{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T22:15:47Z","timestamp":1791324947676,"version":"4.3.1"},"posted":{"date-parts":[[2025,12,8]]},"group-title":"Biochemistry","reference-count":37,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T00:00:00Z","timestamp":1765152000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"name":"Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,12,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Sphingolipids play key roles in cellular systems both as membrane components and as signaling molecules. Their biosynthesis, which occurs in the endoplasmic reticulum (ER), begins with the condensation of an amino acid, typically serine, and a fatty acyl-CoA. Under certain pathological conditions, alanine can be substituted for serine in the condensation reaction, producing 1-deoxysphingolipids, which lack the 1-hydroxyl group on the sphingoid base. Unlike typical sphingolipids, 1-deoxysphingolipids are unable to accept a head group modification, which alters their metabolic processing and prevents their canonical degradation. The accumulation of these \u201cheadless\u201d 1-deoxysphingolipids causes neurotoxicity in various neurological and metabolic disorders. Here, we conducted a genome-wide CRISPR-Cas9 screen to identify pathways leading to 1-deoxysphinganine\u2013induced toxicity in SH-SY5Y cells, a model used to study neurotoxic responses. Our top genetic hits highlighted the pathway involved in synthesizing ceramides with very-long-chain fatty acids (C22\u2013C26). Using CRISPR-Cas9\u2013modified SH-SY5Y cells with loss-of-function (LOF) mutations in the\n                  <jats:italic>TECR<\/jats:italic>\n                  or\n                  <jats:italic>CERS2<\/jats:italic>\n                  genes\u2014both critical for producing very-long-chain ceramides\u2014we validated that this pathway was essential for 1-deoxysphinganine\u2013mediated toxicity. Furthermore, we demonstrated that the ceramide synthesis pathway is required for 1-deoxysphinganine to trigger ER stress, as evidenced by significantly increased expression of the unfolded protein response in WT, but not\n                  <jats:italic>TECR<\/jats:italic>\n                  or\n                  <jats:italic>CERS2<\/jats:italic>\n                  LOF mutant, SH-SY5Y cells exposed to 1-deoxysphinganine. Collectively, these findings identify a specific metabolic pathway for 1-deoxysphinganine leading to very-long-chain 1-deoxyceramide production that culminates in ER stress and toxicity. The findings highlight potential therapeutic targets for neuropathological diseases caused by 1-deoxysphingolipid accumulation.\n                <\/jats:p>","DOI":"10.64898\/2025.12.06.692689","type":"posted-content","created":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T21:15:21Z","timestamp":1765228521000},"source":"Crossref","is-referenced-by-count":1,"title":["1-Deoxysphingolipids Require Very-Long-Chain Ceramide Synthesis to Induce ER Stress and Neurotoxicity"],"prefix":"10.64898","author":[{"given":"Colleen","family":"Byrnes","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin A.","family":"Clarke","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0612-009X","authenticated-orcid":false,"given":"Hongling","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5067-1190","authenticated-orcid":false,"given":"Saurav","family":"Majumder","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2447-4350","authenticated-orcid":false,"given":"Mari","family":"Kono","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0456-1270","authenticated-orcid":false,"given":"Richard L.","family":"Proia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025121005200477000_2025.12.06.692689v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/nrm.2017.107"},{"key":"2025121005200477000_2025.12.06.692689v1.2","doi-asserted-by":"publisher","DOI":"10.1021\/cr2002917"},{"key":"2025121005200477000_2025.12.06.692689v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmet.2021.06.006"},{"key":"2025121005200477000_2025.12.06.692689v1.4","first-page":"512","article-title":"1-Deoxysphingolipids","volume":"1864","year":"2019","journal-title":"Biochim Biophys Acta Mol Cell Biol Lipids"},{"key":"2025121005200477000_2025.12.06.692689v1.5","doi-asserted-by":"crossref","first-page":"735","DOI":"10.3233\/JND-240014","article-title":"Serine Palmitoyltransferase (SPT)-related Neurodegenerative and Neurodevelopmental Disorders","volume":"11","year":"2024","journal-title":"J Neuromuscul Dis"},{"key":"2025121005200477000_2025.12.06.692689v1.6","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M109.092973"},{"key":"2025121005200477000_2025.12.06.692689v1.7","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M110.122259"},{"key":"2025121005200477000_2025.12.06.692689v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-39775-w"},{"key":"2025121005200477000_2025.12.06.692689v1.9","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1815111"},{"key":"2025121005200477000_2025.12.06.692689v1.10","doi-asserted-by":"publisher","DOI":"10.2337\/db14-1325"},{"key":"2025121005200477000_2025.12.06.692689v1.11","doi-asserted-by":"publisher","DOI":"10.1096\/fj.15-272567"},{"key":"2025121005200477000_2025.12.06.692689v1.12","doi-asserted-by":"crossref","first-page":"662","DOI":"10.1111\/jnc.14985","article-title":"Role of 1-Deoxysphingolipids in docetaxel neurotoxicity","volume":"154","year":"2020","journal-title":"J Neurochem"},{"key":"2025121005200477000_2025.12.06.692689v1.13","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbior.2018.10.004"},{"key":"2025121005200477000_2025.12.06.692689v1.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuro.2022.07.008"},{"key":"2025121005200477000_2025.12.06.692689v1.15","doi-asserted-by":"publisher","DOI":"10.1126\/science.1247005"},{"key":"2025121005200477000_2025.12.06.692689v1.16","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-014-0554-4"},{"key":"2025121005200477000_2025.12.06.692689v1.17","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2007.08.008"},{"key":"2025121005200477000_2025.12.06.692689v1.18","doi-asserted-by":"publisher","DOI":"10.1093\/jb\/mvs105"},{"key":"2025121005200477000_2025.12.06.692689v1.19","doi-asserted-by":"crossref","first-page":"e0265539","DOI":"10.1371\/journal.pone.0265539","article-title":"Holocarboxylase synthetase knockout is embryonic lethal in mice","volume":"17","year":"2022","journal-title":"PLoS One"},{"key":"2025121005200477000_2025.12.06.692689v1.20","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M707386200"},{"key":"2025121005200477000_2025.12.06.692689v1.21","doi-asserted-by":"publisher","DOI":"10.1089\/dna.2013.2182"},{"key":"2025121005200477000_2025.12.06.692689v1.22","doi-asserted-by":"crossref","unstructured":"Esaki, K. , Sayano, T. , Sonoda, C. , Akagi, T. , Suzuki, T. , Ogawa, T. , et al. 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