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

项目摘要/摘要 兴奋性突触可塑性的分子和细胞机制引起了人们的兴趣。 几十年来一直是生物学家。除了这些过程在获取和存储 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 参与这种机制的效应基因最近被认为与一系列广泛的 人类神经系统的神经学、精神病学和其他障碍。因此，突触也就不足为奇了 形成、塑性和结构重塑在多个层面受到严格控制。为了更好地理解这一点， 我们已经研究了小的、非编码的microRNA基因，这些基因充当着多功能但选择性的调控因子。 基因的动态表达改变了突触的形态可塑性。通过多个 经过几轮遗传工具开发、筛选和组织特异性分析，我们已经确定了几个高度 在突触后细胞中需要的保守的microRNA，以允许突触的协调重塑 对急性刺激的反应。因为每个microRNA都控制着特定靶标mRNAs的表达，所以我们的 研究已经引导我们找到了几种关键蛋白质，它们的表达必须下调才能允许突触 改建。特别是，我们未发表的对miR-219的分析表明，它控制鸟嘌呤的表达 Ral GTP酶所特有的核苷酸交换因子。虽然这个全环基金(DRalGPS)非常高 保守地说，目前还没有关于果蝇直系同源基因的同行评议出版物。此外，虽然Fly miR-219是 与人miR-219a完全保守，RalGPS中的miR-219反应元件(MRE)也是 这种关系是跨物种保存的，没有得到其他实验室的研究。之前在果蝇上做的关于Ral的工作 幼虫神经肌肉接头(NMJ)描述了一条突触后通路，介导了 树突棘样膜阵列通过募集Sec5和其他外囊被称为突触下网(SSR) 对神经活动作出反应的组件。分析我们未发表的零突变，表达转基因， 而针对dRalGPS的抗体表明，与ral一样，这一ral全环基金对于控制 突触后招募SSR结构的关键决定因素。这些观察结果以及本文件中描述的其他观察结果 建议建立一种工作模型，其中突触的可塑性依赖于收敛的microRNA对 DRalGPS和其他效应器重新编程突触蛋白质组以增加突触而不是稳定。我们 建议结合定点突变和组织特异性分析对这一模型进行严格测试 (目标1)、遗传上位性和蛋白质定位研究(目标2)，以及对目标的全面调控分析 基因，以解决对miR-219和其他突触后microRNA活动的依赖(目标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 In particular, our unpublished analysis of miR-219 suggests that it controls expression of a guanine nucleotide exchange factor (GEF) specific to the Ral GTPase.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).