Ceramide-rich extracellular vesicles as pathogenic biomarkers in traumatic brain injury

Abstract Extracellular vesicles (EVs) contribute to the damage caused by traumatic brain injury (TBI) and can cross the blood-brain barrier (BBB). We analyzed plasma-derived EVs from human TBI patients to identify factors potentially contributing to TBI pathology. EVs were isolated using membrane affinity (ExoEasy) and size exclusion chromatography (iZone), both yielding CD9(+) and CD63(+) EVs with minimal contamination by serum albumin and apolipoprotein. Immunoblotting detected GFAP in TBI but not control EVs, indicating astrocyte-derived EVs crossing the BBB. Proteomic analysis and immunoblotting of EVs from TBI samples identified C-reactive protein and 14-3-3 proteins, which were not detected in control EVs, indicating inflammation associated with TBI. Lipidomic analysis showed ceramide enrichment in TBI EVs, validated by anti-ceramide immunoprecipitation. In a mouse closed head-controlled cortical impact model, brain EVs similarly showed elevated ceramide, confirming ceramide-rich EV release after TBI. Immunocytochemistry localized acid sphingomyelinase (ASM), a ceramide-generating enzyme, to ependymal cilia, suggesting these sites as a potential source of EVs. This was further supported by the detection of ASM in both brain- and plasma-derived EVs, along with the ciliary marker Arl13b in the brain. To assess function, we treated murine neuronal (N2a) cells with TBI EVs. Transcriptomics and STRING analyses revealed enrichment of mitochondrial-associated transcripts. Immunoblotting showed increased p53 and voltage-dependent anion channel 1 (VDAC1), which mediate ceramide-induced apoptosis. Seahorse assays showed that TBI EVs suppressed glycolysis, as indicated by reduced ECAR, while mitochondrial respiration (OCR) remained unchanged. LDH assays further indicated that TBI EVs were more neurotoxic than control EVs. Together, these findings identify ceramide-rich EVs as plasma biomarkers of TBI-induced inflammation, potential mediators of neuronal mitochondrial dysfunction, and pharmacological targets to prevent TBI-induced damage. Competing Interest Statement The authors have declared no competing interest. List of Abbreviations - ASM - acid sphingomyelinase - ApoA1 - Apolipoprotein A1 - BBB - blood–brain barrier - CNS - central nervous system - CRP - C-reactive protein - CHI - closed head injury - DEGs - differentially expressed genes - ECAR - extracellular acidification rate - EVs - extracellular vesicles - GFAP - Glial Fibrillary Acidic Protein - GCS - Glasgow Coma Scale - HK1 - hexokinase 1 - FcR - human Fc receptor - HSA - Human Serum Albumin - IP - immunoprecipitation - IACUC - Institutional Animal Care and Use Committee - LDH - lactate dehydrogenase - LC-MS/MS - liquid chromatography–tandem mass spectrometry - NTA - nanoparticle tracking analysis - OCR - oxygen consumption rate - SEC - size exclusion chromatography - Sph-1P - sphingosine-1-phosphate - TBS-T - Tris-buffered saline - VDAC1 - voltage-dependent anion channel 1