microRNA-Mediated Mechanisms Essential for the Structural Plasticity of Drosophila Glutamatergic Synapses

microRNA介导的果蝇谷氨酸突触结构可塑性所必需的机制

• 批准号: 10701428
• 负责人: David L. Van Vactor
• 金额: $ 59.33万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-27 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10701428
• 关键词: Acute; Address; Antibodies; Architecture; Binding; Binding Sites; Biological Assay; Biological Process; Cell physiology; Cells; Code; Communication; Complex; Data; Dendritic Spines; Dependence; Development; Disease; Drosophila genus; Epigenetic Process; Excitatory Synapse; Exocytosis; Family; Gene Expression; Genes; Genetic; Genetic Epistasis; Glutamate Receptor; Glutamates; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Hippocampus (Brain); Human; Integrins; Intracellular Membranes; Lead; Link; Logic; Mammals; Mass Spectrum Analysis; Mediating; Membrane; Memory; Messenger RNA; MicroRNAs; Modeling; Molecular; Morphogenesis; Morphology; Motor Neurons; Mus; Muscle; Mutagenesis; N-Methyl-D-Aspartate Receptors; Nature; Nervous system structure; Neurologic; Neuromuscular Junction; Neurons; Orthologous Gene; Pathway interactions; Peer Review; Phenotype; Phosphotransferases; Predictive Factor; Prefrontal Cortex; Process; Property; Proteins; Proteome; Publications; Publishing; Regulation; Response Elements; Reticulum; Role; Scaffolding Protein; Sequence Analysis; Signal Transduction; Site; Site-Directed Mutagenesis; Specificity; Structure; Synapses; Synaptic Transmission; System; Testing; Tissues; Translations; Untranslated RNA; Work; conditioned fear; fascinate; filamin; fly; genomic tools; in vivo; neural circuit; neuroadaptation; null mutation; postsynaptic; presynaptic; ral Guanine Nucleotide Exchange Factor; recruit; relating to nervous system; response; screening; sensory input; synaptogenesis; tool; tool development; trafficking; transgene expression

PROJECT SUMMARY / ABSTRACT The molecular and cellular mechanisms underlying the plasticity of excitatory synapses have fascinated biologists for many decades. In addition to the importance of these processes in the acquisition and storage of memories, as well as other adaptations of neural circuits to sensory input or other changing conditions, many of the effector genes that participate in such mechanisms have recently been associated with a wide range of neurological, psychiatric and other disorders of the human nervous system. Thus, it is little surprise that synapse formation, plasticity and structural remodeling are under tight control at many levels. To better understand this, we have investigated small, non-coding microRNA genes that serve as versatile yet selective regulators of dynamic gene expression changes that underly the morphological plasticity of the synapse. Through multiple rounds of genetic tool development, screening, and tissue-specific analysis, we have identified several highly conserved microRNAs that are required in the postsynaptic cell to allow coordinated remodeling of the synapse in response to acute stimulation. Because each microRNA controls the expression of specific target mRNAs, our studies have led us to several key proteins whose expression must be downregulated to allow synapse remodeling. In particular, our unpublished analysis of miR-219 suggests that it controls expression of a guanine nucleotide exchange factor (GEF) specific to the Ral GTPase. Although this GEF (dRalGPS) is very highly conserved, there are no peer reviewed publications on the Drosophila ortholog. Moreover, while fly miR-219 is perfectly conserved with human miR-219a, and the miR-219 response element (MRE) in RalGPS is also conserved across species, this relationship has escaped study by other labs. Prior work on Ral at the Drosophila larval neuromuscular junction (NMJ) delineated a postsynaptic pathway that mediates morphogenesis of a dendritic-spine like membrane array called the subsynaptic reticulum (SSR) by recruiting Sec5 and other Exocyst components in response to neural activity. Analysis of our unpublished null mutation, expression transgenes, and antibodies against dRalGPS show that, like Ral, this Ral GEF is both necessary and sufficient to control the postsynaptic recruitment of key determinants of SSR structure. These and other observations described in this proposal suggest a working model where synapse plasticity depends on convergent microRNA regulation of dRalGPS and other effectors to reprogram the synaptic proteome for synapse addition rather than stability. We propose to rigorously test this model with a combination of site-directed mutagenesis and tissue-specific analysis (Aim 1), genetic epistasis and protein localization studies (Aim 2), and thorough regulatory analysis of the target genes to address their dependence on miR-219 and other postsynaptic microRNA activities (Aim 3).

项目摘要 /摘要 兴奋性突触可塑性背后的分子和细胞机制已着迷 生物学家数十年。除了这些过程在获取和存储中的重要性外 记忆以及神经回路对感官输入或其他变化条件的其他改编，许多 参与此类机制的效应基因最近与广泛的 人类神经系统的神经，精神病和其他疾病。因此，突触毫不奇怪 在许多级别上，形成，可塑性和结构重塑受到严格控制。为了更好地理解这一点， 我们研究了小型非编码microRNA基因，这些基因是多功能但选择性调节剂 动态基因表达会改变突触的形态可塑性。通过多个 遗传工具开发，筛查和组织特异性分析的回合，我们已经确定了几个高度 在突触后细胞中需要的保守microRNA允许对突触进行协调的重塑 响应急性刺激。因为每个microRNA都控制特定靶标mRNA的表达，所以 研究使我们达到了几种关键蛋白，必须下调其表达以允许突触 重塑。特别是，我们对miR-219的未发表分析表明，它控制着鸟嘌呤的表达 RAL GTPase特有的核苷酸交换因子（GEF）。虽然这个GEF（dralgps）非常高 保守的，没有关于果蝇直系同源的同行审查的出版物。而且，飞行mir-219是 与人miR-219a完美保守，RALGPS中的miR-219响应元件（MRE）也是 这种关系跨物种保守，已经逃脱了其他实验室的研究。在果蝇的RAL上进行的先前工作 幼虫神经肌肉连接（NMJ）描述了一种突触后途径，该途径介导 招募SEC5和其他外囊肿，像膜阵列一样的树突状蜘蛛称为膜阵列（SSR） 响应神经活动的成分。分析我们未发表的无效突变，表达转基因， 针对Dralgps的抗体表明，与RAL一样，此RAL GEF既需要且足以控制 SSR结构关键决定因素的突触后募集。这些描述的这些和其他观察 提案提出了一个工作模型，突触可塑性取决于收敛的microRNA调节 Dralgps和其他效应子重新编程突触蛋白组，以增加突触的添加而不是稳定性。我们 建议通过位置定向诱变和组织特异性分析的组合严格测试该模型 （AIM 1），遗传性上毒和蛋白质定位研究（AIM 2），以及对目标的彻底调节分析 基因解决它们对miR-219和其他突触后微洋活性的依赖（AIM 3）。