PrPC-Mediated Ca²⁺/Calcineurin/TFEB Signaling Enhances Autophagic-Lysosomal Function and Anti-inflammatory Astrocyte Transition to Alleviate Cerebral Ischemia-Reperfusion Injury

An increasing number of studies have focused on understanding the role of macroautophagy/autophagy and the autophagy-lysosomal pathway (ALP) in cerebral ischemic injury. Transcription factor EB (TFEB) is a central regulator of genes involved in autophagy and plays a pivotal role in the regulation of the ALP; however, the mechanisms controlling TFEB activity remain incompletely understood. In this study, we investigated the role of cellular prion protein (PrPC)-targeted TFEB in mediating ALP dysfunction and inflammatory phenotypic changes in mouse cortical astrocytes after cerebral ischemia-reperfusion injury (CIRI). Our findings indicate that during the ultra-early phase of CIRI, intracellular Ca2+ levels are low, with inhibited PPP3/calcineurin activity and reactivation of mTOR, leading to TFEB phosphorylation and retention in the cytoplasm. As reperfusion time increases, elevated intracellular Ca2+ levels activate PPP3/calcineurin, resulting in TFEB dephosphorylation, nuclear translocation, and the subsequent induction of autophagy lysosome-associated gene transcription. This process promotes astrocyte survival and shifts the cellular phenotype toward an anti-inflammatory state. Furthermore, increased PrPC expression was observed to maintain intracellular Ca2+ homeostasis and sustain PPP3/calcineurin activation, facilitating continuous TFEB dephosphorylation and nuclear translocation. These results clarify the regulatory role of PrPC in astrocytic autophagy-lysosomal pathways following cerebral ischemia/reperfusion injury, providing new insights for more targeted interventions in ischemic stroke.