Binding of synGAP to PDZ domains of PSD-95 and its role in Intellectual Disability and Autism Spectrum Disorders caused by synGAP haploinsufficiency

SynGAP单倍不足是约2-5%的散发性智力残疾（ID）的原因， 自闭症谱系障碍（ASD）和/或癫痫。SynGAP特异性地位于突触后， 突触后密度（PSD）。它具有加速Ras失活的GTP酶激活（GAP）结构域 和Rap。然而，新的证据，包括我们自己的，表明一个额外的活动，当失去， 对病理学有很大的影响。我们已经证明，SynGAPα1结合所有三个PDZ结构域的 主要PSD支架蛋白PSD-95，并测量了其对每个PDZ结构域的亲和力。SynGAPα1是 其α1亚型在野生型动物中可占据高达15%的PSD 95的PDZ结构域， 从而与其他PDZ结构域配体竞争结合。我们已经证明，在synGAP+/-小鼠（一种动物）中， synGAP单倍不足模型），PSD中synGAP的量减少，而其他synGAP的量减少。 PDZ-结构域结合蛋白增加，包括TARP-2、3、4和8，以及LRRTM 2，两者都是 AMPA型谷氨酸受体（AMPAR）伴侣蛋白。这一组成变化将增加 神经元上突触的兴奋/抑制平衡，并导致异常的脑功能。因此我们 假设synGAP与PSD-95的PDZ结构域结合的减少是ID的主要贡献者， 在SynGAP单倍功能不全中观察到ASD。我们已经发现，通过Ca 2 +/钙调素- 依赖性蛋白激酶II（CaMKII）降低synGAP对PDZ结构域的亲和力。我们推测 synGAP的磷酸化对于LTP诱导早期PSD的重构是重要的。 在目标一，我们将使定量测试的假设，结合synGAP PDZ结构域 通过测量PSD-95的PDZ结构域与PSD-95的PDZ结构域之间的亲和力来调节PSD的组成。 TARP-12、3、4和8、LRRTM 2和NR 2B的羧基末端尾部。我们将表达可溶性融合蛋白 含有每种蛋白质的胞质羧基末端。为了确定每个PDZ结构域的亲和力，我们将 使用Biacore表面等离子体共振检测与重组PDZ结构域的结合， 解决方案”方法，我们完善了与synGAP一起使用。在目标2中，我们将构建计算 MCell中的模型来模拟synGAP和这些蛋白质中的每一种与PSD-95的平衡结合。模型 将利用目标一中测量的参数。我们将构建体外实验模型， 通过将模拟结果与实验结果进行比较来测试它们的概念和参数。然后我们将 在重建的脊柱几何结构内构建空间逼真的模型，以研究空间 脊柱中蛋白质的排列和高密度影响蛋白质之间的结合竞争 到PSD-95在目标三中，我们将使用培养的啮齿类动物神经元来测试以下假设： synGAP驱动原代神经元培养前后PSD组成的急剧变化 LTP的化学诱导。

SynGAP Haploinsufficiency is the cause of ~2-5% of sporadic Intellectual Disability (ID) accompanied by autism spectrum disorder (ASD) and/or epilepsy. SynGAP is specifically located postsynaptically and is located in the postsynaptic density (PSD). It has a GTPase activating (GAP) domain that accelerates inactivation of Ras and Rap. However, new evidence, including our own, suggests an additional activity that, when lost, may contribute significantly to the pathology. We have shown that SynGAPα1 binds to all three PDZ domains of the major PSD scaffold protein, PSD-95, and have measured its affinity for each PDZ domain. SynGAPα1 is abundant in the PSD; its α1 isoform could occupy up to 15% of PSD95's PDZ domains in wild type animals and thus compete with binding of other PDZ-domain ligands. We have shown that in synGAP+/- mice (an animal model for synGAP haploinsufficiency), the amount of synGAP in the PSD is reduced and the amounts of other PDZ-domain binding proteins are increased, including TARP-ɣ2,3,4, and 8, and LRRTM2, both of which are AMPA-type glutamate receptor (AMPAR) chaperone proteins. This change in composition would increase the excitatory/inhibitory balance of synapses onto neurons and contribute to abnormal brain function. Thus, we postulate that reduction of binding of synGAP to PDZ domains of PSD-95 is a major contributor to the ID and ASD observed in SynGAP Haploinsufficiency. We have found that phosphorylation by Ca2+/calmodulin- dependent protein kinase II (CaMKII) decreases the affinity of synGAP for PDZ domains. We postulate that phosphorylation of synGAP is important for reconfiguration of the PSD during early stages of induction of LTP. In Aim One, we will enable quantitative tests of the hypothesis that binding of synGAP to PDZ domains regulates the composition of the PSD by measuring the affinities between PDZ domains of PSD-95 and the carboxyl terminal tails of TARP-ɣ2,3,4, and 8, LRRTM2, and NR2B. We will express soluble fusion proteins containing the cytosolic carboxyl termini of each protein. To determine affinities for each PDZ domain, we will use Biacore surface plasmon resonance detection of binding to recombinant PDZ domains by the “affinity in solution” method that we perfected for use with synGAP. In Aim Two, we will construct computational models in MCell to simulate equilibrium binding of synGAP and each of these proteins to PSD-95. The models will make use of parameters measured in Aim One. We will construct models of in vitro experiments in order to test their concepts and parameters by comparing simulated results to experimental results. We will then construct spatially realistic models within reconstructed spine geometries to study how the spatial arrangement and high densities of proteins in the spine influence competition among the proteins for binding to PSD-95. In Aim Three, we will use cultured rodent neurons to test the hypothesis that phosphorylation of synGAP drives acute changes in the composition of PSDs in primary neuronal cultures before and after chemical induction of LTP.