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Early phase of plasticity-related gene regulation and SRF dependent transcription in the hippocampus.

ABSTRACT: Hippocampal organotypic cultures are a highly reliable in vitro model for studying neuroplasticity: in this paper, we analyze the early phase of the transcriptional response induced by a 20 µM gabazine treatment (GabT), a GABA-Ar antagonist, by using Affymetrix oligonucleotide microarray, RT-PCR based time-course and chromatin-immuno-precipitation. The transcriptome profiling revealed that the pool of genes up-regulated by GabT, besides being strongly related to the regulation of growth and synaptic transmission, is also endowed with neuro-protective and pro-survival properties. By using RT-PCR, we quantified a time-course of the transient expression for 33 of the highest up-regulated genes, with an average sampling rate of 10 minutes and covering the time interval [10?90] minutes. The cluster analysis of the time-course disclosed the existence of three different dynamical patterns, one of which proved, in a statistical analysis based on results from previous works, to be significantly related with SRF-dependent regulation (p-value<0.05). The chromatin immunoprecipitation (chip) assay confirmed the rich presence of working CArG boxes in the genes belonging to the latter dynamical pattern and therefore validated the statistical analysis. Furthermore, an in silico analysis of the promoters revealed the presence of additional conserved CArG boxes upstream of the genes Nr4a1 and Rgs2. The chip assay confirmed a significant SRF signal in the Nr4a1 CArG box but not in the Rgs2 CArG box.

SUBMITTER: Iacono G

PROVIDER: S-EPMC3720722 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Early phase of plasticity-related gene regulation and SRF dependent transcription in the hippocampus.

Iacono Giovanni G Altafini Claudio C Torre Vincent V

PloS one 20130723 7

Hippocampal organotypic cultures are a highly reliable in vitro model for studying neuroplasticity: in this paper, we analyze the early phase of the transcriptional response induced by a 20 µM gabazine treatment (GabT), a GABA-Ar antagonist, by using Affymetrix oligonucleotide microarray, RT-PCR based time-course and chromatin-immuno-precipitation. The transcriptome profiling revealed that the pool of genes up-regulated by GabT, besides being strongly related to the regulation of growth and syna ...[more]

PMID: 23935853

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Project description: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.

Dataset Information

Early phase of plasticity-related gene regulation and SRF dependent transcription in the hippocampus.

Publications

Early phase of plasticity-related gene regulation and SRF dependent transcription in the hippocampus.

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