Integrin-Arg-SHP2 signaling regulates NMDAR function and neuron morphology

整合素-Arg-SHP2信号调节NMDAR功能和神经元形态

• 批准号: 8958719
• 负责人: Aaron Donald Levy
• 金额: $ 2.78万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8958719
• 关键词: Address; Adolescence; Affect; Alleles; Alzheimer' s Disease; Attenuated; Autistic Disorder; Binding; Biochemical; Biotinylation; Cognitive deficits; Confocal Microscopy; Data; Defect; Dendrites; Dendritic Spines; Diagnosis; Disease; Economic Burden; Electron Microscopy; Functional disorder; Genetic; Head; Hippocampus (Brain); Integrins; Knock-in Mouse; Link; Maintenance; Measures; Mediating; Mental Retardation; Mental disorders; Molecular; Mood Disorders; Morphology; Mus; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; NMDA receptor antagonist; Neurodegenerative Disorders; Neurons; Non-Receptor Type 11 Protein Tyrosine Phosphatase; Noonan Syndrome; PTPN11 gene; Pathology; Phosphorylation; Phosphorylation Site; Play; Prosencephalon; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Regulation; Regulatory Pathway; Role; Schizophrenia; Signal Transduction; Site; Slice; Structure; Surface; Synapses; Techniques; Testing; Therapeutic Intervention; Tyrosine Phosphorylation Site; Vertebral column; addiction; adhesion receptor; autism spectrum disorder; density; developmental disease; excitatory neuron; gain of function; insight; mutant; nervous system disorder; neuron loss; public health relevance; receptor function; research study; social; treatment strategy

DESCRIPTION (provided by applicant): Integrin-Arg-SHP2 signaling regulates NMDAR function and neuron morphology. Dendritic spine stability is disrupted in psychiatric and neurological disorders. Regulation of NMDA receptor (NMDAR) activity by integrin adhesion receptors plays a fundamental role in dendritic spine maturation and stability, but the molecular mechanisms by which integrins regulate NMDARs are unknown. We have shown that loss of integrin ?3?1 signaling through the Abl2/Arg non-receptor tyrosine kinase causes widespread dendritic spine loss in late adolescence due to increased GluN2B subunit-mediated NMDA receptor currents. I provide strong evidence that Arg acts through the tyrosine phosphatase SHP2 to control GluN2B phosphorylation and function. NMDA receptor dysfunction is a hallmark of psychiatric, neurodevelopmental and neurological diseases such as schizophrenia, autism, and Alzheimer's disease. Understanding the mechanism by which integrins regulate NMDAR function is critical to understand how synaptic stability is compromised in these disorders and to develop treatment strategies. In this proposal, I will test the hypothesis that integrin-Arg-SHP2 signaling functionally regulates the NMDA receptor to control dendritic spine stability. My first aim is to determine how SHP2 regulates GluN2B phosphorylation and function. In this aim, I will use substrate-trapping mutants of SHP2 to test whether GluN2B is directly dephosphorylated by SHP2 and to determine which site in GluN2B is targeted by SHP2. GluN2B phosphorylation promotes its surface localization. Therefore, I will also use surface biotinylation of NMDARs in cultured hippocampal neurons to measure how genetic and pharmacological loss- or gain-of-function of Arg and SHP2 influence NMDAR surface expression. My second aim is to elucidate an integrin-NMDA receptor regulatory mechanism. GluN2B phosphorylation increases NMDAR-mediated currents, and my preliminary data show that activation of SHP2 decreases GluN2B phosphorylation. To test how SHP2 regulates NMDAR function, I will measure how loss- or gain-of-function of SHP2 activity affects NMDAR-mediated currents in hippocampal slices. I will also test the hypothesis that SHP2 functions as a mechanistic link between integrin-Arg signaling and the NMDAR by assessing whether an SHP2 gain-of-function allele can suppress elevated NMDAR currents in mice lacking integrin ?3?1 or Arg. My third aim is to understand how SHP2 regulates dendritic spine and synapse structure. Our lab has shown that integrin-Arg signaling attenuates NMDAR activity to stabilize spines, and I hypothesize that SHP2 mediates the effects of Arg signaling on NMDAR function. I will use confocal and electron microscopy to quantitatively measure how loss- and gain-of-function of SHP2 activity in mice affects dendritic spine and synapse structure and stability. I will also test whether an activated SHP2 allele can suppress the spine destabilization resulting from loss of integrin ?3?1 or Arg in mice.

描述（由申请人提供）：整合素- arg - shp2信号调节NMDAR功能和神经元形态。树突脊柱稳定性在精神和神经疾病中被破坏。整合素粘附受体对NMDA受体（NMDAR）活性的调控在树突脊柱成熟和稳定中起着重要作用，但整合素调控NMDAR的分子机制尚不清楚。我们已经证明了整合素的损失？由于GluN2B亚单位介导的NMDA受体电流增加，通过Abl2/Arg非受体酪氨酸激酶的1信号通路导致青春期后期广泛的树突脊柱丢失。我提供了强有力的证据，表明精氨酸通过酪氨酸磷酸酶SHP2来控制GluN2B的磷酸化和功能。NMDA受体功能障碍是精神、神经发育和神经系统疾病（如精神分裂症、自闭症和阿尔茨海默病）的标志。了解整合素调节NMDAR功能的机制对于了解这些疾病中突触稳定性如何受到损害以及制定治疗策略至关重要。在本提案中，我将验证整合素- arg - shp2信号传导功能调节NMDA受体以控制树突脊柱稳定性的假设。我的第一个目标是确定SHP2如何调节GluN2B的磷酸化和功能。为此，我将使用SHP2的底物捕获突变体来测试GluN2B是否被SHP2直接去磷酸化，并确定GluN2B中的哪个位点被SHP2靶向。GluN2B磷酸化促进其表面定位。因此，我还将在培养的海马神经元中使用NMDAR的表面生物素化来测量Arg和SHP2的遗传和药理学功能丧失或获得如何影响NMDAR表面表达。我的第二个目标是阐明整合素- nmda受体的调控机制。GluN2B磷酸化增加了nmda介导的电流，我的初步数据显示，激活SHP2会降低GluN2B磷酸化。为了测试SHP2如何调节NMDAR功能，我将测量SHP2活动的功能丧失或功能获得如何影响海马切片中NMDAR介导的电流。我还将通过评估SHP2功能获得等位基因是否可以抑制缺乏整合素的小鼠中升高的NMDAR电流，来验证SHP2作为整合素-精氨酸信号传导与NMDAR之间的机制联系的假设。1或Arg。我的第三个目标是了解SHP2如何调节树突棘和突触结构。我们的实验室已经证明，整合素-精氨酸信号传导减弱NMDAR活性以稳定脊柱，并且我假设SHP2介导精氨酸信号传导对NMDAR功能的影响。我将使用共聚焦显微镜和电子显微镜定量测量小鼠SHP2活性的功能丧失和功能获得如何影响树突脊柱和突触的结构和稳定性。我还将测试激活的SHP2等位基因是否可以抑制因整合素缺失而导致的脊柱不稳定？1或Arg在小鼠中。