microRNA-Regulated Mechanisms Essential for 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。尽管这个独立于RAS的全球环境基金(DRalGPS) 是非常保守的，目前还没有关于果蝇直系同源物的同行评议的出版物。此外，虽然 Fly miR-219与人miR-219a和RalGPS中的miR-219响应元件(MRE)完全保守 在物种间也是保守的，这种关系没有得到其他实验室的研究。在此之前，在 果蝇幼虫神经肌肉接头(NMJ)描述了一条介导形态发生的途径 通过招募Sec5和其他外囊成分来响应神经活动而激活突触下网(SSR)。 对我们未发表的零突变、表达转基因和抗dRalGPS抗体的分析表明， 与Ral一样，这一Ral gef对于控制突触后关键决定因素的招募既是必要的也是充分的 SSR结构。然而，我们对蛋白质复合体的生化分离和随后的遗传分析 RalGPS相关因子表明，RalGPS除了调节Sec5外，还可能调节几种生物输出。 此外，对ArgAert 1复合体的生化分离表明，miR-219负载到miRISC中具有活性- 依赖于，暗示这种细胞骨架效应通路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。我们建议使用站点定向的组合来严格测试该模型 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 对靶基因进行全面的调控分析，以解决它们对miR-219和其他突触的依赖 MicroRNA(目标3)。

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 In particular, our unpublished analysis of miR-219 suggests that it controls expression of a guanine nucleotide exchange factor (GEF) specific to the Ral GTPase.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