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

蛋白磷酸酶 1 亚型和人类突触可塑性的从头突变

• 批准号: 10333322
• 负责人: Karl Francis Wilson Foley
• 金额: $ 5.18万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10333322
• 关键词: AMPA Receptors; Affect; Behavior; Belief; Binding; CREB1 gene; Cell model; Cognitive deficits; Complex; Conceptions; Data; Dendritic Spines; Electrophysiology (science); Fluorescence Resonance Energy Transfer; Genes; Genetic; Genotype; Golgi Apparatus; Hippocampus (Brain); Human; Image; Individual; Intellectual functioning disability; Knock-out; Learning; Long-Term Depression; Long-Term Potentiation; LoxP-flanked allele; Mediating; Memory; Minor; Molecular; Morphology; Mutate; Mutation; N-Methyl-D-Aspartate Receptors; Neurology; Neurons; Nuclear; Pathway interactions; Patients; Peptides; Pharmacology; Phenotype; Phosphorylation; Physicians; Play; Protein Dephosphorylation; Protein Inhibition; Protein Isoforms; Protein phosphatase; Proteins; Regulation; Research; Role; Scaffolding Protein; Scientist; Signal Transduction; Stains; Synapses; Synaptic Transmission; Synaptic plasticity; Technical Expertise; Training; Transgenic Mice; Western Blotting; Work; autism spectrum disorder; base; career; de novo mutation; density; fluorescence lifetime imaging; insight; knowledge base; mouse model; nervous system disorder; neuronal cell body; novel; protein function; sensor; synaptic function; transmission process

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.

项目总结 突触可塑性为学习和记忆提供了一个细胞模型，并为研究提供了一个框架 神经性障碍中的认知缺陷。蛋白磷酸酶1(PP1)是突触可塑性的主要调节因子 它直接作用于参与突触可塑性的突触和核底物，包括AMPA受体 和CREB。PP1有三种神经元亚型，具有不同但重叠的亚细胞 本地化和衬底。每种PP1亚型都在海马区高度表达，但其不同的 函数还没有被研究过。相反，这些异构体在经典研究中被归类在一起 PP1的药理或多肽抑制作用。 由于pp1α和pp1γ1的丰富，推测它们是调节突触可塑性的主要异构体 在树突棘中以及与主要支架蛋白的相互作用中，而pp1β主要存在于 树突轴和胞体，据信起次要作用。令人惊讶的是，人类从头开始的突变是最近的 在pp1β中发现的会导致智力残疾和自闭症样行为的基因。 我用转基因小鼠的基因敲除方法，研究了敲除个体的效果。 在Schaffer侧支CA1处，PP1亚型对突触传递和海马可塑性的影响。我的 初步数据表明，pp1β具有与pp1γ1相反的新功能，而pp1α则扮演一个次要的冗余角色。 此外，我的初步数据显示，PP1在调节基础突触传递方面发挥了作用，这是 否则就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 使用几种方法发现：电生理学、免疫印迹、形态分析和成像。 在目标1中，我将研究每种PP1亚型在调节突触传递和可塑性中的作用 我们的转基因小鼠，允许PP1异构体特异性敲除。在目标2中，我将确定影响 使用基因替代技术研究人类从头开始的pp1β突变对突触传递和可塑性的影响 在这种方法中，野生型pp1β的一个拷贝被突变的pp1β取代，就像在受影响的人类患者中一样。 这个项目的完成将为我提供必要的知识基础和技术技能，以追求 作为一名神经学内科科学家，他的职业生涯很成功。此外，这些发现将有助于我们理解 突触可塑性的分子机制及其与智能障碍的联系。