己糖激酶2通过蛋白激酶活性调节星形胶质细胞外泌体生成参与急性缺血性脑损伤

脑卒中严重危害人类健康。星形胶质细胞糖酵解、外泌体参与了脑损伤过程。前期发现，糖酵解酶HK2通过代谢外效应引起神经元损伤和小胶质细胞炎性反应；抑制星形胶质细胞HK2阻断缺氧诱导的外泌体生成，并显著减轻急性脑卒中损伤，表明HK2通过外泌体引起急性缺血性脑损伤。但有待阐明HK2对星形胶质细胞外泌体生成的调节机制。预实验显示HK2结合外泌体脂质生成途径关键酶中性鞘磷脂酶（nSMase），HK2酶活性缺失取消其磷酸化，强烈提示HK2通过蛋白激酶活性直接磷酸化nSMase，促进外泌体生成。本项目拟研究HK2对星形胶质细胞外泌体脂质生成途径的调节，确证HK2直接磷酸化nSMase并鉴定磷酸化修饰位点、分析nSMase激活后对产物神经酰胺和外泌体生成的影响。应用星形胶质细胞HK2基因敲除小鼠确证外泌体脑卒中急性期的损伤作用。这将揭示HK2蛋白激酶相关急性缺血性脑损伤机制，为靶向HK2的药物研发提供依据。

Cerebral stroke is a serious hazard to human health. The glycolysis and exosomes of astrocytes are involved in brain injury. We previously identified the glycolytic enzyme hexokinase 2 (HK2) as a critical damage molecule in acute ischemic stroke, which caused neuronal injury and microglial inflammation via the effects other than metabolic function. The inhibition of HK2 in astrocytes significantly reduced the production of exosomes which selectively induced by hypoxia and alleviated acute ischemic brain injury, indicating HK2 caused brain damages by promoting the biogenesis of astrocytes-derived exosomes. However, the regulatory mechanism of HK2 on astrocytes-derived exosomes remains to be elucidated. Our preliminary experiments have shown that HK2 interacted with the neutral sphingomyelinase (nSMase), the key enzyme in the lipid pathway of exosomes biogenesis, and the absence of HK2 enzymic activity abrogated its phosphorylation. These results strongly suggest that HK2 causes acute ischemic brain injury through directly phosphorylating nSMase via its novel activity of protein kinase and promoting the production and release of astrocyte-derived exosomes. This project majorly aims to study the regulation of HK2 via its protein kinase activity on the lipid pathway of exosomes biogenesis. We will further confirm the direct phosphorylation modification of nSMase by HK2, identify its phosphorylation site, and analyze the production of downstream ceramide and the exosomes after the activation of nSMase. Finally, using astrocyte HK2 gene knockout mice we will confirm the damaging effect of astrocytes-derived exosomes in the acute phase of ischemic stroke. This project will reveal a new pathological mechanism of acute ischemic stroke relate to HK2 protein kinase activity and supply a basis for the development of the drugs targeting HK2.