ER-localized ceramide accumulation contributes to replicative senescence.

Ceramides regulate diverse cellular processes through compartment-specific accumulation. While mitochondrial ceramide accumulation promotes apoptosis, its regulation and function during senescence remain incompletely understood. Here, we integrate lipidomics, transcriptomics, Raman spectroscopy, and biochemical characterizations to define sphingolipid remodeling in replicative senescence. Senescent cells exhibit elevated ceramide levels and depletion of very-long-chain sphingomyelins, despite unaltered sphingomyelin synthase 1 expression, implicating impaired ceramide-sphingomyelin turnover. Pharmacological inhibition of ceramide transfer protein (CERT), the ER-to-Golgi ceramide transporter, phenocopies sphingolipid remodeling and enhances senescence, suggesting disrupted ceramide trafficking as a driver of senescence. Raman spectroscopy suggests ceramide accumulation localized to the ER. In parallel, analysis of ER-enriched fractions confirms increased ceramide levels in ER fractions of senescent cells. Mechanistically, ceramide accumulation at the ER can contribute to ER stress. These findings identify altered ceramide trafficking as a contributor to ER stress and highlight ER-localized ceramide as a critical component of senescence-associated sphingolipid remodeling.