糖代谢重编程在小胶质细胞持续活化中的作用和调控机制研究

A hallmark of multiple chronic central nervous system disorders is the sustained activation of microglia at the disease sites, where they contribute to the pathogenesis and progression of the diseases. While mechanisms triggering microglia activation have received extensive attention, little attention has been paid about the maintenance of the activated state. ATP induces rapid activation of microglia, but the downstream mechanisms underlying the sustained microglia activation remains unknown. Recent studies have revealed that metabolic reprogramming, by promoting glycolysis and suppressing oxidative phosphorylation, is involved in the peripheral immune cell activation, maintenance and inflammation. We have found that ATP induces the rapid activation of mTOR, a master of cellular metabolic regulator, and upregulates the key glycolytic enzyme hexokinase 2 (HK2). We therefore hypothesize that metabolic reprogramming via ATP-induced mTOR activation and HK2 upregulation may promote glycolysis and anabolic metabolism, thereby meeting the high anabolic demands and facilitating the maintenance of sustained activated microglia. This grant proposal will use metabolic flux analysis, RNAi, tissue-specific targeting and pharmacological tools, in combination with in vivo two-photon imaging to investigate the role of HK2-mediated metabolic reprogramming in the sustained activation of microglia at cellular, physiological and pathological levels. A systematic study on the sustained activation of microglia from the perspective of key metabolic enzymes will not only provide new insights into the mechanisms underlying the sustained activation of microglia, but helps to elucidate the pathogenesis of CNS disorders in which the sustained microglial activation is implicated.

小胶质细胞的持续活化是多种慢性CNS疾病的病理特征。既往多关注触发小胶质细胞活化的分子机制，对活化的维持所知甚少。ATP能快速激活小胶质细胞，但下游维持活化的机制不明。新近研究显示外周免疫细胞可通过促进糖酵解、抑制氧化磷酸化的代谢重编程，持续供给细胞能量和生物前体来维持活化、促进炎症。我们发现ATP能诱导小胶质细胞快速激活mTOR，并上调促糖酵解酶HK2。我们推测ATP 诱导mTOR激活和HK2上调可促进活化小胶质细胞的糖酵解和合成代谢，利于活化细胞的稳定和维持。因此，本项目拟通过代谢分析、RNAi、基因敲除和活体显微成像技术，从细胞、活体和病理模型三方面研究糖代谢重编程在维持小胶质细胞活化中的作用与调控机制。项目以糖代谢关键酶为切入点探讨小胶质细胞持续活化的机制，将为稳定和维持小胶质细胞活化的调控机制提供新的思路，为阐明相关疾病的分子病理机制提供新的线索和理论依据。

小胶质细胞是脑中重要的免疫细胞，其突起能够不间断地对中枢神经系统微环境进行监测，也能够快速响应微环境的刺激。小胶质细胞的活动状态对于维持中枢神经系统的正常发育以及稳态具有非常重要的意义。小胶质细胞的动态监测微环境、快速响应刺激以及维持活化状态需要特殊的代谢模式，但是维持其独特动力学和功能的代谢模式一直不清。我们的研究首先发现了原代小胶质细胞的激活伴随葡萄糖和脂质代谢途径相关基因的改变，通过代谢流的检测发现，脂多糖（LPS）作为典型的病原体相关分子模式，可以促进原代小胶质细胞升高糖酵解代谢途径，抑制氧化磷酸化。而ATP，典型的损伤相关分子模式，无菌激活小胶质细胞，则促进糖酵解和氧化磷酸化升高。此外，LPS和ATP刺激都可以激活小胶质细胞中雷帕霉素（mTOR）信号通路和糖代谢关键酶II型己糖激酶。阻断mTOR信号通路和HK2介导的糖酵解途径，都可以降低LPS和ATP刺激引起的糖酵解水平、促炎细胞因子和活性氧（ROS）的产生。此外，我们还构建了条件性敲除小胶质细胞中HK2的小鼠，通过双光子显微镜观察到，在激光损伤组织介导的小胶质细胞迁移模型中，敲除了小胶质细胞中的hk2可以显著降低小胶质细胞对损伤的响应以及迁移。我们首次发现，小胶质细胞不同的活化状态具有不同的糖代谢模式，其中mTOR和HK2介导的糖酵解途径对于维持小胶质细胞的快速响应和活化状态具有重要的作用。因此，我们的研究揭示了mTOR和HK2介导的糖酵解途径在调控小胶质细胞的活化状态中的重要作用，有望成为调控中枢神经系统疾病小胶质细胞活化状态的重要靶点。