“…2; lower panel). Consistent with the qPCR results, the previously published RNA-Seq studies by Piechota et al [24] showed higher levels of expression of Nr3c2 , Pgr and Esr1 in neurons and higher levels of expression of Nr3c1 in astrocytes. RNA-seq data published by Zhang et al [13] were similar to qPCR results with two differences—astrocytes exhibited higher levels of Nr3c2 , and mRNA of Esr1 was undetected.Fig.…”
Section: Resultssupporting
confidence: 90%
“…Astrocytes showed higher levels of abundance of glucocorticoid receptor ( Nr3c1 ) ( p = 7.4 × 10 −7 ). In addition, to compare mRNA levels between the receptors we used the available RNA-seq data from previous experiments by Piechota et al [24] (Fig. 2; middle panel) and by Zhang et al [13] (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Significant differences in the expression between astrocytes and neurons are indicated by asterisks ( t test, *** p < 0.001). Bar graphs ( bottom left ) summarizing RNA-seq-based measurement of gene expression of steroid nuclear receptors [24]. Bar graphs summarizing RNA-seq-based measurement of gene expression of steroid nuclear receptors as reported by [13].…”
BackgroundThe mechanisms of steroids actions in the brain mainly involve the binding and nuclear translocation of specific cytoplasmic receptors. These receptors can act as transcription factors and regulate gene expression. However, steroid-dependent transcriptional regulation in different types of neural cells is not yet fully understood. The aim of this study was to evaluate and compare transcriptional alterations induced by various steroid receptor agonists in primary cultures of astrocytes and neurons from mouse brain.ResultsWe utilized whole-genome microarrays (Illumina Mouse WG-6) and quantitative PCR analyses to measure mRNA abundance levels. To stimulate gene expression we treated neuronal and astroglial cultures with dexamethasone (100 nM), aldosterone (200 nM), progesterone (200 nM), 5α-dihydrotestosterone (200 nM) and β-Estradiol (200 nM) for 4 h. Neurons were found to exhibit higher levels of expression of mineralocorticoid receptor, progesterone receptor and estrogen receptor 2 than astrocytes. However, higher mRNA level of glucocorticoid receptor mRNA was observed in astrocytes. We identified 956 genes regulated by steroids. In astrocytes we found 381 genes altered by dexamethasone and 19 altered by aldosterone. Functional classification of the regulated genes indicated their putative involvement in multiple aspects of cell metabolism (up-regulated Slc2a1, Pdk4 and Slc45a3) and the inflammatory response (down-regulated Ccl3, Il1b and Tnf). Progesterone, dihydrotestosterone and estradiol did not change gene expression in astrocytes. We found no significant changes in gene expression in neurons.ConclusionsThe obtained results indicate that glial cells might be the primary targets of transcriptional action of steroids in the central nervous system. Substantial changes in gene expression driven by the glucocorticoid receptor imply an important role for the hypothalamic–pituitary–adrenal axis in the hormone-dependent regulation of brain physiology. This is an in vitro study. Hence, the model may not accurately reflect all the effects of steroids on gene expression in neurons in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-017-0352-5) contains supplementary material, which is available to authorized users.
