Phosphate Under IntraceUular Conditions: Formation of Brushite from an Amorphous Precursor in the Absence of ATP. Calcif Tissue Int. 1985.","DOI":"10.1007\/BF02553710"},{"key":"2025090914550958000_2025.08.04.668590v2.16","doi-asserted-by":"crossref","unstructured":"Duvvuri B , Lood C. Mitochondrial Calcification. Immunometabolism (United States). Lippincott Williams and Wilkins; 2021.","DOI":"10.20900\/immunometab20210008"},{"key":"2025090914550958000_2025.08.04.668590v2.17","first-page":"339","article-title":"Mitochondrial matrix granules in soft tissues","volume":"124","year":"1979","journal-title":"I. Elemental composition by x-ray microanalysis. Exp Cell Res"},{"key":"2025090914550958000_2025.08.04.668590v2.18","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2990.2008.00970.x"},{"key":"2025090914550958000_2025.08.04.668590v2.19","doi-asserted-by":"crossref","unstructured":"Jennings RB . Historical perspective on the pathology of myocardial ischemia\/reperfusion injury. Circ Res. 2013. p. 428\u201338.","DOI":"10.1161\/CIRCRESAHA.113.300987"},{"key":"2025090914550958000_2025.08.04.668590v2.20","doi-asserted-by":"crossref","unstructured":"Halestrap AP , Clarke SJ , Javadov SA . Mitochondrial permeability transition pore opening during myocardial reperfusion - A target for cardioprotection. Cardiovasc Res. 2004. p. 372\u201385.","DOI":"10.1016\/S0008-6363(03)00533-9"},{"key":"2025090914550958000_2025.08.04.668590v2.21","doi-asserted-by":"crossref","unstructured":"Rottenberg H , Hoek JB . The mitochondrial permeability transition: Nexus of aging, disease and longevity. Cells. MDPI; 2021. p. 1\u201323.","DOI":"10.3390\/cells10010079"},{"key":"2025090914550958000_2025.08.04.668590v2.22","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2021.04.027"},{"key":"2025090914550958000_2025.08.04.668590v2.23","doi-asserted-by":"publisher","DOI":"10.1038\/cr.2014.135"},{"key":"2025090914550958000_2025.08.04.668590v2.24","doi-asserted-by":"publisher","DOI":"10.1111\/febs.16756"},{"key":"2025090914550958000_2025.08.04.668590v2.25","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2005.01.024"},{"key":"2025090914550958000_2025.08.04.668590v2.26","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.3978-12.2013"},{"key":"2025090914550958000_2025.08.04.668590v2.27","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.0531-04.2004"},{"key":"2025090914550958000_2025.08.04.668590v2.28","doi-asserted-by":"crossref","unstructured":"Lemieux H , Blier PU , Gnaiger E . Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: Electron flow through the Q-junction in permeabilized fibers. Sci Rep. 2017;7.","DOI":"10.1038\/s41598-017-02789-8"},{"key":"2025090914550958000_2025.08.04.668590v2.29","doi-asserted-by":"crossref","unstructured":"Wu GH , Smith-Geater C , Galaz-Montoya JG , Gu Y , Gupte SR , Aviner R , et al. CryoET reveals organelle phenotypes in huntington disease patient iPSC-derived and mouse primary neurons. Nat Commun. 2023;14.","DOI":"10.1038\/s41467-023-36096-w"},{"key":"2025090914550958000_2025.08.04.668590v2.30","doi-asserted-by":"crossref","unstructured":"Fischer TD , Dash PK , Liu J , Waxham MN . Morphology of mitochondria in spatially restricted axons revealed by cryo-electron tomography. PLoS Biol. 2018;16.","DOI":"10.1101\/273052"},{"key":"2025090914550958000_2025.08.04.668590v2.31","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.1c03075"},{"key":"2025090914550958000_2025.08.04.668590v2.32","doi-asserted-by":"crossref","unstructured":"Habraken WJEM , Tao J , Brylka LJ , Friedrich H , Bertinetti L , Schenk AS , et al. Ion-association complexes unite classical and non-classical theories for the biomimetic nucleation of calcium phosphate. Nat Commun. 2013;4.","DOI":"10.1038\/ncomms2490"},{"key":"2025090914550958000_2025.08.04.668590v2.33","doi-asserted-by":"publisher","DOI":"10.1038\/1881144a0"},{"key":"2025090914550958000_2025.08.04.668590v2.34","doi-asserted-by":"crossref","unstructured":"Uoselis L , Nguyen TN , Lazarou M. Mitochondrial degradation: Mitophagy and beyond. Mol Cell. Cell Press; 2023. p. 3404\u201320.","DOI":"10.1016\/j.molcel.2023.08.021"},{"key":"2025090914550958000_2025.08.04.668590v2.35","doi-asserted-by":"crossref","unstructured":"Zhao X , Lei Y , Zheng J , Peng J , Li Y , Yu L , et al. Identification of markers for migrasome detection. Cell Discov. Nature Publishing Groups; 2019.","DOI":"10.1038\/s41421-019-0093-y"},{"key":"2025090914550958000_2025.08.04.668590v2.36","doi-asserted-by":"crossref","unstructured":"Jasielec JJ , Filipek R , Do\u0142owy K , Lewenstam A . Precipitation of inorganic salts in mitochondrial matrix. Membranes (Basel). 2020;10.","DOI":"10.3390\/membranes10050081"},{"key":"2025090914550958000_2025.08.04.668590v2.37","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1016\/j.actbio.2022.03.003","article-title":"Nuclear and cellular, micro and nano calcification in Alzheimer\u2019s disease patients and correlation to phosphorylated Tau","volume":"143","year":"2022","journal-title":"Acta Biomater"},{"key":"2025090914550958000_2025.08.04.668590v2.38","doi-asserted-by":"publisher","DOI":"10.1111\/neup.12742"},{"key":"2025090914550958000_2025.08.04.668590v2.39","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-019-0591-8"},{"key":"2025090914550958000_2025.08.04.668590v2.40","doi-asserted-by":"publisher","DOI":"10.1002\/PRO.3943"},{"key":"2025090914550958000_2025.08.04.668590v2.41","doi-asserted-by":"crossref","first-page":"1514","DOI":"10.1017\/S1431927622006109","article-title":"Object Segmentation on Cryo-electron Tomography Data","volume":"28","year":"2022","journal-title":"Microscopy and Microanalysis"},{"key":"2025090914550958000_2025.08.04.668590v2.42","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2006.05.009"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.08.04.668590","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:03:25Z","timestamp":1782277405000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.08.04.668590"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,6]]},"references-count":42,"URL":"https:\/\/doi.org\/10.1101\/2025.08.04.668590","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1186\/s13041-025-01272-0","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2025,8,6]]},"subtype":"preprint"}}