PI(3,5)P2 synthesis in AMPA receptor trafficking and synaptic function

AMPA 受体运输和突触功能中的 PI(3,5)P2 合成

• 批准号: 8465947
• 负责人: AMBER Joyce MCCARTNEY
• 金额: $ 2.89万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8465947
• 关键词: AMPA Receptors; Accounting; Acute; Back; Biochemical; Biochemistry; Biological; Biological Neural Networks; Birth; Brain; Cell membrane; Cell surface; Cells; Cessation of life; Charcot-Marie-Tooth Disease; Chronic; Clinical; Cues; Defect; Dendrites; Development; Disease; Early Endosome; Electrophysiology (science); Endosomes; Epilepsy; Etiology; Gene Expression; Gene Silencing; Generations; Genetic; Glutamate Receptor; Goals; Hippocampus (Brain); Human; Immunofluorescence Immunologic; Knockout Mice; Lead; Life; Link; Lipids; Lysosomes; Maintenance; Mediating; Membrane; Membrane Protein Traffic; Molecular; Mus; Mutant Strains Mice; Nerve Degeneration; Neurons; Neurotransmitter Receptor; Outcome; Perinatal; Peripheral Nervous System Diseases; Phenocopy; Phosphatidylinositols; Phospholipids; Phosphorylation; Phosphotransferases; Physiology; Play; Proteins; RNA Interference; Recycling; Regulation; Research; Role; Route; Seizures; Signal Transduction; Signaling Molecule; Sorting - Cell Movement; Structure; Synapses; Synaptic Transmission; Syndrome; System; Testing; Time; Vesicle; Wild Type Mouse; base; genetic manipulation; genetic regulatory protein; human disease; insight; late endosome; mortality; nervous system disorder; neural circuit; novel; phosphatidylinositol 3,5-diphosphate; postsynaptic; prevent; receptor; receptor density; receptor recycling; research study; response; small hairpin RNA; synaptic function; trafficking

DESCRIPTION (provided by applicant): Normal brain function requires dynamic trafficking of membrane proteins, such as neurotransmitter receptors, for maintaining and altering excitatory synaptic strength. Defects in receptor trafficking could result in a host of pathological outcomes, including destabilization of activity in seizure-prone neural circuits, such as the hippocampus. Upon internalization, AMPA-type glutamate receptors follow ubiquitous vesicle trafficking routes whereby receptors may recycle back to the cell-surface or be targeted to the lysosome for degradation. While it is known that the fate of AMPA receptors may be determined by synaptic activity, little is known about the intracellular signaling cues that determine if receptors will be recycled or degraded. However, a key sorting point is known to exist at the level of the early endosome, where changes in both protein and lipid composition control the identity and dynamics of endocytic trafficking compartments. The specific objective of this proposal is to determine the role of phophatidylinositide (3,5)-bisphosphate (PtdIns(3,5)P2 or PI(3,5)P2) signaling in sorting internalized AMPA receptors and how this sorting may impact synaptic function. PI(3,5)P2 is generated on internal membranes, through phosphorylation of PI3P by phosphoinositide 5-kinase (PIKfyve), and is thought to be critical for the transition from early to late endosomes. My central hypothesis is that PI(3,5)P2 synthesis is a major intracellular signal that determines postendocytic sorting of AMPARs to either recycling endosomes for reinsertion at the cell membrane or to late endosomes/lysosomes for degradation. This hypothesis will be tested in the following two aims: 1) Determine the role of PI(3,5)P2 synthesis in regulating post-endocytic sorting of AMPA receptors; 2) Determine if PI(3,5)P2 synthesis contributes to activity-dependent control of synaptic efficacy. Under Aim 1, changes in AMPA receptor sorting will be assessed via biochemistry and immunofluorescence in cultured neurons from wild-type and mutant mice in which the essential PIKfyve regulator, Vac14-/-, is deleted. Under Aim 2, changes in postsynaptic strength will be assessed via electrophysiology in combina- tion with cell-specific manipulations of PIKfyve function by RNAi-mediated gene silencing or expression of constitutively active PIKfyve. The experiments outlined in this proposal are expected to reveal novel links between AMPA receptor trafficking and PI(3,5)P2 signaling. By characterizing a unique role for PI(3,5)P2 signaling in regulation of synaptic strength, this project will advance our understanding of intracellular signaling cues that direct intracellular sorting of AMPA receptors, which may yield important insights into the etiology and treatment of epilepsy.

描述（由申请人提供）：正常的大脑功能需要膜蛋白（例如神经递质受体）的动态运输，以维持和改变兴奋性突触强度。受体运输的缺陷可能会导致一系列病理结果，包括海马体等易于癫痫发作的神经回路的活动不稳定。内化后，AMPA 型谷氨酸受体遵循普遍存在的囊泡运输路线，受体可以循环回到细胞表面或靶向溶酶体进行降解。虽然我们知道 AMPA 受体的命运可能由突触活动决定，但对于决定受体是否被回收或降解的细胞内信号传导线索却知之甚少。然而，已知一个关键的分选点存在于早期内体水平，其中蛋白质和脂质组成的变化控制内吞运输区室的身份和动态。该提案的具体目标是确定磷脂酰肌醇 (3,5)-二磷酸（PtdIns(3,5)P2 或 PI(3,5)P2）信号传导在内化 AMPA 受体分选中的作用，以及这种分选如何影响突触功能。 PI(3,5)P2 是通过磷酸肌醇 5-激酶 (PIKfyve) 磷酸化 PI3P 在内膜上生成的，并且被认为对于从早期内体到晚期内体的转变至关重要。我的中心假设是，PI(3,5)P2 合成是一种主要的细胞内信号，决定 AMPAR 的内吞后分选，以回收核内体以重新插入细胞膜或晚期核内体/溶酶体以进行降解。该假设将在以下两个目标中得到检验：1）确定PI(3,5)P2合成在调节AMPA受体内吞后分选中的作用； 2) 确定 PI(3,5)P2 合成是否有助于突触功效的活动依赖性控制。在目标 1 下，将通过野生型和突变型小鼠（其中必需的 PIKfyve 调节因子 Vac14-/- 被删除）培养的神经元中的生物化学和免疫荧光来评估 AMPA 受体分选的变化。在目标 2 下，将通过电生理学结合通过 RNAi 介导的基因沉默或组成型活性 PIKfyve 表达对 PIKfyve 功能的细胞特异性操作来评估突触后强度的变化。该提案中概述的实验预计将揭示 AMPA 受体运输与 PI(3,5)P2 信号传导之间的新联系。通过表征 PI(3,5)P2 信号在突触强度调节中的独特作用，该项目将增进我们对指导 AMPA 受体细胞内排序的细胞内信号线索的理解，这可能会对癫痫的病因和治疗产生重要的见解。