Molecular Mechanisms of Postsynaptic AMPA Receptor Localization

突触后 AMPA 受体定位的分子机制

• 批准号: 9093834
• 负责人: JOHANNES W HELL
• 金额: $ 49.44万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-26 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9093834
• 关键词: AMPA Receptors; Actinin; Acute; Affect; Alzheimer' s Disease; Autistic Disorder; Binding; Calmodulin; Central Nervous System Diseases; Chemicals; Co-Immunoprecipitations; Complex; DLG4 gene; Data; Engineering; Epilepsy; Exhibits; F-Actin; Fluorescence Microscopy; Foundations; Frequencies; Functional disorder; Glutamate Receptor; Health; Hippocampus (Brain); Homologous Gene; Injection of therapeutic agent; Link; Maintenance; Mediating; Mental Depression; Molecular; Monitor; Mutate; Mutation; N-Methylaspartate; N-terminal; Neurons; Peptides; Phenocopy; Physiological; Point Mutation; Post-Traumatic Stress Disorders; Process; Proteins; Psyche structure; Role; Schizophrenia; Site; Stroke; Structure; Synapses; Synaptic Transmission; Testing; Translating; Vertebral column; Work; alpha Actinin; knock-down; mutant; nervous system disorder; overexpression; postsynaptic; prenylation; prevent; AMPA Receptors; Actinin; Acute; Affect; Alzheimer' s Disease; Autistic Disorder; Binding; Calmodulin; Central Nervous System Diseases; Chemicals; Co-Immunoprecipitations; Complex; DLG4 gene; Data; Engineering; Epilepsy; Exhibits; F-Actin; Fluorescence Microscopy; Foundations; Frequencies; Functional disorder; Glutamate Receptor; Health; Hippocampus (Brain); Homologous Gene; Injection of therapeutic agent; Link; Maintenance; Mediating; Mental Depression; Molecular; Monitor; Mutate; Mutation; N-Methylaspartate; N-terminal; Neurons; Peptides; Phenocopy; Physiological; Point Mutation; Post-Traumatic Stress Disorders; Process; Proteins; Psyche structure; Role; Schizophrenia; Site; Stroke; Structure; Synapses; Synaptic Transmission; Testing; Translating; Vertebral column; Work; alpha Actinin; knock-down; mutant; nervous system disorder; overexpression; postsynaptic; prenylation; prevent

DESCRIPTION (provided by applicant): AMPAR and spine dysfunction or dysregulation underlies many CNS diseases including depression, autism, PTSD, epilepsy, and stroke-induced neuronal damage. Precise postsynaptic localization of AMPARs is critical for fast synaptic transmission. It depends on PSD-95 and its interaction with auxiliary AMPAR subunits called TARPs. Despite its central role in targeting AMPARs, it is unknown how PSD-95 itself is anchored at the postsynapse. Our preliminary data suggest that A) a-actinin binds to the first 13 residues of the N-terminus of PSD-95; B) knock-down (KD) of a-actinin reduces postsynaptic PSD-95 content and mEPSCs, the latter phenocopying KD of PSD-95; C) mutating either Lys10 or Lys11 to Glu (K10E, K11E) specifically impairs PSD- 95 binding to a-actinin and postsynaptic targeting of PSD-95 and of AMPARs; D) peptide PSD95(1-13) displaces PSD-95 from a-actinin; E) injection of PSD95(1-13) decreases mEPSC amplitude. We hypothesize that a-actinin is critical for postsynaptic anchoring of PSD-95 and thereby AMPAR-TARP complexes. Proving this hypothesis will fundamentally advance our understanding of postsynaptic AMPAR localization. A), B), and D) are final data. Aims 1 and 2 will further scrutinize C) and E), i.e., whether mutating K10E and K11E or injecting PSD95(1-13) affect synaptic PSD-95 and AMPAR taregting using fluorescence microscopy and mEPSC. NMR structural analysis will identify residues in a-actinin that are important for PSD-95 binding. KD of endogenous a-actinin and replacement with either WT or mutant a-actinin will show whether mutant a-actinin is not able to rescue the KD effect on PSD-95, in contrast to our rescue with WT a-actinin. Aim 3 is to test whether NMDA-induced Ca2+ influx displaces PSD-95 from a-actinin and thereby from postsynaptic sites along with AMPARs via calmodulin (CaM). We found that Ca2+/CaM binds to the N-terminus of PSD-95 to dislodge a-actinin. Our structural NMR analysis of a complex between Ca2+/CaM and the first 71 aa of PSD-95 identified Y12 in PSD-95 as critical for CaM binding. Mutating Y12 to Glu (Y12E) prevents Ca2+/CaM binding without affecting a-actinin binding or postsynaptic localization of PSD-95. NMDA-induced Ca2+ influx displaces a portion of WT but not Y12E PSD-95 from spines. In fact, Y12E exhibits a large increase rather than decrease in spines upon Ca2+ influx. We will test whether this mutation and other manipulations unmask a mechanism that leads to postsynaptic accumulation of PSD-95 and AMPARs rather than their decrease. Such a decrease is usually seen following 5 min NMDA treatment and is referred to as chemical LTD. This exciting new direction will elucidate how Ca2+ influx can cause LTD rather than LTP. Page 1

