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).

项目概要/摘要 兴奋性突触可塑性的分子和细胞机制令人着迷 几十年来的生物学家。除了这些过程在采集和存储方面的重要性之外， 记忆，以及神经回路对感觉输入或其他变化条件的其他适应，许多 参与此类机制的效应基因最近与广泛的 神经、精神和人类神经系统的其他疾病。因此，突触的出现也就不足为奇了 形成、可塑性和结构重塑在许多层面上都受到严格控制。为了更好地理解这一点， 我们研究了小的、非编码的 microRNA 基因，它们可以作为多功能且选择性的调节因子 动态基因表达变化是突触形态可塑性的基础。通过多个 经过几轮遗传工具的开发、筛选和组织特异性分析，我们已经鉴定了几个高度 突触后细胞中允许协调突触重塑所需的保守 microRNA 对急性刺激作出反应。因为每个 microRNA 控制特定目标 mRNA 的表达，我们的 研究使我们发现了几种关键蛋白质，其表达必须下调才能允许突触 重塑。特别是，我们未发表的 miR-219 分析表明它控制鸟嘌呤的表达 Ral GTPase 特异的核苷酸交换因子 (GEF)。虽然这个独立于 Ras 的 GEF (dRalGPS) 非常高度保守，没有关于果蝇直系同源物的同行评审出版物。此外，虽然 果蝇 miR-219 与人 miR-219a 以及 RalGPS 中的 miR-219 响应元件 (MRE) 完全保守 这种关系在物种间也是保守的，但其他实验室没有研究过这种关系。先前对 Ral 的研究 果蝇幼虫神经肌肉接头（NMJ）描绘了介导形态发生的途径 突触亚网 (SSR) 通过招募 Sec5 和其他 Exocyst 成分来响应神经活动。 对我们未发表的无效突变、表达转基因和 dRalGPS 抗体的分析表明， 与 Ral 一样，Ral GEF 对于控制关键决定因素的突触后募集来说是必要且充分的 SSR结构。然而，我们对蛋白质复合物的生化分离和随后的遗传分析 RalGPS 相关因素表明，除了 sec5 之外，RalGPS 还可能介导多种生物输出。 此外，Argonaut 1 复合物的生化分离表明 miR-219 加载到 miRISC 中具有活性- 依赖，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。我们建议结合站点定向来严格测试该模型 诱变和组织特异性分析（目标 1）、遗传上位性和蛋白质定位研究（目标 2）以及 对靶基因进行彻底的调控分析，以解决其对 miR-219 和其他突触的依赖性 microRNA（目标 3）。