ER-Localized Ceramide Accumulation Due to Disrupted CERT Transport Contributes to Replicative Senescence

Ceramides are bioactive sphingolipids that are central to cellular metabolism, signaling, and stress responses. Synthesized de novo in the endoplasmic reticulum (ER) through a tightly regulated enzymatic pathway, ceramides can also be generated from hydrolysis complex sphingolipids, including hexosylceramides and sphingomyelins, and the salvage pathway. Their dynamic turnover across distinct cellular compartments results in spatial regulation of ceramide levels and its functional consequences. Mitochondrial ceramide accumulation, in particular, is a critical factor of apoptosis, where ceramides can disrupt membrane integrity. In addition to apoptosis, ceramides are implicated in replicative senescence. However, the mechanisms of ceramide regulation and function in senescence remain incompletely understood. In this study, we employed comparative lipid profiling, gene expression analysis and Raman spectroscopy to dissect sphingolipid regulatory networks that might be responsible for ceramide regulation and localization in senescence. We found that while both apoptotic and senescent cells exhibit elevated ceramide levels, only senescent cells display a marked depletion of sphingomyelins, especially very-long-chain species. Despite transcriptional upregulation of sphingomyelin synthase 1, enzymatic activity remained unchanged, suggesting impaired ceramide transport. Pharmacological inhibition of Ceramide Transfer Protein (CERT), the ER-to-Golgi ceramide transporter, phenocopied the senescent sphingolipid profile and enhanced senescence markers. Furthermore, Raman spectroscopy revealed ceramide accumulation at the ER—but not the mitochondria—in senescent cells, consistent with a non-apoptotic phenotype. Together, our results identify disrupted ceramide turnover to sphingomyelin as a key feature of senescence and highlight the importance of subcellular ceramide localization in determining cell fate.