Single-Molecule Imaging of Ubiquitination Dynamics in Neurons

神经元泛素化动力学的单分子成像

• 批准号: 10817362
• 负责人: GARY J BASSELL
• 金额: $ 43.04万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10817362
• 关键词: Affect; Biochemical; Biological Assay; Biological Models; Biological Process; Biology; Complement; Complex; Cytoplasmic Granules; DLG4 gene; DNA Sequence Alteration; Defect; Dendrites; Dendritic Spines; Detection; Disease; Endocytosis; Event; FMR1; FMRP; FXR1 gene; Fluorescence; Fractionation; Fragile X Syndrome; Future; GTPBP1 gene; Gene Expression Process; Glutamate Receptor; Health; Human; Image; Impairment; Induced pluripotent stem cell derived neurons; Inherited; Intellectual functioning disability; Knockout Mice; Link; Mass Spectrum Analysis; Mediating; Metabotropic Glutamate Receptors; Methods; Microscopy; Mus; Mutation; Nature; Neurons; Process; Protein Biosynthesis; Proteins; RNA; RNA-Binding Proteins; Reporter; Research; Role; Scaffolding Protein; Stimulus; Synapses; Synaptic plasticity; System; Testing; Treatment Efficacy; Ubiquitination; Venus; Visualization; autism spectrum disorder; cell fixing; density; design; efficacy evaluation; human model; inhibitor; knock-down; link protein; molecular imaging; mouse model; multicatalytic endopeptidase complex; nervous system disorder; new technology; pharmacologic; postsynaptic; prevent; proteostasis; response; single molecule; small hairpin RNA; synaptic function; tool; ubiquitin-protein ligase

The subcellular dynamics of ubiquitination has important roles in many biological processes from gene expression to synaptic function and impairments may directly result and/or contribute to neurological disease. While past studies using biochemical fractionation or microscopy on fixed cells suggest the importance of the ubiquitination-proteasome system (UPS) in synaptic protein homeostasis and neuronal function, the methods used do not allow for the direct visualization of subcellular localization and dynamics of ubiquitination. The transient nature of the ubiquitinated products hinders their detection and prevents the analysis of the rate and how the rate of protein ubiquitination may be affected by synaptic plasticity and/or altered in neurological diseases. We have recently developed a new single molecule method, Single Molecule Ubiquitination Mediated Fluorescence Complementation (SM-UbFC), using split-Venus-based fluorescent reporters to visualize and quantify the subcellular dynamics of ubiquitination in live neurons. This method will be applied to elucidate new mechanisms of synapse and RNA biology in health and disease using cultured mouse cortical neurons and human iPSC derived neurons. Aim-1 tests the hypothesis that plasticity-inducing stimuli regulate ubiquitination of synaptic scaffolding proteins and glutamate receptor subunits directly in dendritic spines, which is altered in fragile x syndrome. We will further develop, optimize and apply SM-UbFC to detect dynamic changes in the ubiquitination of synaptic proteins in mouse and human iPSC derived neurons in response to plasticity inducing stimuli. Aim- 2 tests the hypothesis that the Cdh1-APC E3 ligase complex regulates RNA granules at synapses by ubiquitination of multiple RNA binding proteins, focusing on candidates we have recently identified in a mass spectrometry analysis of the Cdh1 interactome. These include FMRP, FXR1P and Caprin-1. We will further develop, optimize and apply SM-UbFC to detect dynamic changes in the ubiquitination of these RNA binding proteins in RNA granules using mouse cortical neurons and human iPSC derived neurons. We will further test the hypothesis that RNA granule dynamics are altered in FMR1 KO neurons or by disease linked mutations in Caprin-1. The model systems selected in Aims1+2 will uncover new mechanisms of ubiquitination dynamics regulating the postsynaptic density and glutamate receptors (Aim-1) and RNA granules localized to synapses (Aim-2). The SM-UbFC method is anticipated to be useful for future studies to elucidate mechanisms regulating ubiquitination dynamics and understanding how they may go awry in neurological diseases leading to dysregulation of synaptic protein homeostasis. This sensitive method provides a potential tool to assess efficacy of therapeutic strategies to correct defects in ubiquitination dynamics in neurological disease.

泛素化的亚细胞动力学在基因的许多生物过程中具有重要作用 突触功能的表达和损伤可能直接导致和/或促成神经系统疾病。 虽然过去使用生化分级分离或显微镜对固定细胞进行的研究表明了 泛素化蛋白酶体系统（UPS）在突触蛋白稳态和神经元功能中的作用，方法 使用的方法不允许直接可视化亚细胞定位和泛素化动力学。这 泛素化产物的瞬时性质阻碍了它们的检测并妨碍了速率和 蛋白质泛素化率如何受到突触可塑性的影响和/或神经系统改变 疾病。我们最近开发了一种新的单分子方法，单分子泛素化 介导荧光互补 (SM-UbFC)，使用基于分裂金星的荧光报告基因 可视化和量化活神经元中泛素化的亚细胞动力学。该方法将应用于 使用培养的小鼠皮质阐明突触和 RNA 生物学在健康和疾病中的新机制 神经元和人类 iPSC 衍生的神经元。 Aim-1 测试可塑性诱导刺激调节的假设 直接在树突棘中突触支架蛋白和谷氨酸受体亚基的泛素化， 这在脆性 X 综合征中发生了改变。我们将进一步开发、优化和应用SM-UbFC来检测动态 小鼠和人类 iPSC 衍生神经元中突触蛋白泛素化的变化 可塑性诱导刺激。 Aim-2检验Cdh1-APC E3连接酶复合物调节RNA的假设 通过泛素化多种 RNA 结合蛋白在突触中形成颗粒，重点关注我们拥有的候选者 最近在 Cdh1 相互作用组的质谱分析中发现。其中包括 FMRP、FXR1P 和 Caprin-1。我们将进一步开发、优化和应用SM-UbFC来检测动态变化 使用小鼠皮质神经元和人 iPSC 对 RNA 颗粒中的这些 RNA 结合蛋白进行泛素化 派生神经元。我们将进一步检验 FMR1 KO 中 RNA 颗粒动力学发生改变的假设 神经元或 Caprin-1 中与疾病相关的突变。 Aims1+2 中选择的模型系统将揭示新的 泛素化动力学调节突触后密度和谷氨酸受体的机制 (Aim-1) 和定位于突触的 RNA 颗粒 (Aim-2)。 SM-UbFC 方法预计将在未来有用 研究阐明调节泛素化动力学的机制并了解它们如何出错 在导致突触蛋白稳态失调的神经系统疾病中。这种灵敏的方法 提供了一个潜在的工具来评估纠正泛素化缺陷的治疗策略的功效 神经系统疾病的动力学。