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单倍体不足是~2%-5%的散发性智能障碍(ID)的原因，伴随着 自闭症谱系障碍(ASD)和/或癫痫。SynGAP专门位于突触后，并位于 突触后密度(PSD)。它有一个GTP酶激活(GAP)结构域，可以加速RAS的失活 还有说唱。然而，包括我们自己在内的新证据表明，还有一种额外的活动，一旦丢失，可能 对病理有很大的贡献。我们已经证明SynGAPPDZ1与α的所有三个PDZ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 IS 在PSD中含量丰富；其α1亚型可占据野生动物PSD95‘S PDZ结构域的15% 从而与其他PDZ结构域配体的结合竞争。我们已经证明，在SynGAP+/-小鼠(一种动物)中 SynGAP单倍性不足模型)，PSD中的SynGAP量减少，其他 PDZ结构域结合蛋白增加，包括TARP-ɣ2，3，4和8，以及LRRTM2，两者都是 AMPA型谷氨酸受体(AMPAR)伴侣蛋白。这一成分的变化将增加 突触在神经元上的兴奋性/抑制性平衡，导致大脑功能异常。因此，我们 假设SynGAP与PSD-95的PDZ结构域的结合减少是ID和 在SynGAP单倍体功能不全中观察到ASD。我们发现，钙离子/钙调素的磷酸化- 依赖蛋白激酶II(CaMKII)降低SynGAP与PDZ结构域的亲和力。我们假定 在LTP诱导的早期阶段，SynGAP的磷酸化对于PSD的重新配置是重要的。 在第一个目标中，我们将实现对SynGAP与PDZ结构域结合的假设的定量测试 通过测量PSD-95的PDZ结构域与PSD-95的亲和力来调节PSD的组成 TARP-ɣ2、3、4和8、LRRTM2和NR2B的羧基末端尾巴。我们将表达可溶的融合蛋白 含有每种蛋白质的胞质羧基末端的。为了确定每个PDZ域的亲和力，我们将 用Biacore表面等离子体共振检测与重组PDZ结构域的结合 Solution“方法，我们针对SynGAP的使用进行了完善。在目标二中，我们将构建计算 在Mcell中建立模型，模拟SynGAP和每种蛋白质与PSD-95的平衡结合。模特们 将利用在目标一号中测量的参数。我们将构建体外实验模型，以便 通过将模拟结果与实验结果进行比较来检验它们的概念和参数。到时候我们会的 在重建的脊椎几何图形中构建空间逼真模型，以研究空间 脊椎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。在第三个目标中，我们将使用培养的啮齿动物神经元来验证这样的假设： SynGAP导致原代神经元培养前后PSD组成的急剧变化 LTP的化学诱导。