Abstract WP046: Astrocyte-Derived Migrasomes as Novel Mediators of Homeostatic Regulation Under Ischemic Stress

Background: Astrocytes are key regulators of central nervous system homeostasis, but how they adapt to ischemic injury is unclear. Migrasomes (Migs）are large (0.5–3 μm) vesicular organelles budding from retraction fibers of migrating cells. They act as “signaling and disposal stations,” packaging cellular contents—including damaged mitochondria—for release, mediating intercellular communication and tissue remodeling. Dysregulated Mig biogenesis is linked to oxidative stress, metabolic imbalance, and neurodegeneration. However, their role in astrocyte adaptation to ischemia–reperfusion remains unknown. Methods: Primary astrocytes were subjected to OGD/R to model ischemic stress. Migs were characterized by SEM/TEM, TSPAN4-GFP labeling, and confocal microscopy with WGA and mitochondrial probes. Mig production was quantified by nanoparticle tracking analysis (NTA). Bleomycin was used to inhibit Mig formation, and effects on astrocyte viability, ROS, and superoxide (SOX) levels were measured. Proteomic profiling at 3, 6, 15, and 24 h of reoxygenation was performed, with subcellular localization and KEGG pathway analyses. Results: OGD/R markedly increased Migs number and size, with frequent mitochondrial inclusion. Actin stress fibers were disrupted in TSPAN4-GFP–labeled astrocytes post-OGD/R. Bleomycin-mediated inhibition of Mig reduced astrocyte viability and elevated ROS/SOX, implicating Migs in oxidative stress control. NTA confirmed significant production increases post-OGD/R. Proteomics identified dynamic shifts in protein expression, with enrichment in cytosolic, mitochondrial, and plasma membrane proteins. Early reoxygenation (3 h) upregulated carbohydrate metabolism pathways; prolonged reoxygenation (6–15 h) caused progressive downregulation of oxidative phosphorylation, synaptic vesicle cycle, mTOR signaling, and multiple neurodegeneration-related pathways. Mitochondrial-related protein diversity increased over time, suggesting enhanced mitochondrial extrusion and metabolic adaptation. Conclusion: This is the first evidence that ischemic stress stimulates astrocyte Mig biogenesis and reshapes their functional proteome. Migs appear to safeguard astrocyte homeostasis by facilitating mitochondrial clearance, mitigating oxidative injury, and modulating energy metabolism. Understanding Mig biology opens new avenues for targeting astrocyte-mediated neuroprotection in stroke and other neurological disorders.