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

描述(申请人提供)：整合素-Arg-SHP2信号调节NMDAR功能和神经元形态。在精神和神经疾病中，树突棘的稳定性被破坏。整合素黏附受体对NMDAR活性的调节对树突棘的成熟和稳定起着基础性作用，但整合素调节NMDAR的分子机制尚不清楚。我们已经证明，通过ABL2/Arg非受体酪氨酸激酶失去整合素NMDA3？1信号会导致青春期晚期广泛的树突棘丢失，这是由于GluN2B亚单位介导的α受体电流增加所致。我提供了强有力的证据表明，Arg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、神经发育和神经疾病的标志，如精神分裂症、自闭症和阿尔茨海默病。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是如何受损的并制定治疗策略至关重要。在这项提案中，我将检验整合素-Arg-SHP2信号功能调节NMDA受体以控制树突棘稳定性的假设。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是否直接被SHP2去磷酸化，并确定SHP2针对GluN2B中的哪个位点。GluN2B的磷酸化促进其表面定位。因此，我还将利用培养的海马神经元中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表面的表达。我的第二个目标是阐明整合素-NMDA受体的调节机制。GluN2B的磷酸化增加了NMDAR介导的电流，我的初步数据显示，SHP2的激活降低了GluN2B的磷酸化。为了测试SHP2如何调节NMDAR功能，我将测量SHP2活性的丧失或功能恢复如何影响海马片中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α3？1或Arg的小鼠升高的NMDAR电流，来测试SHP2作为整合素-Arg信号和NMDAR之间的机制链接的假设。我的第三个目标是了解SHP2是如何调控树突棘和突触结构的。我们的实验室已经证明整合素-Arg信号减弱了NMDAR的活性以稳定脊柱，我假设SHP2介导了Arg信号对NMDAR功能的影响。我将使用共聚焦显微镜和电子显微镜来定量测量小鼠SHP2活性的丧失和功能恢复如何影响树突棘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由于整合素α3ç1或Arg的丢失而导致的小鼠脊柱不稳定。

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 α3ß1 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 α3ß1 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 α3ß1 or Arg in mice.