microRNA-Regulated Mechanisms Essential for Structural Plasticity of Drosophila Glutamatergic Synapses

microRNA 调控机制对于果蝇谷氨酸突触的结构可塑性至关重要

• 批准号: 10792326
• 负责人: David L. Van Vactor
• 金额: $ 52.75万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-26 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10792326
• 关键词: Actins; Acute; Address; Antibodies; Architecture; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Biological Process; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communication; Complex; Cues; Cytoskeleton; Data; Dendritic Spines; Dependence; Development; Disease; Drosophila genus; Endocytosis; Excitatory Synapse; Exocytosis; Experimental Genetics; Family; Gene Expression; Genes; Genetic; Genetic Epistasis; Glutamate Receptor; Glutamates; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Hippocampus; Human; 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; Neurologic; Neuromuscular Junction; Neurons; Orthologous Gene; Output; Pathway interactions; Peer Review; Phenotype; Phosphotransferases; Predictive Factor; Prefrontal Cortex; Process; Property; Proteins; Publications; Response Elements; Reticulum; Role; Sequence Analysis; Signal Transduction; Site-Directed Mutagenesis; Specificity; Structure; Synapses; Synaptic Transmission; Synaptic plasticity; System; Testing; Tissues; Translations; Untranslated RNA; Vertebral column; Work; conditioned fear; fascinate; fly; gene conservation; genetic analysis; genomic tools; in vivo; neural; neural circuit; neuroadaptation; novel; null mutation; postsynaptic; presynaptic; protein complex; ral Guanine Nucleotide Exchange Factor; recruit; response; screening; sensory input; synaptogenesis; tool development; trafficking; transgene expression

PROJECT SUMMARY / ABSTRACT The molecular and cellular mechanisms underlying 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 Ras-independent 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 pathway that mediates morphogenesis 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. However, our biochemical isolation of protein complexes and subsequent genetic analysis of RalGPS-associated factors suggests that RalGPS may mediate several biological outputs in addition to sec5. Moreover, biochemical isolation of Argonaut 1 complexes suggests that miR-219 loading into miRISC is activity- dependent, implicating this cytoskeletal effector pathway is part of an acute synapse plasticity mechanism yet to be studied in our system. 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 synaptic microRNA (Aim 3).

项目摘要 /摘要 兴奋性突触可塑性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）。尽管这种与RAS无关的GEF（Dralgps） 非常保守，没有关于果蝇直系同源的同行评审出版物。而且， Fly miR-219与人miR-219a完美保守，而miR-219响应元件（MRE）在ralgps中 在整个物种之间也是保守的，这种关系逃脱了其他实验室的研究。在RAL上的事先工作 果蝇幼虫神经肌肉连接（NMJ）描述了介导形态发生的途径 通过募集SEC5和其他外囊肿成分来响应神经活动，次突触网（SSR）。 对我们未发表的无效突变，表达转基因和针对Dralgps的抗体的分析表明， 像RAL一样，此RAL GEF既需要且足以控制关键决定因素的突触后募集 SSR结构。然而，我们的蛋白质复合物的生化分离以及随后的遗传分析 RALGPS相关的因子表明，除SEC5外，Ralgps还可以介导几种生物输出。 此外，Argonaut 1配合物的生化分离表明，将MiR-219载入Mirisc是活性 - 依赖性，暗示这种细胞骨架效应途径是急性突触可塑性机制的一部分 在我们的系统中进行研究。我们建议用以网站定向的组合进行严格测试此模型 诱变和组织特异性分析（AIM 1），遗传上毒和蛋白质定位研究（AIM 2）和 对目标基因的彻底调节分析，以解决其对miR-219和其他突触的依赖性 microRNA（目标3）。