描述（由申请人提供）：AMPAR和脊柱功能障碍或功能障碍是许多中枢神经系统疾病的基础，包括抑郁症，自闭症，PTSD，癫痫和中风引起的神经元损害。 AMPAR的精确突触后定位对于快速突触传播至关重要。它取决于PSD-95及其与称为TARPS的辅助AMPAR亚基的相互作用。尽管它在靶向AMPAR中的核心作用，但尚不清楚PSD-95本身如何锚定在后震荡之后。我们的初步数据表明a）a-肌动蛋白与PSD-95 N末端的前13个残基结合； b）a-肌动蛋白的敲低（KD）减少了突触后的PSD-95含量和MEPSC，后者的pSD-95 kD kD; c）将lys10或lys11突变为GLU（K10E，K11E）特异性损害了PSD-95与A-肌动蛋白和PSD-95和AMPARS的突触后靶向的结合； D）肽PSD95（1-13）从A-肌动蛋白中取代PSD-95； e）注射PSD95（1-13）会降低MEPSC振幅。我们假设A-肌动蛋白对于PSD-95的突触后锚定及其AMPAR-TARP复合物至关重要。证明这一假设将从根本上提高我们对突触后AMPAR定位的理解。 a），b）和d）是最终数据。目标1和2将进一步仔细检查c）和e），即是使用荧光显微镜和MEPSC进行突变的K10E和K11E或注射PSD95（1-13）影响突触的PSD-95和AMPAR对。 NMR结构分析将鉴定A-肌动蛋白中的残基对PSD-95结合很重要。与WT A-肌动蛋白营救相比，内源性A-肌动蛋白的KD和用WT或突变体A-肌动蛋白替代的KD将表明突变体A-肌动蛋白是否无法挽救KD对PSD-95的效应。 AIM 3是测试NMDA诱导的Ca2+流入是否从A-肌动蛋白中取代PSD-95，从而通过钙调蛋白（CAM）从突触后位点以及AMPAR以及AMPAR中取代PSD-95。我们发现Ca2+/CAM与PSD-95的N末端结合以消除A-肌动蛋白。我们对Ca2+/CAM与PSD-95的第一个71 AA之间的复合物的结构NMR分析，在PSD-95中识别为Y12对于CAM结合至关重要。将Y12突变为GLU（Y12E）可防止CA2+/CAM结合，而不会影响PSD-95的A-肌动蛋白结合或突触后定位。 NMDA诱导的Ca2+涌入的一部分是WT的一部分，而不是从棘突中取代Y12E PSD-95。实际上，Y12E在Ca2+流入时刺显示刺的大幅增长而不是减少。我们将测试该突变和其他操纵是否揭示导致PSD-95和AMPAR的突触后积累的机制，而不是减少。在5分钟NMDA处理后，通常会看到这种减少，并称为Chemical Ltd。这个令人兴奋的新方向将阐明CA2+流入如何引起LTD而不是LTP。第1页