The miRNA Pathway Controls Rapid Changes in Activity-Dependent Synaptic Structure at the Drosophila melanogaster Neuromuscular Junction

Abstract

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.

Transmission, Development, and Plasticity of Synapses

“…mir-8 is postsynaptic and regulates levels of the effector protein Enabled (Ena), an actin regulator (Loya et al 2009(Loya et al , 2014. mir-8 was also identified in a screen for miRNAs that are differentially regulated by activity (Nesler et al 2013). This same study demonstrated that mir-8 regulates wg expression in vitro, supporting a role for mir-8 in synaptic plasticity.…”

Presynaptic CamKII regulates activity-dependent axon terminal growth

“…Similarly, we have shown that spaced stimulation rapidly downregulates levels of five miRNAs in Drosophila larval ventral ganglia (Nesler et al, 2013). We demonstrated that three of these miRNAs (miRs-8, -289, and -958) control rapid presynaptic bouton growth at the larval NMJ.…”

“…Stocks were obtained from the following sources: Canton-S, w 1118 (Iso31), UAS-CamKII Ala , UAS-CamKII R3(WT) , UAS-CamKII T287A , UAS-CamKII T287D , and C380-Gal4 (Bloomington Drosophila Stock Center); UAS-CamKII v38930 and UAS-CamKII v47280 long hairpin RNAi lines (Vienna Drosophila Resource Center) (Dietzl et al, 2007); UAS-CamKII:EYFP-CamKII 3’UTR and UAS-CamKII:EYFP-NUT 3’UTR were gifts from S. Kunes (Ashraf et al, 2006); UAS-mCherry: miR-289 pri-miRNA was from Barbee lab stocks (Nesler et al, 2013). …”

“…The acute spaced synaptic depolarization assay was done exactly as we have previously described (Nesler et al, 2013). Where indicated, cyclohexamide (100 mM) was added to normal HL3 haemolymph-like dissection buffer during the entire rest phase.…”

“…While several other signaling mechanisms have been implicated in this process (Ataman et al, 2008; Koon et al, 2011; Korkut et al, 2009; Korkut et al, 2013) little is known about the role of presynaptic CamKII. Furthermore, even less is known about the upstream mechanisms that are involved in the control of activity-dependent presynaptic bouton outgrowth and, more specifically, precisely how these upstream mechanisms are linked to local presynaptic signaling events (Freeman et al, 2011; Nesler et al, 2013; Pradhan et al, 2012). …”