Regulation of Synapse Morphogenesis in Drosophila

描述（由申请人提供）：调节突触形式和功能的分子通路功能障碍导致许多神经系统疾病，包括癫痫、自闭症和精神发育迟滞。我们的目标是探索介导突触发育和形态发生的分子机制。这些基础知识对于我们理解神经系统疾病和未来治疗工具的概念非常重要。使用果蝇神经肌肉接头（NMJ）作为遗传模型系统，我们发现miR- 8是高度保守的miR-200 microRNA（miRNAs）家族的成员，对于突触的正常生长和复杂性是必需的。缺乏miR-8的动物在不同发育阶段显示NMJ缺陷。在幼虫阶段，当NMJ在多种刺激和调控途径的控制下急剧扩张时，肌肉细胞中需要miR-8来促进突触前末梢的生长。我们的分析表明，miR-8是所需的正常结构的细胞基质，定义的突触下网状（SSR）的NMJ，类似于突触后密度标记的PSD-95在哺乳动物中的结构。多重筛选以确定下游效应物表明，miR-8调节涉及突触发生和细胞骨架生物学的几个靶基因的表达。为了更好地定义miR-8下游的机制，我们已经表明，突触后抑制肌动蛋白相关蛋白Enabled（Ena）在控制NMJ生长在晚期幼虫阶段，与本地化的SSR Ena一致的重要作用。Ena被预测为miR-8的直接靶点，并且在细胞运动和细胞连接形成期间控制细胞骨架结构和动力学方面，但其在突触中的作用尚不清楚。初步数据还表明，尽管miR-8在中枢神经系统（CNS）中表达，但其活性在神经元中相对于其他组织受到某种程度的抑制。此外，遗传上位性揭示了miR-8是由限制突触形态发生的关键突触前通路（脆性X智力迟钝基因，FMR 1）的激活诱导的NMJ扩增所需的，这表明突触后miR-8的上游涉及某种类型的跨突触通信。总之，这些发现揭示了一种调节突触发育的迷人机制，以及利用强大且定义明确的模型系统来理解miRNA控制突触形式和功能的逻辑的绝佳机会。然而，需要进行许多额外的研究来确定miR-8在NMJ发挥作用所需的发育、细胞和分子机制。 公共卫生相关性：最近的见解表明，microRNA在突触发育过程中提供了必要的调节机制。控制突触形态发生的分子和通路的功能障碍导致神经系统疾病，如精神发育迟滞和自闭症，然而，我们对上游调控机制的理解是有限的。我们将确定果蝇miR-8促进突触形成的策略，作为更好地定义突触microRNA上游和下游通路逻辑的模型。

DESCRIPTION (provided by applicant): Dysfunction in the molecular pathways that regulate synapse form and function leads to a number of neurological disorders, including epilepsy, autism and mental retardation. Our goal is to explore the molecular machinery that mediates synapse development and morphogenesis. This fundamental knowledge will be important for our understanding of neurological disease and for the conception of future therapeutic tools. Using the Drosophila neuromuscular junction (NMJ) as a genetic model system, we have discovered that miR- 8, a member of the highly conserved miR-200 family of microRNAs (miRNAs), is essential for the normal growth and complexity of the synapse. Animals lacking miR-8 display NMJ defects at different stages of development. During larval stages, when NMJs dramatically expand under control of multiple stimuli and regulatory pathways, miR-8 is required in muscle cells to promote the growth of presynaptic terminals. Our analysis suggests that miR-8 is required for the normal architecture of the cytomatrix which defines subsynaptic reticulum (SSR) of the NMJ, a structure analogous to the postsynaptic density marked by PSD-95 in mammals. Multiple screens to define downstream effectors suggest that miR-8 regulates the expression of several target genes implicated in synaptogenesis and cytoskeletal biology. To better define the mechanisms downstream of miR-8, we have shown that postsynaptic repression of the actin-associated protein Enabled (Ena) plays an important role in controlling NMJ growth in late larval stages, consistent with the localization of Ena to the SSR. Ena is predicted to be a direct target of miR-8, and controls aspects of cytoskeletal structure and dynamics during cell movement and cell junction formation, but its role(s) at the synapse are not understood. Preliminary data also indicate that although miR-8 is expressed in the central nervous system (CNS), its activity is somehow suppressed in neurons relative to other tissues. Moreover, genetic epistasis reveals that miR-8 is required for NMJ expansion induced by activation of a key presynaptic pathway that limits synapse morphogenesis (the Fragile-X Mental Retardation gene, FMR1), suggesting that some type of trans- synaptic communication is involved upstream of postsynaptic miR-8. Together, these findings reveal a fascinating mechanism that regulates synapse development, and a wonderful opportunity to exploit a powerful and well-defined model system to understand the logic of miRNA control over synapse form and function. However, many additional studies will be required to define the developmental, cellular and molecular mechanisms required for miR-8 to exert its effects at the NMJ. PUBLIC HEALTH RELEVANCE: Recent insights reveal that microRNAs provide essential regulatory mechanisms during synapse development. Dysfunction in the molecules and pathways that control synaptic morphogenesis leads to neurological disorders such as mental retardation and autism, however, our understanding of the regulatory mechanisms upstream is limited. We will determine the strategy by which Drosophila miR-8 promotes synapse formation as a model to better define the logic of pathways upstream and downstream of synaptic microRNAs.