Roles of Phosphoinositides at the Synapse

描述（由申请人提供）：磷酸肌苷，如PI(4,5) p2 (PIP2)，调节肌动蛋白动力学和囊泡运输的各个方面。然而，对PIP2在囊泡运输、定位和合成调控中的确切作用尚不清楚。突触为探索这些问题提供了一个很好的模型。因此，本项目的目标是探索PIP2在突触囊泡运输和肌动蛋白动力学中的突触前作用。为此，我将研究改变它们在活体突触中的可用性的后果。通过向七鳃鳗巨型网状脊髓突触注射特异性PIP2结合蛋白模块来减少内源性PIP2，或注射扰乱PIP2代谢酶的试剂，可以改变PIP2的可用性。荧光成像、电生理学和电子显微镜的结合将用于检查这些试剂对肌动蛋白动力学和突触囊泡运输的影响。这种方法将首次提供对突触中PIP2功能的全面分析。De Camilli实验室已经确定并描述了在突触处代谢PIP2的酶。其中一种酶PIP2-磷酸酶synaptojanin的遗传和急性损伤会改变肌动蛋白细胞骨架并破坏突触中的突触囊泡循环，这表明PIP2在这些过程中起重要作用。磷酸肌肽代谢的改变与阿尔茨海默病的发病机制以及高血压、葡萄糖代谢和脂质代谢等多种人类疾病有关。因此，我的研究可能对人类健康有广泛的影响。

DESCRIPTION (provided by applicant): Phosphoinositides, such as PI(4,5)P 2 (PIP2), regulate various aspects of actin dynamics and vesicle trafficking. However, the precise contributions of PIP2 to vesicle trafficking, its localization, and regulation by synthesis is not well-understood. The synapse offers a good model in which to explore these questions. Therefore, the goal of this project is to explore the presynaptic roles of the PIP2 in synaptic vesicle trafficking and actin dynamics. To do so, I will examine the consequences of altering their availability in living synapses. PIP2 availability will be altered by injecting into lamprey giant reticulospinal synapses either specific, PIP2 binding protein modules to decrease endogenous PIP2 or by injecting reagents that perturb PIP2-metabolizing enzymes. A combination of fluorescence imaging, electrophysiology, and electron microscopy will be used to examine the consequences of these reagents on actin dynamics and synaptic vesicle trafficking in vivo. This approach will provide the first comprehensive analysis of PIP2 functions at the synapse. The De Camilli lab has already identified and characterized enzymes that metabolize PIP2 at the synapse. Genetic and acute impairment of one of these enzymes, the PIP2-phosphatase synaptojanin, alters actin cytoskeleton and disrupts synaptic vesicle recycling in synapses, suggesting an important role for PIP2 in these processes. Altered phosphoinositide metabolism has been implicated in the pathogenesis of Alzheimer's disease, as well as a variety of human disorders of hypertension, glucose metabolism, and lipid metabolism. Thus, my studies may have wide implications for human health.