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A role for CaV1 and calcineurin signaling in depolarization-induced changes in neuronal DNA methylation.

ABSTRACT: Direct manipulations of neuronal activity have been shown to induce changes in DNA methylation (DNAm), although little is known about the cellular signaling pathways involved. Using reduced representation bisulfite sequencing, we identify DNAm changes associated with moderate chronic depolarization in dissociated rat hippocampal cultures. Consistent with previous findings, these changes occurred primarily in the vicinity of loci implicated in neuronal function, being enriched in intergenic regions and underrepresented in CpG-rich promoter regulatory regions. We subsequently used 2 pharmacological interventions (nifedipine and FK-506) to test whether the identified changes depended on 2 interrelated signaling pathways known to mediate multiple forms of neuronal plasticity. Both pharmacological manipulations had notable effects on the extent and magnitude of depolarization-induced DNAm changes indicating that a high proportion of activity-induced changes are likely to be mediated by calcium entry through L-type CaV1 channels and/or downstream signaling via the calcium-dependent phosphatase calcineurin.

SUBMITTER: Hannon E

PROVIDER: S-EPMC4659419 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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A role for Ca<sub>V</sub>1 and calcineurin signaling in depolarization-induced changes in neuronal DNA methylation.

Hannon Eilis E Chand Annisa N AN Evans Mark D MD Wong Chloe C Y CC Grubb Matthew S MS Mill Jonathan J

Neuroepigenetics 20150625

Direct manipulations of neuronal activity have been shown to induce changes in DNA methylation (DNAm), although little is known about the cellular signaling pathways involved. Using reduced representation bisulfite sequencing, we identify DNAm changes associated with moderate chronic depolarization in dissociated rat hippocampal cultures. Consistent with previous findings, these changes occurred primarily in the vicinity of loci implicated in neuronal function, being enriched in intergenic regio ...[more]

PMID: 26702400

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Project description:Simple Summary When neurons are activated, two waves of gene expression changes occur to achieve neuronal adaptation to their environment. Currently, little is known about the changes in post-transcriptional modifications of neuronal transcripts in response to environmental stimuli. This study aims to investigate the dynamics of m5C modification on neuronal mRNAs upon depolarization using potassium chloride. We found that neuronal mRNAs carrying m5C modifications are enriched for genes with important neuronal functions. The numbers of m5C sites and methylated transcripts increased at 2 h after neuronal depolarization and slightly dropped at 6 h. Interestingly, for differentially methylated genes, a negative correlation between the levels of gene expression and RNA methylation is more prevalent. We anticipate our findings may shed new lights on post-transcriptional regulation in activated neurons, and the transcriptomic plus epi-transcriptomic datasets generated in this study would provide a valuable resource to the scientific community. Abstract Neuronal activity is accomplished via substantial changes in gene expression, which may be accompanied by post-transcriptional modifications including RNA cytosine-5 methylation (m5C). Despite several reports on the transcriptome profiling of activated neurons, the dynamics of neuronal mRNA m5C modification in response to environmental stimuli has not been explored. Here, we provide transcriptome-wide maps of m5C modification, together with gene expression profiles, for mouse cortical neurons at 0 h, 2 h, and 6 h upon membrane depolarization. Thousands of differentially expressed genes (DEGs) were identified during the neuronal depolarization process. In stimulated neurons, the majority of early response genes were found to serve as expression regulators of late response genes, which are involved in signaling pathways and diverse synaptic functions. With RNA bisulfite sequencing data, a union set of 439 m5C sites was identified with high confidence, and approximately 30% of them were shared by neurons at all three time points. Interestingly, over 41% of the m5C sites showed increased methylation upon neuronal activation and were enriched in transcripts coding for proteins with synaptic functions. In addition, a modest negative correlation was observed between RNA expression and methylation. In summary, our study provided dynamic transcriptome-wide landscapes of RNA m5C methylation in neurons, and revealed that mRNA m5C methylation is associated with the regulation of gene expression.

Project description:ObjectiveAn increase of neuronal Cav 1.3 L-type calcium channels (LTCCs) has been observed in various animal models of epilepsy. However, LTCC inhibitors failed in clinical trials of epileptic treatment. There is compelling evidence that paroxysmal depolarization shifts (PDSs) involve Ca2+ influx through LTCCs. PDSs represent a hallmark of epileptiform activity. In recent years, a probable epileptogenic role for PDSs has been proposed. However, the implication of the two neuronal LTCC isoforms, Cav 1.2 and Cav 1.3, in PDSs remained unknown. Moreover, Ca2+ -dependent nonspecific cation (CAN) channels have also been suspected to contribute to PDSs. Nevertheless, direct experimental support of an important role of CAN channel activation in PDS formation is still lacking.MethodsPrimary neuronal networks derived from dissociated hippocampal neurons were generated from mice expressing a dihydropyridine-insensitive Cav 1.2 mutant (Cav 1.2DHP-/- mice) or from Cav 1.3-/- knockout mice. To investigate the role of Cav 1.2 and Cav 1.3, perforated patch-clamp recordings were made of epileptiform activity, which was elicited using either bicuculline or caffeine. LTCC activity was modulated using the dihydropyridines Bay K 8644 (agonist) and isradipine (antagonist).ResultsDistinct PDS could be elicited upon LTCC potentiation in Cav 1.2DHP-/- neurons but not in Cav 1.3-/- neurons. In contrast, when bicuculline led to long-lasting, seizure-like discharge events rather than PDS, these were prolonged in Cav 1.3-/- neurons but not in Cav 1.2DHP-/- neurons. Because only the Cav 1.2 isoform is functionally coupled to CAN channels in primary hippocampal networks, PDS formation does not require CAN channel activity.SignificanceOur data suggest that the LTCC requirement of PDS relates primarily to Cav 1.3 channels rather than to Cav 1.2 channels and CAN channels in hippocampal neurons. Hence, Cav 1.3 may represent a new therapeutic target for suppression of PDS development. The proposed epileptogenic role of PDSs may allow for a prophylactic rather than the unsuccessful seizure suppressing application of LTCC inhibitors.

Dataset Information

A role for CaV1 and calcineurin signaling in depolarization-induced changes in neuronal DNA methylation.

Publications

A role for Ca<sub>V</sub>1 and calcineurin signaling in depolarization-induced changes in neuronal DNA methylation.

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