Molecular Mechanisms of Postsynaptic AMPA Receptor Localization

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、癫痫和中风诱导的神经元损伤。AMPAR的精确突触后定位对于快速突触传递至关重要。它依赖于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中起着核心作用，但目前尚不清楚PSD-95本身如何锚定在突触后。我们的初步研究结果表明：A）α-辅肌动蛋白与PSD-95 N端的前13个残基结合，B）α-辅肌动蛋白的敲低（KD）降低了突触后PSD-95含量和mEPSC，后者表型复制PSD-95的KD; C）将Lys 10或Lys 11突变为Glu（K10 E，K11 E）特异性地损害PSD-95与α-辅肌动蛋白的结合以及PSD-95和AMPAR的突触后靶向; E）注射PSD 95（1-13）降低mEPSC振幅。我们假设α-辅肌动蛋白对于PSD-95的突触后锚定以及AMPAR-TARP复合物是至关重要的。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）和D）为最终数据。目标1和2将进一步审查C）和E），即，无论是突变K10 E和K11 E还是注射PSD 95（1-13），使用荧光显微镜和mEPSC影响突触PSD-95和AMPAR taregting。NMR结构分析将鉴定α-辅肌动蛋白中对PSD-95结合重要的残基。内源性α-辅肌动蛋白的KD和用WT或突变体α-辅肌动蛋白替换将显示突变体α-辅肌动蛋白是否不能拯救对PSD-95的KD作用，与我们用WT α-辅肌动蛋白的拯救相反。目的3是测试NMDA诱导的Ca 2+内流是否从α-辅肌动蛋白中置换PSD-95，从而通过钙调蛋白（CaM）与AMPAR沿着从突触后位点置换PSD-95。我们发现，Ca ~（2+）/CaM结合PSD-95的N-末端，以驱逐α-辅肌动蛋白。我们对Ca 2 +/CaM和PSD-95的前71个氨基酸之间的复合物的结构NMR分析确定PSD-95中的Y12对CaM结合至关重要。将Y12突变为Glu（Y12 E）阻止Ca 2 +/CaM结合，而不影响PSD-95的α-辅肌动蛋白结合或突触后定位。NMDA诱导的Ca 2+内流从棘置换WT的一部分，但不置换Y12 E PSD-95。事实上，Y12 E在Ca 2+内流时表现出刺的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。这样的减少通常是看到5分钟NMDA治疗后，被称为化学LTD。这一令人兴奋的新方向将阐明如何Ca 2+流入可能会导致LTD，而不是LTP。第1页

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