智障关联激酶PAK在内源性大麻素通路中底物的鉴定及其功能研究

Mental retardation related gene PAK (p21-activated kinase) family play important roles in neurodevelopment, synaptic transmission and plasticity. Various genetic and pharmacological manipulations on PAK have successfully reversed multiple neurobiological and behavioral deficits in transgenic mouse models of Autism. We have found that sustained kinase activity of PAK1, which is the major form of PAK expressed in the brain, is critical for the maintenance of endocannabinoid signaling and GABAergic synaptic transmission and plasticity. In order to identify PAK1 substrates that may regulate these processes, we performed iTRAQ-based phosphoproteomic analysis on the PAK1 knockout brain. We discovered a group of candidate protein substrates of PAK1, including MAGL (Monoacylglycerol lipase). MAGL is the major lipase that is responsible for the degradation of endocannabinoid 2-AG. Its phosphorylation level and lipase activity is significantly reduced in PAK1 knockout mice. In this project, we will first examine the kinase-substrate relation between PAK1 and MAGL, then utilize a battery of approaches, including electrophysiology, biochemical assay and behavior analysis, to investigate the regulation and underlying mechanisms of PAK1-mediated MAGL phosphorylation on its lipase activity, endocannabinoid signaling, GABAergic transmission and animal social behaviors. This study may provide new insights into regulatory machinery on the endocannabinoid system and PAK-mediated regulation on brain functions.

智力障碍关联基因PAK在神经发育、突触传递与可塑性等方面都有重要的调节作用。采用分子和药物手段对其活性的操控可反转多种孤独症模式小鼠的神经生物学及社交行为缺陷。我们的前期工作发现PAK家族在大脑中的主要亚型PAK1持续的激酶活性对维持内源性大麻素通路及GABA能突触传递的正常水平至关重要，但具体机制不明。现有对大脑中PAK1激酶底物认识的匮乏严重制约了相关分子机制研究的深入开展。通过对PAK1敲除小鼠的脑组织进行基于iTRAQ技术的全蛋白组磷酸化水平定量分析，我们已经发掘出了一批PAK1激酶的候选底物。其中主控内源性大麻素2-AG降解的单酰基甘油脂肪酶MAGL的磷酸化水平及功能在PAK1缺失后显著降低。本项目拟对该候选底物进行细化鉴定，并深入研究PAK1介导的MAGL磷酸化与其酯酶功能的关联以及该作用对内源性大麻素通路、GABA能突触传递和动物社交行为的调控作用。

PAK（p21-activated kinase）激酶是一族在真核生物中广泛存在且进化高度保守的丝氨酸/苏氨酸激酶。在大脑中，PAK被认为是一种智力障碍关联基因，在神经发育、突触传递与可塑性等方面都有重要的调节作用。前人采用分子和药物手段对其活性的操控可反转多种孤独症模式小鼠的神经生物学及社交行为缺陷，但目前对PAK在高级脑功能如社交行为和情感等方面的作用机制知之甚少。自本项目执行以来，项目组通过对PAK1敲除小鼠的脑组织进行基于iTRAQ技术的全蛋白组磷酸化水平定量分析，发掘出了一批PAK1激酶的候选底物并进行了细化鉴定，并进行了电生理记录、酶活及生化分析、荧光免疫成像、行为学检测等研究，实验结果表明：PAK可以通过动态调节环氧化酶2(Cyclooxygenase, COX-2)的亚细胞定位来限制其对大脑中重要的内源性大麻素AEA (anandamide)的降解，控制突触前递质释放的抑制效应，维持海马区GABA能神经传递的正常强度及脑功能。该成果为寻找治疗孤独症的药物、疗法提供了新的思路。此外，PAK信号通路及内源性大麻素系统在人体中各脏器中广泛存在，且在肿瘤及炎症发生中也起着重要作用，上述关于PAK对内源性大麻素调控机制的成果可为相关研究提供参考。