Abstract

microRNAs (miRNAs) are major regulators of protein synthesis in the brain. A major goal is to identify changes in miRNA expression underlying protein synthesis-dependent forms of synaptic plasticity such as long-term potentiation (LTP). Previous analyses focused on changes in miRNA levels in total lysate samples. Here, we asked whether changes in total miRNA accurately reflect changes in the amount of miRNA bound to Argonaute protein within the miRNA-induced silencing complex (miRISC). Ago2 immunoprecipitation was used to isolate RISC-associated miRNAs following high-frequency stimulation (HFS)-induced LTP in the dentate gyrus of anesthetized rats. Using locked-nucleic acid-based PCR cards for high-throughput screening and independent validation by quantitative TaqMan RT-PCR, we identified differential regulation of Ago2-associated and total miRNA expression. The ratio of Ago2/total miRNA expression was regulated bidirectionally in a miRNA-specific manner and was largely dependent on N-methyl-D-aspartate receptor (NMDA) activation during LTP induction. The present results identify miRNA association with Ago2 as a potential control point in activity-dependent synaptic plasticity in the adult brain. Finally, novel computational analysis for targets of the Ago2-associated miRNAs identifies 21 pathways that are enriched and differentially targeted by the miRNAs including axon guidance, mTOR, MAPK, Ras, and LTP.

Highlights

  • Stable forms of activity-dependent synaptic plasticity require coordinated gene transcription and bursts of protein synthesis and degradation

  • VALIDATION OF ARGONAUTE-2 IMMUNOPRECIPITATION Dentate gyrus lysates were immunoprecipitated with an Ago2specific antibody or control, non-immune IgG, and expression of the brain-specific miRNAs, miR-151, and miR-347, was examined by qPCR. miR-151 and miR-347 levels were enriched more than 100-fold in the Ago2 IP (Figure 1A)

  • HIGH-THROUGHPUT miRNA EXPRESSION PROFILING Brief bursts of high-frequency stimulation (HFS) applied to the medial perforant path of anesthetized rats induced stable long-term potentiation (LTP) of the field EPSP (fEPSP) slope (Figure 2A)

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Summary

Introduction

Stable forms of activity-dependent synaptic plasticity require coordinated gene transcription and bursts of protein synthesis and degradation. The precursor miRNA is exported to the cytoplasm where a second RNase III enzyme, Dicer, generates a mature double-stranded miRNA intermediate. One of these strands, the guide strand, is recognized and bound by the protein Argonaute (Ago), which further functions in recruitment of the multi-protein, miRNA-induced silencing complex (miRISC) (Bartel, 2004). Once assembled on target bound miRNA, the miRISC inhibits protein synthesis by repressing translation, promoting mRNA decay, or some combination of the two processes (Filipowicz et al, 2008; Huntzinger and Izaurralde, 2011; Béthune et al, 2012; Djuranovic et al, 2012)

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