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

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 研究已经引导我们找到了几种关键蛋白质，它们的表达必须被下调，才能使突触 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）。虽然这个全球环境基金（dRalGPS）非常高， 由于基因保守，目前还没有关于果蝇直系同源物的同行评审出版物。此外，虽然苍蝇miR-219是 与人miR-219 a完全保守，并且RalGPS中的miR-219响应元件（MRE）也是 这种关系在不同物种间都是保守的，其他实验室没有进行过研究。之前在果蝇实验室对Ral的研究 幼虫神经肌肉接头（NMJ）描绘了一个突触后通路，介导的形态发生， 树突棘样膜阵列称为突触下网状结构（SSR），通过募集Sec 5和其他外囊 对神经活动的反应。分析我们未发表的无效突变，表达转基因， 抗dRalGPS的抗体表明，与Ral一样，这种Ral GEF对于控制 SSR结构的关键决定因素的突触后募集。本报告中描述的这些和其他观察结果 一项提案提出了一个工作模型，其中突触可塑性取决于聚合的microRNA调节， dRalGPS和其他效应器重新编程突触蛋白质组用于突触添加而不是稳定性。我们 我建议结合定点诱变和组织特异性分析来严格测试这个模型 (Aim 1），遗传上位性和蛋白定位研究（目的2），并对靶点进行彻底的调控分析 基因，以解决其对miR-219和其他突触后microRNA活性的依赖性（Aim 3）。

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 In particular, our unpublished analysis of miR-219 suggests that it controls expression of a guanine nucleotide exchange factor (GEF) specific to the Ral GTPase.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).