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: 8202383
• 负责人: AMBER Joyce MCCARTNEY
• 金额: $ 3.19万
• 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 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8202383
• 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. PUBLIC HEALTH RELEVANCE: Neurotransmitter receptors are constantly cycling into and out of synapses through a system of endosomal membranes that are highly regulated by signaling molecules, and proper trafficking of these receptors is known to be important for the normal development, maintenance, and plasticity of neural circuits in the brain. Defects in receptor trafficking will likely destabilize activity in neuronal networks, which could contribute to a variety of neurological disorders, including predisposing particularly vulnerable circuits, such as the hippocampus, to epilepsy. This project will test the hypothesis that a specific lipid signaling molecule, PI(3,5)P2, plays a critical role in receptor trafficking and controlling the strength of synapses in neurons of the hippocampus, and may provide important insights into a broad range of human diseases, including epilepsy.

描述（由申请人提供）：正常的大脑功能需要动态运输膜蛋白，例如神经递质受体，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许多病理结局，包括在癫痫发作的神经回路（例如海马）中的活性破坏。内在化后，AMPA型谷氨酸受体遵循无处不在的囊泡运输路线，从而受体可以回收回细胞表面或靶向溶酶体以降解。虽然已知AMPA受体的命运可以通过突触活性确定，但对确定受体是否会被回收或降解的细胞内信号提示知之甚少。然而，已知关键排序点在早期内体的水平上存在，其中蛋白质和脂质组成的变化都控制了内吞运输室的身份和动力学。该提案的具体目标是确定磷脂酰肌醇（3,5） - 磷酸（PTDINS（3,5）P2或PI（3,5）P2）信号传导在分类内部AMPA受体中的信号传导以及此分类如何影响突触功能。 PI（3,5）P2是通过磷酸肌醇5-激酶（Pikfyve）对PI3P磷酸化生成的，被认为对于从早期内体到晚期到晚期至晚期至关重要。我的中心假设是，PI（3,5）P2合成是一个主要的细胞内信号，它决定了AMPAR的后内环细胞分选，以使内体回收内体或在细胞膜晚期内体/溶酶体中的重新插入。该假设将在以下两个目的中进行检验：1）确定PI（3,5）P2合成在调节AMPA受体的内吞后分选中的作用； 2）确定PI（3,5）P2合成是否有助于活性依赖于突触功效的控制。在AIM 1下，将通过生物化学和来自野生型和突变小鼠的培养神经元的免疫荧光来评估AMPA受体分选的变化，其中所必需的pikfyve调节剂VAC14 - / - 被删除。在AIM 2下，将通过RNAi介导的基因沉默或组成性活性Pikfyve的表达与细胞特异性操纵相结合，通过电生理学与细胞特异性操纵来评估突触后强度的变化。预计该提案中概述的实验将揭示AMPA受体运输与PI（3,5）P2信号之间的新联系。通过表征PI（3,5）P2信号在调节突触强度中的独特作用，该项目将促进我们对直接细胞内分类AMPA受体的细胞内信号传导提示的理解，这可能会产生对病因学和癫痫病的重要见解。 公共卫生相关性：神经递质受体不断通过信号分子高度调节的内体膜系统循环和从突触中循环，并且已知这些受体的适当运输对大脑中神经电路的正常发育，维持和可变性很重要。受体运输的缺陷可能会破坏神经元网络中的活性，这可能导致各种神经系统疾病，包括使特别脆弱的电路（如海马）症状症状，引起癫痫病。该项目将检验以下假设：特定的脂质信号分子PI（3,5）P2在受体运输和控制海马神经元突触的强度中起着至关重要的作用，并且可以为包括癫痫在内的广泛人类疾病提供重要的见解。