Dynamic Regulation of Vesicular Trafficking by Altered Sterol Homeostasis

项目总结 一个对正常神经发育和神经元功能至关重要的基本细胞过程是由网状蛋白介导的。 内吞作用。在中枢神经系统内，网状蛋白介导的内吞作用与胞吐作用在功能上是偶联的。 突触小泡用于神经传递，是小泡受体脱敏所必需的。而胆固醇 已知耗竭可以显著抑制细胞骨架蛋白介导的信号转导，其具体机制和 膜动力学和网状蛋白介导的内吞途径的基本要求尚不清楚。 有趣的是，常染色体隐性胆固醇合成障碍，以细胞替代为特征 胆固醇为甾醇中间体，构成一组严重影响神经的畸形综合征 系统的开发和功能。我们的初步数据显示，网状蛋白介导的内吞作用表现出 具有高度的脂类专一性，功能正常。描述改变的类固醇的神经学后果 网状蛋白活性的动态平衡将提供关于囊泡运输的新的机制数据 神经发育和神经功能。我们对这项建议的长远目标是界定 类固醇替代对囊泡运输和神经元功能的细胞影响。目标一号将利用现场直播- CRISPR-Cas9编辑的人诱导多能干细胞(IPSCs)的细胞成像研究 改变了笼蛋白介导的内吞作用中的固醇动态平衡。将监测内源性网状蛋白的动态 在实时和由活细胞共聚焦成像、光漂白后荧光恢复(FRAP)、 和全内反射荧光(TIRF)显微镜。目标2将通过以下方式确定分子机制 哪种胆固醇合成抑制通过原子力扰乱了笼蛋白介导的内吞事件 显微镜和偏振全内反射荧光显微镜动态监测 细胞硬度、膜弯曲和网状蛋白组装。目标3将定义甾醇的功能效应 在神经突触内的囊泡转运和网状蛋白介导的内吞作用上的替代使用Patch-... 钳夹电生理学和高分辨率显微分析分化的患者来源的ipscs。 这些研究将直接有助于理解分子筛蛋白介导的内吞作用和功能 类固醇前体对哺乳动物囊泡运输过程的干扰 Synapse。这项工作将为中断的网状蛋白介导的交易提供关键证据 在胆固醇合成障碍中观察到的神经缺陷，并可能支持 在与胆固醇改变相关的更常见的神经系统疾病中，笼状蛋白介导的内吞作用 水平，如精神分裂症、亨廷顿病和阿尔茨海默病。

PROJECT SUMMARY A fundamental cellular process critical for normal neurodevelopment and neuronal function is clathrin-mediated endocytosis. Within the CNS, clathrin-mediated endocytosis is functionally coupled to the exocytosis of synaptic vesicles for neurotransmission and essential for vesicular receptor desensitization. While cholesterol depletion is known to dramatically inhibit clathrin-mediated signaling, the specific mechanisms and requirements underlying membrane dynamics and clathrin-mediated endocytic pathways are unknown. Interestingly, autosomal recessive disorders of cholesterol synthesis, characterized by substitution of cellular cholesterol for sterol intermediates, constitute a group of malformation syndromes that severely affect nervous system development and function. Our preliminary data demonstrates clathrin-mediated endocytosis exhibits a high degree of lipid specificity to function normally. Delineating the neurological consequences of altered sterol homeostasis on clathrin activity will provide novel mechanistic data regarding vesicular trafficking in the context of neurodevelopment and neuronal function. Our long-term objective for this proposal is to delineate the cellular consequences of sterol substitution on vesicular trafficking and neuronal function. Aim 1 will utilize live- cell imaging of CRISPR-Cas9-edited human induced pluripotent stem cells (iPSCs) to define the impact of altered sterol homeostasis on clathrin-mediated endocytosis. Endogenous clathrin dynamics will be monitored in real-time and quantified by live cell confocal imaging, fluorescence recovery after photobleaching (FRAP), and total internal reflection fluorescence (TIRF) microscopy. Aim 2 will determine the molecular mechanism by which cholesterol synthesis inhibition disrupts clathrin-mediated endocytic events through atomic force microscopy and polarized total internal reflection fluorescence (polTIRF) microscopy to dynamically monitor cell stiffness, membrane bending and clathrin assembly. Aim 3 will define the functional effects of sterol substitution on vesicular trafficking and clathrin-mediated endocytosis within the neural synapse using patch- clamp electrophysiology and high-resolution microscopic analysis of differentiated patient-derived iPSCs. These studies will directly contribute to the understanding of clathrin-mediated endocytosis and functional implications of disruption of this process by sterol precursors on vesicular trafficking at the mammalian synapse. This work will provide critical evidence for disrupted clathrin-mediated trafficking underlying the neurological deficits observed in cholesterol synthesis disorders and may support a role for dysfunction of clathrin-mediated endocytosis in more common neurological conditions associated with altered cholesterol levels, such as schizophrenia, Huntington's disease, and Alzheimer's disease.