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

synGAP 与 PSD-95 的 PDZ 结构域的结合及其在 synGAP 单倍体不足引起的智力障碍和自闭症谱系障碍中的作用

• 批准号: 10115810
• 负责人: MARY B KENNEDY
• 金额: $ 43.25万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10115810
• 关键词: AMPA Receptors; Acute; Affinity; Animal Model; Animals; Basic Science; Binding; Binding Proteins; Biochemical; Chemicals; Chimeric Proteins; Collaborations; Competitive Binding; Complex; Computer Models; DLG4 gene; Defect; Detection; Disease; Epilepsy; Equilibrium; Experimental Models; Genes; Genetic; Geometry; Glutamate Receptor; Goals; Guanosine Triphosphate Phosphohydrolases; In Situ; In Vitro; Individual; Intellectual functioning disability; Ligands; Measurable; Measures; Memory; Methods; Modeling; Mole the mammal; Molecular; Molecular Chaperones; Mus; Mutate; Mutation; N-Methyl-D-Aspartate Receptors; Neurons; Normal Range; Pathology; Pharmacologic Substance; Phosphorylation; Play; Prosencephalon; Protein Binding Domain; Protein Isoforms; Proteins; Recombinant Proteins; Recombinants; Rodent; Role; Scaffolding Protein; Surface Plasmon Resonance; Synapses; Synaptic plasticity; Tail; Testing; Transfection; Tube; Vertebral column; Work; autism spectrum disorder; brain abnormalities; calmodulin-dependent protein kinase II; density; design; experimental study; improved functioning; in vitro Model; postsynaptic; presynaptic density protein 95; programs; repaired

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.

Syngap单倍不足是造成偶发性智障（ID）约2-5％的原因 自闭症谱系障碍（ASD）和/或癫痫。 Syngap是专门位于突触后的，位于 在突触后密度（PSD）中。它具有GTPase激活（GAP）域，可加速RAS的失活 和说唱。但是，包括我们自己在内的新证据提出了一项额外的活动，当丢失时， 对病理做出了重大贡献。我们已经表明，syngapα1与所有三个PDZ结构域结合 主要的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 PSD丰富；它的α1同工型可以占据野生型动物中PSD95 PDZ域的15％，并且 因此，与其他PDZ结构域配体的结合竞争。我们已经表明，在Syngap +/-小鼠中（动物 Syngap单倍体不足的模型），PSD中的合成量减少，其他的量 PDZ结合蛋白增加了，包括TARP-ɣ2,3,4和8和LRRTM2，这两个都是 AMPA型谷氨酸受体（AMPAR）伴侣蛋白。这种构图的变化将增加 突触到神经元上的兴奋/抑制平衡，并导致脑功能异常。那，我们 假设Syngap与PSD-95的PDZ域的结合减少是ID的主要因素和 在Syngap单倍体弥补中观察到ASD。我们发现Ca2+/钙调蛋白 - 依赖性蛋白激酶II（CAMKII）降低了Syngap对PDZ结构域的亲和力。我们假设 Syngap的磷酸化对于在LTP诱导的早期阶段重新配置PSD很重要。 在目标中，我们将对合成域与PDZ域结合的假设进行定量测试 通过测量PSD-95和PSD-95和 TARP-ɣ2,3,4和8，LRRTM2和NR2B的羧基末端尾巴。我们将表达固体融合蛋白 包含每种蛋白质的胞质羧基末端。为了确定每个PDZ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“我们完善的方法可与Syngap一起使用。在AIM二中，我们将构建计算 MCELL中的模型以模拟Syngap的等效结合，并模拟这些蛋白质与PSD-95的同等结合。模型 将利用AIM ONE中测得的参数。我们将构建体外实验的模型 通过将模拟结果与实验结果进行比较来测试他们的概念和参数。然后我们会 在重建的脊柱几何形状中构建空间逼真的模型，以研究空间 脊柱中的排列和高密度的蛋白质影响蛋白质之间的竞争 到PSD-95。在AIM第三，我们将使用培养的啮齿动物神经元来检验以下假设。 Syngap在原代神经元培养物中和之后的原代神经元培养物中PSD组成的急性变化 LTP的化学诱导。