Very long chain fatty acids drive 1-deoxy-Sphingolipid toxicity

1-deoxy-sphingolipids (1-deoxySLs) are atypical sphingolipids synthesized by the serine palmitoyltransferase (SPT) when L-alanine is used instead of its canonical substrate L-serine. Increased 1-deoxySLs are associated with sensory neuropathies such as Hereditary Sensory and Autonomic Neuropathy type 1 (HSAN1) and diabetic polyneuropathy (DPN). Despite their known cellular, mitochondrial, and neurotoxic effects, the mechanisms underlying their toxicity remain poorly understood. Using a CRISPR interference (CRISPRi) screening approach, we identified CERS2, ELOVL1, ACACA, HSD17B12, and PTPLB as key mediators of 1-deoxySL-induced toxicity. All genes are integral to the biosynthesis of very long-chain (VLC) fatty acids and VLC-ceramides. We validated these findings through genetic knockdown experiments, cytotoxicity assays, and stable isotope-resolved lipidomics via LC-MS/MS. Pharmacological inhibition of ELOVL1 using a preclinical tested compound alleviated the cellular, mitochondrial, and neuronal toxicity induced by 1-deoxySLs. Supplementation experiments combining 1-deoxySLs with various VLC fatty acids revealed that 1-deoxyDHceramide conjugated to nervonic acid (m18:0/24:1) is the principal toxic specie. Further mechanistic studies showed that m18:0/24:1 induces apoptosis through the mitochondrial permeability transition pore (mPTP) formation. Inhibition of BAX or blocking mPTP formation with cyclosporin A effectively prevented toxicity. In conclusion, our findings demonstrate that 1-deoxyDHCeramides conjugated to nervonic acid are the primary mediators of 1-deoxySL toxicity, acting through mitochondrial dysfunction and BAX-dependent apoptosis.