Protein phosphatase 1 isoforms and human de novo mutations in synaptic plasticity

项目摘要 突触可塑性为学习和记忆提供了一个细胞模型， 神经系统疾病中的认知缺陷。蛋白磷酸酶1（PP 1）是突触可塑性的主要调节因子 直接作用于参与突触可塑性的突触和核基质，包括AMPA受体 和CREB。PP 1在神经元中有三种亚型，它们的亚细胞结构不同但相互重叠。 定位和基质。每个PP 1亚型在海马体中高度表达，但它们的独特性 功能还没有研究过。相反，在经典的研究中， PP 1的药理学或肽抑制。 由于PP 1 α和PP 1 γ1的富集，推测它们是调节突触可塑性的主要亚型 在树突棘和与主要支架蛋白的相互作用，而PP 1 β，主要发现在树突棘， 树突轴和索马被认为起次要作用。令人惊讶的是，人类的新生突变最近 在PP 1 β中发现了导致智力残疾和自闭症样行为的基因。 使用基因敲除的方法与floxed转基因小鼠，我研究了敲除个体的影响， PP 1亚型对海马中突触传递和可塑性的影响，在Schaffer侧支-CA 1。我 初步数据表明，PP 1 β的一个新作用是对抗PP 1 γ1，而PP 1 α则起着次要的、多余的作用。 此外，我的初步数据表明PP 1在调节基础突触传递中的作用， 否则会被传统的药理学方法所掩盖。在这个项目中，我将复制和扩展这些 研究结果使用几种方法：电生理学，免疫印迹，形态学分析和成像。 在目标1中，我将研究每个PP 1亚型在调节突触传递和可塑性中的作用， 我们的floxed转基因小鼠，允许PP 1亚型特异性敲除。在目标2中，我将确定 使用遗传替代技术研究人类从头PP 1 β突变对突触传递和可塑性的影响 方法，其中野生型PP 1 β的一个拷贝被突变的PP 1 β取代，如在受影响的人类患者中。 完成这个项目将为我提供必要的知识基础和技术技能，以追求一个 作为一名神经病学的物理学家和科学家取得了成功。此外，这些发现将使我们了解 突触可塑性的分子机制及其与智力残疾的联系。

PROJECT SUMMARY Synaptic plasticity presents a cellular model for learning and memory and provides a framework to study cognitive deficits in neurological disorders. Protein phosphatase 1 (PP1) is a major regulator of synaptic plasticity that acts directly on synaptic and nuclear substrates involved in synaptic plasticity, including AMPA receptors and CREB, respectively. There are three neuronal isoforms of PP1 with different but overlapping subcellular localizations and substrates. Each PP1 isoform is highly expressed in the hippocampus, but their distinct functions have not been studied. Rather, the isoforms have been grouped together in classic studies that use pharmacologic or peptide inhibition of PP1. PP1α and PP1γ1 are assumed to be the primary isoforms regulating synaptic plasticity due to their enrichment in the dendritic spine and interactions with major scaffolding proteins, whereas PP1β, found primarily in the dendritic shaft and soma, is believed to play a minor role. Surprisingly, human de novo mutations were recently discovered in PP1β that cause intellectual disability and autism-like behaviors. Using a genetic knockout approach with floxed transgenic mice, I investigated the effect of knocking out individual PP1 isoforms on synaptic transmission and plasticity in the hippocampus, at Schaffer collateral-CA1. My preliminary data suggest a novel role for PP1β that opposes PP1γ1, while PP1α plays a minor, redundant role. Additionally, my preliminary data demonstrates a role of PP1 in regulating basal synaptic transmission, which is otherwise obscured by classic pharmacological approaches. In this project, I will replicate and expand these findings using several approaches: electrophysiology, immunoblotting, morphological analysis, and imaging. In Aim 1, I will investigate the role of each PP1 isoform in regulating synaptic transmission and plasticity using our floxed transgenic mice, which allow for PP1 isoform-specific knockout. In Aim 2, I will determine the effect of human de novo PP1β mutations on synaptic transmission and plasticity using a genetic replacement approach, in which one copy of wildtype PP1β is replaced with mutated PP1β, as in affected human patients. Completion of this project will provide me with the knowledge base and technical skills necessary to pursue a successful career as a physician-scientist in neurology. Moreover, these findings will inform our understanding of the molecular mechanisms of synaptic plasticity and their connection to intellectual disability.