Glycolytic Enzyme HK2 Phosphorylates nSMase1 to Promote Astrocytic Exosomes Biogenesis Contributing to Acute Ischemic Stroke Injury

Abstract Stroke represents a significant threat to public health, with insufficient understanding of its pathological mechanisms hindering therapeutic advancements. Exosomes are implicated in both the injury and recovery processes of ischemic stroke. While enhanced glycolysis is linked to exosome biogenesis, its regulatory mechanisms remain largely unexplored. Here, an increase in the number of exosomes within the cerebrospinal fluid (CSF) and plasma in acute ischemic stroke is observed. Hypoxia selectively enhanced the exosomes biogenesis in astrocytes. Through RNAi screening, we identified the glycolytic enzyme hexokinase 2 (HK2) as a key promoter of hypoxia‐induced exosomes. HK2 facilitates the formation of intraluminal vesicles (ILVs), the precursors of exosomes, without affecting their degradation in lysosomes. HK2 directly phosphorylates neutral sphingomyelinase 1 (nSMase1), a critical enzyme involved in the lipid biogenesis pathway of exosomes. Moreover, hypoxia‐induced astrocytic exosomes disrupt cerebrovascular endothelial tight junction proteins. Astrocyte‐specific knockdown of HK2 significantly reduces exosomes release and alleviates brain injury caused by middle cerebral artery occlusion (MCAO). Notably, MCAO markedly increased the phosphorylation of nSMase1, which is effectively abolished by astrocytic HK2 knockdown. In conclusion, this study reveals a non‐metabolic role of HK2 in exosomes biogenesis via its protein kinase activity, offering a potential therapeutic target for stroke.