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

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和脊柱功能障碍或失调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伤。AMPARs的突触后精确定位对快速突触传递至关重要。它依赖于PSD-95及其与辅助AMPAR亚基tarp的相互作用。尽管PSD-95在靶向ampar中起着核心作用，但尚不清楚PSD-95本身如何锚定在突触后。我们的初步数据表明，A) A -肌动蛋白结合在PSD-95 n端前13个残基上；B) a-actin敲低（KD）减少突触后PSD-95含量和mepsc，后者表型复制PSD-95的KD；C)将Lys10或Lys11突变为Glu （K10E, K11E）特异性地损害PSD-95与a- actitin的结合以及PSD-95和AMPARs的突触后靶向；D)肽PSD95（1-13）取代a- actitin中的PSD-95；E)注射PSD95（1-13）降低mEPSC振幅。我们假设a-肌动蛋白对PSD-95突触后锚定以及AMPAR-TARP复合物至关重要。证明这一假设将从根本上推进我们对突触后AMPAR定位的理解。A)， B)， D)为最终数据。Aims 1和2将进一步检查C)和E)，即使用荧光显微镜和mEPSC检测突变K10E和K11E或注射PSD95（1-13）是否会影响突触PSD-95和AMPAR靶向。核磁共振结构分析将确定a-actin中对PSD-95结合很重要的残基。内源性a- actitin的KD和用WT或突变的a- actitin替代将显示突变的a- actitin是否不能挽救KD对PSD-95的影响，与我们用WT - actitin的挽救相反。目的3是测试nmda诱导的Ca2+内流是否通过钙调蛋白（CaM）将PSD-95从a-actin中取代，从而从突触后位点和ampar中取代。我们发现Ca2+/CaM结合到PSD-95的n端以去除a-肌动蛋白。我们对Ca2+/CaM与PSD-95的前71 aa之间的复合物进行了结构核磁共振分析，发现PSD-95中的Y12对CaM结合至关重要。将Y12突变为Glu （Y12E）可阻止Ca2+/CaM结合，而不影响a-actin结合或PSD-95的突触后定位。nmda诱导的Ca2+内流移位了部分WT，但不移位脊柱中的Y12E PSD-95。事实上，在Ca2+内流时，Y12E在脊柱中表现出大量增加而不是减少。我们将测试这种突变和其他操作是否揭示了导致PSD-95和ampar突触后积累而不是减少的机制。这种减少通常在NMDA处理5分钟后出现，被称为化学限制。这个令人兴奋的新方向将阐明Ca2+内流如何导致LTD而不是LTP。第1页