Microglia-derived iron-overloaded exosomes induce neuronal ferroptosis and aggravate neurological impairment after subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a devastating stroke subtype often leading to poor neurological outcomes. Iron homeostasis imbalance is a key contributor to cognitive dysfunction in neurological diseases. Extracellular vesicles, including exosomes (EXs), are crucial mediators of intercellular communication. This study investigated the role of EXs in post-SAH iron metabolism and neurological impairment. We isolated EXs from various neural cells (microglia, astrocytes, endothelial cells, neurons; n = 4-6 independent isolations) in vitro after SAH mimicked by oxyhemoglobin (OxyHb) or hemin. We found that microglial EXs (MC-EXs) were significantly enriched in iron and potently impaired neuronal viability. Using specific inhibitors and fluorescence imaging, we demonstrated that neurons internalize MC-EXs primarily via dynamin-dependent, clathrin-, caveolae-, and lipid raft-mediated endocytosis. Combining transcriptomic analysis with in vivo and in vitro SAH models, we discovered that iron-overloaded MC-EXs induce neuronal ferroptosis. In mice, intranasal administration of MC-EXs (10⁹ particles/day for 3 days, n = 10/group) exacerbated SAH-induced motor, sensory, and cognitive deficits. Bioinformatic analysis and experimental validation (including C3 siRNA knockdown) identified the complement C3/C5/NF-κB pathway as a key molecular mechanism through which iron-overloaded MC-EXs trigger ferroptosis. This research provides evidence for a novel EX-mediated mechanism for iron toxicity in SAH, highlighting MC-EXs and the C3/C5/NF-κB axis as potential therapeutic targets.