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.
Highlights
The establishment of long-lasting changes in synapse structure and function requires the rapid regulation of spatial and temporal gene expression in response to neural stimulation
We initially focused on genes with annotated functions in the control of axon physiology and/or neurite outgrowth that were found in one of two groups: 1) messenger RNA (mRNA) targeted for co-regulation by miRs-8 and -289 that map to a neuron-related annotation cluster (Table 1); or 2) all mRNAs targeted for co-regulation by miRs-8, -289, and -958 (Table 2)
When the wg reporter was co-transfected with miRs-8, -289, or -958 we found that only miR-8 was capable of repression (Figure 6B; miR-8 = 24%; p,0.0001)
Summary
The establishment of long-lasting changes in synapse structure and function requires the rapid regulation of spatial and temporal gene expression in response to neural stimulation. MiRNAs are abundant small regulatory RNAs that postranscriptionally repress the expression of target mRNAs, usually by binding to sequences in their 39 UTRs. With the exception of the ‘‘seed sequence’’ (positions 2–8 of the miRNA), miRNAs bind to target mRNAs with only partial complementarity. With the exception of the ‘‘seed sequence’’ (positions 2–8 of the miRNA), miRNAs bind to target mRNAs with only partial complementarity This allows each individual miRNA to bind to and, potentially, coordinate or fine-tune the expression of 10s to 100s of target mRNAs [2]. MiRNAs are involved in the control of diverse cellular processes ranging from dendrite spine formation and/or function to the control of synaptic plasticity [3]. Dysregulation of key neuronal miRNA expression is associated with several human neurological disorders [4]
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