From injury to degeneration: delayed lipid-mediated neurotoxicity in a human astrocyte traumatic brain injury in-a-dish model

Abstract Traumatic brain injury (TBI) is a major cause of long-term neurological disability, and repeated mild TBI (rmTBI) is increasingly linked to chronic neurodegeneration. Clinical evidence suggests that rmTBI creates a prolonged window of vulnerability to further injury. However, the astrocyte-intrinsic mechanisms that convert repeated mechanical stress into delayed neuronal injury remain poorly defined. In particular, whether rmTBI induces a distinct lipid-mediated neurotoxic programme in human astrocytes is unknown. Using a human astrocyte in vitro platform of rmTBI, we deliver controlled repeated mild injuries to astrocytes. Repeated injury induces a distinctive astrocytic phenotype characterised by cytoskeletal remodelling, reactive oxygen species accumulation, biphasic volume changes, extracellular matrix remodelling and lipid droplet formation. Shotgun lipidomics of astrocyte-conditioned medium reveals selective enrichment of bioactive diacylglycerols (DG) following rmTBI. When applied to human cortical neurons, this conditioned medium impairs neuronal function and morphology, increases apoptosis and activates protein kinase C delta (PKCδ). Pharmacological inhibition of PKC attenuates the neurotoxic effect of rmTBI astrocyte-conditioned medium. These findings support a model in which human astrocytes retain a form of ‘injury memory’ through sustained phenotypic and lipid metabolic remodelling. By implicating the DG-PKCδ axis in delayed neurotoxicity, the study identifies PKC signalling as a tractable target for limiting the long-term consequences of rmTBI.