Abstract
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.
Highlights
Mounting evidence supports a role for Arc as a vertebratespecific gene specialized for mediating activity-dependent synaptic plasticity [1,2,3,4]
Arc synthesis is required for different forms of protein synthesis-dependent synaptic plasticity including long-term potentiation (LTP), long-term depression (LTD), and homeostatic plasticity, as well as postnatal development of the visual cortex and multiple forms of long-term memory [5,6,7,8,9]
A panel of 7 microRNAs predicted not to bind the Arc 39UTR was used as a normalization control
Summary
Mounting evidence supports a role for Arc as a vertebratespecific gene specialized for mediating activity-dependent synaptic plasticity [1,2,3,4]. Arc synthesis is required for different forms of protein synthesis-dependent synaptic plasticity including long-term potentiation (LTP), long-term depression (LTD), and homeostatic plasticity, as well as postnatal development of the visual cortex and multiple forms of long-term memory [5,6,7,8,9]. Animal disease models link dysregulation of Arc protein expression to Angelman mental retardation syndrome, Alzheimer’s disease, syndromic autism, seizure development, and anxiety-like behavior [10,11,12,13,14]. The present study sought to elucidate a possible role for microRNAs as modulators of Arc
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