Molecular Mechanisms of Postsynaptic AMPA Receptor Localization
突触后 AMPA 受体定位的分子机制
基本信息
- 批准号:9093834
- 负责人:
- 金额:$ 49.44万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AMPA ReceptorsActininAcuteAffectAlzheimer&aposs DiseaseAutistic DisorderBindingCalmodulinCentral Nervous System DiseasesChemicalsCo-ImmunoprecipitationsComplexDLG4 geneDataEngineeringEpilepsyExhibitsF-ActinFluorescence MicroscopyFoundationsFrequenciesFunctional disorderGlutamate ReceptorHealthHippocampus (Brain)Homologous GeneInjection of therapeutic agentLinkMaintenanceMediatingMental DepressionMolecularMonitorMutateMutationN-MethylaspartateN-terminalNeuronsPeptidesPhenocopyPhysiologicalPoint MutationPost-Traumatic Stress DisordersProcessProteinsPsyche structureRoleSchizophreniaSiteStrokeStructureSynapsesSynaptic TransmissionTestingTranslatingVertebral columnWorkalpha Actininknock-downmutantnervous system disorderoverexpressionpostsynapticprenylationprevent
项目摘要
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
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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JOHANNES W HELL其他文献
JOHANNES W HELL的其他文献
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Molecular Mechanisms of Postsynaptic AMPA Receptor Localization
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