Department of Biomedicine and K.G. Jebsen Centre for Research on Neuropsychiatric Disorders, University of Bergen, Bergen, Norway.
PLoS One. 2012;7(7):e41688. doi: 10.1371/journal.pone.0041688. Epub 2012 Jul 26.
Expression of activity-regulated cytoskeleton associated protein (Arc) is crucial for diverse types of experience-dependent synaptic plasticity and long-term memory in mammals. However, the mechanisms governing Arc-specific translation are little understood. Here, we asked whether Arc translation is regulated by microRNAs. Bioinformatic analysis predicted numerous candidate miRNA binding sites within the Arc 3'-untranslated region (UTR). Transfection of the corresponding microRNAs in human embryonic kidney cells inhibited expression of an Arc 3'UTR luciferase reporter from between 10 to 70% across 16 microRNAs tested. Point mutation and deletion of the microRNA-binding seed-region for miR-34a, miR-326, and miR-19a partially or fully rescued reporter expression. In addition, expression of specific microRNA pairs synergistically modulated Arc reporter expression. In primary rat hippocampal neuronal cultures, ectopic expression of miR-34a, miR-193a, or miR-326, downregulated endogenous Arc protein expression in response to BDNF treatment. Conversely, treatment of neurons with cell-penetrating, peptide nucleic acid (PNA) inhibitors of miR-326 enhanced Arc mRNA expression. BDNF dramatically upregulated neuronal expression of Arc mRNA and miR-132, a known BDNF-induced miRNA, without affecting expression of Arc-targeting miRNAs. Developmentally, miR-132 was upregulated at day 10 in vitro whereas Arc-targeting miRNAs were downregulated. In the adult brain, LTP induction in the dentate gyrus triggered massive upregulation of Arc and upregulation of miR-132 without affecting levels of mature Arc-targeting miRNAs. Turning to examine miRNA localization, qPCR analysis of dentate gyrus synaptoneurosome and total lysates fractions demonstrated synaptic enrichment relative to small nucleolar RNA. In conclusion, we find that Arc is regulated by multiple miRNAs and modulated by specific miRNA pairs in vitro. Furthermore, we show that, in contrast to miR-132, steady state levels of Arc-targeting miRNAs do not change in response to activity-dependent expression of Arc in hippocampal neurons in vitro or during LTP in vivo.
活性调节细胞骨架相关蛋白（Arc）的表达对于哺乳动物各种类型的经验依赖性突触可塑性和长时记忆至关重要。然而，调控 Arc 特异性翻译的机制还知之甚少。在这里，我们想知道 Arc 翻译是否受 microRNA 调控。生物信息学分析预测，Arc 3'UTR 中有大量候选 microRNA 结合位点。在人胚肾细胞中转染相应的 microRNA，在 16 种测试的 microRNA 中，Arc 3'UTR 荧光素酶报告基因的表达被抑制了 10%至 70%。miR-34a、miR-326 和 miR-19a 的 microRNA 结合种子区的点突变和缺失部分或完全挽救了报告基因的表达。此外，特定 microRNA 对的表达协同调节 Arc 报告基因的表达。在原代大鼠海马神经元培养物中，外源性表达 miR-34a、miR-193a 或 miR-326 可下调 BDNF 处理后内源性 Arc 蛋白的表达。相反，用穿透细胞的肽核酸（PNA）抑制 miR-326 处理神经元可增强 Arc mRNA 的表达。BDNF 可显著上调神经元 Arc mRNA 和 miR-132 的表达，miR-132 是一种已知的 BDNF 诱导的 microRNA，而不影响 Arc 靶向 microRNA 的表达。在发育过程中，miR-132 在体外第 10 天上调，而 Arc 靶向 microRNA 下调。在成年大脑中，齿状回的 LTP 诱导会引发 Arc 的大量上调和 miR-132 的上调，而不影响成熟的 Arc 靶向 microRNA 的水平。进一步研究 microRNA 的定位，通过 qPCR 分析齿状回突触小体和总裂解物部分，显示相对于小核仁 RNA 突触富集。总之，我们发现 Arc 受多种 microRNA 调控，并在体外受特定的 microRNA 对调节。此外，我们发现与 miR-132 不同，在体外海马神经元中 Arc 表达的活性依赖性表达或体内 LTP 过程中，Arc 靶向 microRNA 的稳态水平不会发生变化。
