The miRNA pathway controls rapid changes in activity-dependent synaptic structure at the Drosophila melanogaster neuromuscular junction (miRNA array). Drosophila melanogaster

It is widely accepted that long-term changes in synapse structure and function are mediated by rapid activity-dependent gene transcription and new protein synthesis. A growing amount of evidence suggests that the microRNA (miRNA) pathway plays an important role in coordinating these processes. Despite recent advances in this field, there remains a critical need to identify specific activity-regulated miRNAs as well as their key messenger RNA (mRNA) targets. To address these questions, we used the larval Drosophila melanogaster neuromuscular junction (NMJ) as a model synapse in which to identify novel miRNA-mediated mechanisms that control activity-dependent synaptic growth. First, we developed a screen to identify miRNAs differentially regulated in the larval CNS following spaced synaptic stimulation. Surprisingly, we identified five miRNAs (miRs-1, -8, -289, -314, and -958) that were significantly downregulated by activity. Neuronal misexpression of three miRNAs (miRs-8, -289, and -958) suppressed activity-dependent synaptic growth suggesting that these miRNAs control the translation of biologically relevant target mRNAs. Functional annotation cluster analysis revealed that putative targets of miRs-8 and -289 are significantly enriched in clusters involved in the control of neuronal processes including axon development, pathfinding, and growth. In support of this, miR-8 regulated the expression of a wingless 3’UTR (wg 3’ untranslated region) reporter in vitro. Wg is an important presynaptic regulatory protein required for activity-dependent axon terminal growth at the fly NMJ. In conclusion, our results are consistent with a model where key activity-regulated miRNAs are required to coordinate the expression of genes involved in activity-dependent synaptogenesis. Overall design: larval CNS of UAS-ChR2 x C380-Gal4 following synaptic stimulation

学界普遍认为，突触结构与功能的长期改变由快速的活动依赖型基因转录及新蛋白质合成所介导。越来越多的证据表明，微小RNA（miRNA）通路在协调上述过程中发挥着重要作用。尽管该领域近年来已取得诸多进展，但目前仍亟需鉴定特定的活动调控型miRNA及其关键信使RNA（mRNA）靶标。
为解决上述问题，我们以黑腹果蝇（Drosophila melanogaster）幼虫神经肌肉接头（NMJ）作为模型突触，用于挖掘调控活动依赖型突触生长的新型miRNA介导机制。首先，我们开发了一套筛选体系，用于鉴定经间隔突触刺激后，幼虫中枢神经系统（CNS）中差异表达的miRNA。令人意外的是，我们筛选得到5个表达量被活动显著下调的miRNA（miRs-1、-8、-289、-314及-958）。
对3个miRNA（miRs-8、-289及-958）进行神经元异位表达后，其可抑制活动依赖型突触生长，提示这些miRNA可调控具有生物学相关性的靶标mRNA的翻译过程。功能注释聚类分析显示，miRs-8与miRs-289的推定靶标显著富集于参与神经元过程调控的聚类集合中，涵盖轴突发育、路径导向及生长等过程。
为验证该结论，我们在体外实验中证实，miR-8可调控无翅基因（wg）3'非翻译区（3' untranslated region）报告基因的表达。Wg是果蝇NMJ处活动依赖型轴突终末生长所必需的重要突触前调控蛋白。
综上，本研究结果与下述模型相符：关键的活动调控型miRNA是协调参与活动依赖型突触发生的基因表达所必需的。
整体实验设计：经突触刺激后的UAS-ChR2 × C380-Gal4幼虫中枢神经系统（CNS）