Investigation of the adhesion-GPCR BAI2 in excitatory synapse development

我们感兴趣的是突触如何在大脑中形成。这项建议的目的是 研究这一新发现背后的机制，即脑特异性血管生成 抑制物2(BAI2)在培养的神经元中过表达时增加突触数量。BAI2是 粘附性G蛋白偶联受体家族的成员，由一个大的N- 末端胞外(粘连)结构域，然后是七个跨膜GPCR域。 这一新发现扩展了我们发表的在关键基因中发现功能突变的工作 BAI2胞外区在小鼠正向诱变高活性筛选中的作用 异种细胞表面BAI2表达减少。我们的假设是细胞内的 BAI2的C末端对于我们观察到的突触数量的增加是必不可少的。 具体地说，有两个基序--一个与肌动蛋白细胞骨架的关键调节因子结合的结构域 和PDZ结合序列--这可能是早期突触形成的关键介质。我们 将使用分子生物学方法与超分辨率显微镜相结合 在存在或不存在BAI2的情况下分离海马神经元来检验这一假说。 有趣的是，BAI2的一个新的C末端突变导致结构性受体信号转导 在一名患有进行性痉挛瘫痪和其他疾病的人类患者身上发现 症状。因此，这些研究的结果将为深入了解BAI2在突触中的功能提供依据 发展以及导致人类疾病的功能障碍。

We are interested in how synapses form in the brain. The objective of this proposal is to investigate the mechanism underlying the novel finding that Brain-Specific Angiogenesis Inhibitor 2 (BAI2) increases synapse number when overexpressed in cultured neurons. BAI2 is a member of adhesion G-protein coupled receptor (GPCR) family, comprised of a large N- terminal extracellular (adhesive) domain followed by a seven transmembrane GPCR domain. This novel finding extends our published work that identified a functional mutation in a critical extracellular region of BAI2 underlying a forward mutagenesis hyperactivity screen in mice that reduced surface expression of BAI2 in heterologous cells. Our hypothesis is that the intracellular C-terminus of BAI2 is indispensable for the increase in synapse number that we observed. Specifically, there are two motifs—a domain that binds a key regulator of the actin cytoskeleton and a PDZ binding sequence—that may be critical mediators of early synapse formation. We will employ a molecular biology approach coupled with super-resolution microscopy in dissociated hippocampal neurons in the presence or absence of BAI2 to test this hypothesis. Intriguingly, a de novo C-terminal mutation of BAI2 that leads to constitutive receptor signaling was identified in a human patient suffering from progressive spastic paraparesis and other symptoms. Thus, results of these studies will provide insight into BAI2 function in synapse development as well as dysfunction that leads to human disease.