Dynamic Regulation of Vesicular Trafficking by Altered Sterol Homeostasis

改变甾醇稳态对囊泡运输的动态调节

基本信息

批准号：
10093155
负责人：
Ruthellen Hope Anderson
金额：
$ 5.1万
依托单位：
UNIVERSITY OF SOUTH DAKOTA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-02-20 至 2022-02-19
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10093155
关键词：
ATP binding cassette transporter 1 Affect Alzheimer&apos s Disease Atomic Force Microscopy CRISPR/Cas technology Cell Adhesion Cell membrane Cell model Cell physiology Cells Cellular Membrane Cholesterol Cholesterol Synthesis Inhibition Clathrin Clathrin Light Chains Cognition Communication Coupled Data Development Disease Electrophysiology (science)Endocytosis Enzymes Event Exhibits Exocytosis Fluorescence Fluorescence Microscopy Fluorescence Recovery After Photobleaching Functional disorder Genes Human Huntington Disease Intellectual functioning disability Knock-out Label Learning Lipids Measures Mediating Membrane Membrane Lipids Memory Microcephaly Microscopic Microscopy Modeling Molecular Monitor Mutation Nervous System Physiology Neurologic Neurologic Deficit Neurologic Dysfunctions Neurons Neurophysiology - biologic function Pathway interactions Patients Process Rare Diseases Recycling Regulation Resolution Role Schizophrenia Self-Injurious Behavior Signal Transduction Specificity Squalene Sterols Structure Synapses Synaptic Vesicles Synaptic plasticity Syndrome Testing Time Transmission Electron Microscopy Vesicle Work autistic behaviour clinically relevant confocal imaging desensitization experimental study induced pluripotent stem cell inhibitor/antagonist insight live cell imaging malformation nervous system development neurodevelopment neurotransmission novel patch clamp receptor relating to nervous system scaffold stem cell model sterol homeostasis synaptic function synaptogenesis synthetic enzyme trafficking translocase

项目摘要

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.

项目概要对于正常神经发育和神经元功能至关重要的基本细胞过程是网格蛋白介导的内吞作用。在中枢神经系统内，网格蛋白介导的内吞作用在功能上与胞吐作用耦合突触小泡对于神经传递和小泡受体脱敏至关重要。而胆固醇已知耗尽可显着抑制网格蛋白介导的信号传导，其具体机制和膜动力学和网格蛋白介导的内吞途径的需求尚不清楚。有趣的是，胆固醇合成的常染色体隐性遗传疾病，其特征是细胞取代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。我们的初步数据表明网格蛋白介导的内吞作用表现出高度的脂质特异性才能正常发挥作用。描述甾醇改变的神经系统后果网格蛋白活性的稳态将提供有关囊泡贩运的新机制数据神经发育和神经元功能。我们此提案的长期目标是界定甾醇替代对囊泡运输和神经元功能的细胞影响。目标 1 将利用 live- 对 CRISPR-Cas9 编辑的人类诱导多能干细胞 (iPSC) 进行细胞成像，以确定其影响改变网格蛋白介导的内吞作用的甾醇稳态。将监测内源性网格蛋白动态通过活细胞共聚焦成像、光漂白后荧光恢复 (FRAP) 进行实时定量，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胆固醇合成抑制通过原子力破坏网格蛋白介导的内吞事件显微镜和偏振全内反射荧光 (polTIRF) 显微镜进行动态监测细胞硬度、膜弯曲和网格蛋白组装。目标 3 将定义甾醇的功能作用使用 patch- 替代神经突触内的囊泡运输和网格蛋白介导的内吞作用对分化的患者来源的 iPSC 进行钳位电生理学和高分辨率显微分析。这些研究将直接有助于理解网格蛋白介导的内吞作用和功能甾醇前体破坏这一过程对哺乳动物囊泡贩运的影响突触。这项工作将为破坏网格蛋白介导的贩运提供关键证据在胆固醇合成障碍中观察到的神经缺陷，可能支持胆固醇功能障碍的作用在与胆固醇改变相关的更常见神经系统疾病中网格蛋白介导的内吞作用水平，如精神分裂症、亨廷顿舞蹈症和阿尔茨海默病。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis.

甾醇降低有效网格蛋白介导的内吞作用所需的膜弯曲和裂变的能量屏障。

DOI：
发表时间：
2022-02-08
期刊：
影响因子：
8.8
作者：
Anderson, Ruthellen H;Sochacki, Kem A;Vuppula, Harika;Scott, Brandon L;Bailey, Elizabeth M;Schultz, Maycie M;Kerkvliet, Jason G;Taraska, Justin W;Hoppe, Adam D;Francis, Kevin R
通讯作者：
Francis, Kevin R

Generation of a CLTA reporter human induced pluripotent stem cell line, CRMi001-A-1, using the CRISPR/Cas9 system to monitor endogenous clathrin trafficking.

使用 CRISPR/Cas9 系统监测内源性网格蛋白运输，生成 CLTA 报告基因人类诱导多能干细胞系 CRMi001-A-1。

DOI：
发表时间：
2018
期刊：
影响因子：
1.2
作者：
Anderson, Ruthellen H;Kerkvliet, Jason G;Otta, Jaelin J;Ross, Alan D;Leiferman, Patricia C;Hoppe, Adam D;Francis, Kevin R
通讯作者：
Francis, Kevin R

Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis.

甾醇降低有效网格蛋白介导的内吞作用所需的膜弯曲和裂变的能量屏障。

DOI：
发表时间：
2021
期刊：
影响因子：
8.8
作者：
Anderson, Ruthellen H;Sochacki, Kem A;Vuppula, Harika;Scott, Brandon L;Bailey, Elizabeth M;Schultz, Maycie M;Kerkvliet, Jason G;Taraska, Justin W;Hoppe, Adam D;Francis, Kevin R
通讯作者：
Francis, Kevin R