Molecular Mechanisms of Postsynaptic AMPA Receptor Localization

描述(由申请人提供)：AMPAR和脊柱功能障碍或调节失调是许多中枢神经系统疾病的基础，包括抑郁症、自闭症、创伤后应激障碍、癫痫和中风引起的神经元损伤。准确的突触后AMPAR定位是突触快速传递的关键。它依赖于PSD-95及其与称为TARP的辅助AMPAR亚单位的相互作用。尽管PSD-95在靶向AMPAR方面发挥了核心作用，但它本身是如何锚定在突触后的尚不清楚。我们的初步数据表明，A)a-Actinin与PSD-95 N末端的前13个残基结合；B)a-Actinin的敲除(KD)减少突触后PSD-95的含量和mEPSCs，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-Actinin的结合以及PSD-95和AMPARs的突触后靶向；D)肽PSD95(1-13)取代PSD-95从a-Actinin；E)注射PSD95(1-13)降低mEPSC的幅度。我们假设α-肌动蛋白对于突触后锚定PSD-95和AMPAR-TARP复合体是至关重要的。证明这一假说将从根本上促进我们对突触后AMPAR定位的理解。A)、B)和D)是最终数据。AIMS 1和2将进一步研究C)和E)，即突变K10E和K11E或注射PSD95(1-13)是否影响突触的PSD-95和AMPAR的转录，使用荧光显微镜和mEPSC。核磁共振结构分析将确定a-肌动蛋白中对PSD-95结合重要的残基。内源性a-actinin的Kd以及用WT或突变体a-actinin替换将表明突变体a-actinin是否不能挽救PSD-95上的KD效应，而不是我们用WT a-actinin拯救。目的3是测试NMDA诱导的钙内流是否通过钙调素(CaM)取代α-肌动蛋白的PSD-95，从而与AMPAR一起取代突触后部位的PSD-95。我们发现Ca~(2+)/CaM结合到PSD-95的N-末端以清除α-肌动蛋白。我们对Ca~(2+)/CaM与PSD-95的前71个氨基酸之间的络合物的结构核磁共振分析表明，PSD-95中的Y12是CaM结合的关键。将Y12突变为Glu(Y12E)可在不影响α-肌动蛋白结合或PSD-95突触后定位的情况下阻止钙/CaM结合。NMDA诱导的Ca~(2+)内流取代了WT的一部分，但不能取代Y12E PSD-95的脊椎。事实上，当钙离子内流时，Y12E的脊椎不是减少，而是增加很多。我们将测试这种突变和其他操作是否揭示了导致突触后PSD-95和AMPAR积累而不是它们减少的机制。这种下降通常出现在NMDA治疗5分钟后，被称为化学有限公司。这一令人兴奋的新方向将阐明钙离子内流如何导致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