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）：正常的脑功能需要膜蛋白（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，包括海马体等易受损伤神经回路的活动不稳定。在内化后，AMPA型谷氨酸受体遵循普遍存在的囊泡运输路线，由此受体可以再循环回到细胞表面或靶向溶酶体进行降解。虽然已知AMPA受体的命运可能由突触活性决定，但对决定受体是否被回收或降解的细胞内信号传导线索知之甚少。然而，一个关键的分选点是已知的存在于早期内体的水平，其中蛋白质和脂质组成的变化控制的身份和动态的内吞运输隔间。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）信号转导在分选内化AMPA受体中的作用，以及这种分选如何影响突触功能。PI（3，5）P2通过磷酸肌醇5-激酶（PIKfyve）磷酸化PI 3 P在内膜上产生，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合成是决定AMPAR的胞内后分选的主要细胞内信号，以回收内体用于重新插入细胞膜或晚期内体/溶酶体用于降解。该假设将在以下两个目标中进行测试：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合成是否有助于突触功效的活性依赖性控制。在目标1下，将通过生物化学和免疫荧光在来自野生型和突变小鼠的培养神经元中评估AMPA受体分选的变化，其中缺失了必需的PIKfyve调节因子Vac 14-/-。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的表达对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有望揭示AMPA受体运输和PI（3，5）P2信号传导之间的新联系。通过表征PI（3，5）P2信号在调节突触强度中的独特作用，该项目将推进我们对指导AMPA受体细胞内分选的细胞内信号线索的理解，这可能会对癫痫的病因学和治疗产生重要的见解。 公共卫生相关性：神经递质受体通过信号分子高度调节的内体膜系统不断循环进出突触，并且已知这些受体的适当运输对于大脑中神经回路的正常发育、维持和可塑性是重要的。受体运输的缺陷可能会使神经元网络的活动不稳定，这可能导致各种神经系统疾病，包括使特别脆弱的回路（如海马体）易患癫痫。该项目将测试一种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强度方面发挥关键作用的假设，并可能为包括癫痫在内的广泛的人类疾病提供重要见解。