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).

项目总结/摘要 兴奋性突触可塑性的分子和细胞机制已经吸引了 生物学家几十年来除了这些过程在获取和存储的重要性之外， 记忆，以及神经回路对感官输入或其他变化条件的其他适应，许多 参与这种机制的效应基因最近已经与广泛的 神经、精神和人类神经系统的其它病症。因此，突触 形成、可塑性和结构重塑在许多水平上受到严格控制。为了更好地理解这一点， 我们已经研究了小的非编码microRNA基因，这些基因作为多功能但选择性的调节因子， 动态基因表达变化是突触形态可塑性的基础。通过多 经过一轮又一轮的遗传工具开发、筛选和组织特异性分析，我们已经确定了几个高度 突触后细胞中允许突触协调重塑所需的保守microRNA 对急性刺激的反应。由于每个microRNA控制特定靶mRNA的表达，我们的 研究让我们找到了几种关键蛋白质，它们的表达必须下调才能形成突触 重塑特别是，我们未发表的对miR-219的分析表明，它控制着鸟嘌呤的表达， Ral GTP酶特异性的核苷酸交换因子（GEF）。尽管这个独立于RAS的全球环境基金（dRalGPS） 是非常高度保守的，没有关于果蝇直系同源物的同行评议出版物。此外，虽然 果蝇miR-219与人miR-219 a完全保守，并且RalGPS中的miR-219应答元件（MRE） 在物种间也是保守的，这种关系已经逃脱了其他实验室的研究。之前在Ral上的工作 果蝇幼虫神经肌肉接头（NMJ）描绘了一条介导形态发生的通路， 通过募集Sec 5和其他外囊组分来响应神经活动，从而调节突触下网状结构（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， 与Ral一样，Ral GEF对于控制关键决定因子的突触后募集是必要的，也是充分的 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相关的因素表明，RalGPS可能介导几个生物输出，除了sec 5。 此外，Argonaut 1复合物的生物化学分离表明，miR-219加载到miRISC中是活性的。 依赖性，暗示这种细胞骨架效应器途径是尚未发现的急性突触可塑性机制的一部分 在我们的系统中学习。我们建议严格测试这个模型与网站导向的组合， 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，以解决它们对miR-219和其他突触的依赖性。 microRNA（Aim 3）。