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 CNS 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。 AMPAR 的精确突触后定位对于快速突触传递至关重要。它取决于 PSD-95 及其与称为 TARP 的辅助 AMPAR 亚基的相互作用。尽管 PSD-95 在靶向 AMPAR 方面发挥着核心作用，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) α-辅肌动蛋白与 PSD-95 N 末端的前 13 个残基结合； B)α-辅肌动蛋白的敲除(KD)减少突触后PSD-95含量和mEPSC，后者模仿PSD-95的表型KD； C)将Lys10或Lys11突变为Glu(K10E、K11E)特异性损害PSD-95与α-肌动蛋白的结合以及PSD-95和AMPAR的突触后靶向； D) 肽PSD95(1-13)从α-辅肌动蛋白中置换PSD-95； E) 注射 PSD95(1-13) 会降低 mEPSC 幅度。我们假设α-辅肌动蛋白对于 PSD-95 以及 AMPAR-TARP 复合物的突触后锚定至关重要。证明这一假设将从根本上增进我们对突触后 AMPAR 定位的理解。 A)、B) 和 D) 是最终数据。目标 1 和 2 将进一步审查 C) 和 E)，即使用荧光显微镜和 mEPSC 突变 K10E 和 K11E 或注射 PSD95(1-13) 是否会影响突触 PSD-95 和 AMPAR 靶向。 NMR 结构分析将鉴定α-辅肌动蛋白中对 PSD-95 结合很重要的残基。内源性α-辅肌动蛋白的KD和用WT或突变型α-辅肌动蛋白替换将显示突变型α-辅肌动蛋白是否无法挽救PSD-95上的KD效应，这与我们用WT a-肌动蛋白的挽救相反。目标 3 是测试 NMDA 诱导的 Ca2+ 流入是否会通过钙调蛋白 (CaM) 取代α-肌动蛋白上的 PSD-95，从而与 AMPAR 一起从突触后位点上取代 PSD-95。我们发现 Ca2+/CaM 与 PSD-95 的 N 末端结合以去除 α-辅肌动蛋白。我们对 Ca2+/CaM 与 PSD-95 前 71 个氨基酸之间的复合物进行结构 NMR 分析，确定 PSD-95 中的 Y12 对于 CaM 结合至关重要。将 Y12 突变为 Glu (Y12E) 可防止 Ca2+/CaM 结合，而不影响 a-肌动蛋白结合或 PSD-95 的突触后定位。 NMDA 诱导的 Ca2+ 流入会取代脊柱中的一部分 WT，但不会取代 Y12E PSD-95。事实上，Y12E 在 Ca2+ 流入时表现出刺的大幅增加而不是减少。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 5 分钟 NMDA 处理后出现，被称为化学 LTD。这个令人兴奋的新方向将阐明 Ca2+ 流入如何导致 LTD 而不是 LTP。第1页