Investigating the synaptic pathology of Autism

项目总结 导致自闭症(ASD)风险的基因突变通常发生在构成信号转导网络的基因中 将突触传递与基因表达的下游变化联系起来。然而，动态的网络- 这些复杂和互连的信号网络在正常或疾病状态下的规模行为很差 明白了。这是第一次申请高生产率的R01赠款，该赠款建立了一个量化的多元化 免疫共沉淀或QMI面板用于研究20个成员的蛋白质相互作用网络的动态活性 (PIN)，由谷氨酸受体、支架和信号转导分子组成；基因突变 编码所有目标蛋白在基因上与自闭症有关。使用QMI，我们发现这个PIN 通过改变响应中协调交互模块的组成和强度来编码信息 接收到的信号。此外，我们发现导致自闭症的突变有破坏突触PIN的风险。 通过使它们呈现一种类似于野生型神经元的状态的网络状态 经历了动态平衡的调整。这导致网络响应变化的动态范围减小 对随后的刺激，并导致基础谷氨酸水平的系统水平紊乱(反映在中断 E/I平衡)。在第二个周期中，我们关注的问题是，我们能否使ASD PIN正常化，以及这是否会 正常化与表型的功能挽救相关吗？在目标1中，我们将重点放在通过FYN实现标准化 在突触可塑性下游的FMR1/y小鼠中，我们发现它是一个异常调节的网络中心 投入。初步数据显示，抑制过度活跃的FYN信号可使过度活跃的蛋白质恢复正常 合成和行为；我们建议进行广泛的分子、细胞和行为分析 探讨FYN抑制治疗FMR1缺乏症表型的可能性。在目标2中，我们扩展了我们的 对第二个对突触可塑性至关重要的PIN的网络尺度分析，即mTOR网络。我们用 MTOR的药理和遗传抑制以模拟通过mTOR PIN的信息流 突触可塑性，并确定内稳态伸缩所需的途径的哪些组成部分，在 体内或体外。在目标3中，我们专注于通过操纵突触活动来实现PIN的标准化。我们尝试着-- 测量携带ASD突变的小鼠的突触PIN，并测量这种治疗是否能够恢复正常 PIN活性和神经元进行正常内平衡调节的能力。关键的是，这最后一个实验 将揭示发育机制下游神经元活动水平的改变是否会导致 突触和mTOR信号转导，或者相反，如果信号转导中持续的缺陷是独立的， 或者甚至是致命性的，改变了基础活动水平。总体而言，此次续签将继续我们对 ASD危险基因扰乱突触信号转导的分子网络机制。

PROJECT SUMMARY Genetic mutations that confer autism (ASD) risk often occur in genes that comprise signal transduction networks that link synaptic transmission to downstream changes in gene expression. However, the dynamic, network- scale behavior of these complex and interconnected signaling networks in normal or disease states is poorly understood. This is the first renewal application of a highly productive R01 grant that built a quantitative multiplex co-immunoprecipitation or QMI panel to study the dynamic activity of a 20-member protein interaction network (PIN), consisting of glutamate receptors, scaffolds, and signal transduction molecules; mutations in the genes encoding all target proteins have been genetically linked to autism. Using QMI, we discovered that this PIN encodes information by varying the composition and intensity of modules of coordinated interactions in response to incoming signals. Moreover, we found that mutations that contribute to autism risk disrupt synaptic PINs by causing them to assume a network state that resembles the state of a wildtype neuron that has undergone homeostatic scaling. This results in a reduced dynamic range of the network to change in response to subsequent stimuli, and leads to a systems-level disturbance in basal glutamate tone (as reflected in disrupted E/I balance). In the second cycle we focus on the question of, can we normalize ASD PINs, and will this normalization correlate with functional rescue of phenotypes? In Aim 1, we focus on normalization via FYN kinase, which we identified as a dysregulated network hub in FMR1-/y mice downstream of synaptic plasticity inputs. Preliminary data demonstrate that inhibition of hyperactive FYN signaling normalizes hyperactive protein synthesis and behavior; we propose to perform an extensive battery of molecular, cellular and behavioral assays to investigate the potential of FYN inhibition to treat the phenotypes of FMR1 deficiency. In Aim 2, we extend our network-scale analysis to a second PIN critical to synaptic plasticity, the mTOR network. We use pharmacological and genetic inhibition of mTOR to model information flow through the mTOR PIN during synaptic plasticity, and to establish which components of the pathway are required for homeostatic scaling, in vivo or in vitro. In Aim 3, we focus on PIN normalization by manipulating synaptic activity. We attempt to `un- scale' the synaptic PIN of ASD-mutation-carrying mice, and measure if this treatment is able to restore normal PIN activity and the ability of the neuron to undergo normal homeostatic scaling. Critically, this last experiment will reveal whether altered levels of neuronal activity downstream of developmental mechanisms cause disrupted synaptic and mTOR signal transduction, or conversely if ongoing deficits in signal transduction are independent, or even causative, of altered basal activity levels. Overall, this renewal would continue our investigations into the molecular network mechanisms by which ASD risk genes disrupt synaptic signal transduction.