“…2; lower panel). Consistent with the qPCR results, the previously published RNA-Seq studies by Piechota et al [24] showed higher levels of expression of Nr3c2 , Pgr and Esr1 in neurons and higher levels of expression of Nr3c1 in astrocytes. RNA-seq data published by Zhang et al [13] were similar to qPCR results with two differences—astrocytes exhibited higher levels of Nr3c2 , and mRNA of Esr1 was undetected.Fig.…”
Section: Resultssupporting
confidence: 90%
“…Astrocytes showed higher levels of abundance of glucocorticoid receptor ( Nr3c1 ) ( p = 7.4 × 10 −7 ). In addition, to compare mRNA levels between the receptors we used the available RNA-seq data from previous experiments by Piechota et al [24] (Fig. 2; middle panel) and by Zhang et al [13] (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Significant differences in the expression between astrocytes and neurons are indicated by asterisks ( t test, *** p < 0.001). Bar graphs ( bottom left ) summarizing RNA-seq-based measurement of gene expression of steroid nuclear receptors [24]. Bar graphs summarizing RNA-seq-based measurement of gene expression of steroid nuclear receptors as reported by [13].…”
BackgroundThe mechanisms of steroids actions in the brain mainly involve the binding and nuclear translocation of specific cytoplasmic receptors. These receptors can act as transcription factors and regulate gene expression. However, steroid-dependent transcriptional regulation in different types of neural cells is not yet fully understood. The aim of this study was to evaluate and compare transcriptional alterations induced by various steroid receptor agonists in primary cultures of astrocytes and neurons from mouse brain.ResultsWe utilized whole-genome microarrays (Illumina Mouse WG-6) and quantitative PCR analyses to measure mRNA abundance levels. To stimulate gene expression we treated neuronal and astroglial cultures with dexamethasone (100 nM), aldosterone (200 nM), progesterone (200 nM), 5α-dihydrotestosterone (200 nM) and β-Estradiol (200 nM) for 4 h. Neurons were found to exhibit higher levels of expression of mineralocorticoid receptor, progesterone receptor and estrogen receptor 2 than astrocytes. However, higher mRNA level of glucocorticoid receptor mRNA was observed in astrocytes. We identified 956 genes regulated by steroids. In astrocytes we found 381 genes altered by dexamethasone and 19 altered by aldosterone. Functional classification of the regulated genes indicated their putative involvement in multiple aspects of cell metabolism (up-regulated Slc2a1, Pdk4 and Slc45a3) and the inflammatory response (down-regulated Ccl3, Il1b and Tnf). Progesterone, dihydrotestosterone and estradiol did not change gene expression in astrocytes. We found no significant changes in gene expression in neurons.ConclusionsThe obtained results indicate that glial cells might be the primary targets of transcriptional action of steroids in the central nervous system. Substantial changes in gene expression driven by the glucocorticoid receptor imply an important role for the hypothalamic–pituitary–adrenal axis in the hormone-dependent regulation of brain physiology. This is an in vitro study. Hence, the model may not accurately reflect all the effects of steroids on gene expression in neurons in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-017-0352-5) contains supplementary material, which is available to authorized users.
“…All animals were decapitated 2 h after treatment, and brains were then extracted and dissected with needles under a binocular. Drug doses that produce robust and comparable gene expression alterations were selected based on our previous experience [ 2 , 3 , 13 ]. Doses were selected to provide a reasonable comparison of the effects of each drug on the molecular level.…”
Section: Methodsmentioning
confidence: 99%
“…Treatment with psychotropic drugs aims to restore the normal function of the affected circuits; long-term therapeutic benefits may depend on the ability of drugs to restore normal plasticity. Consistent with this notion, commonly used psychotropic drugs robustly induce molecular effects on the brain associated with neuroplastic alterations [ 2 , 3 ].…”
BackgroundThe long-term effects of psychotropic drugs are associated with the reversal of disease-related alterations through the reorganization and normalization of neuronal connections. Molecular factors that trigger drug-induced brain plasticity remain only partly understood. Doublecortin-like kinase 1 (Dclk1) possesses microtubule-polymerizing activity during synaptic plasticity and neurogenesis. However, the Dclk1 gene shows a complex profile of transcriptional regulation, with two alternative promoters and exon splicing patterns that suggest the expression of multiple isoforms with different kinase activities.ResultsHere, we applied next-generation sequencing to analyze changes in the expression of Dclk1 gene isoforms in the brain in response to several psychoactive drugs with diverse pharmacological mechanisms of action. We used bioinformatics tools to define the range and levels of Dclk1 transcriptional regulation in the mouse nucleus accumbens and prefrontal cortex. We also sought to investigate the presence of DCLK1-derived peptides using mass spectrometry. We detected 15 transcripts expressed from the Dclk1 locus (FPKM > 1), including 2 drug-regulated variants (fold change > 2). Drugs that act on serotonin receptors (5-HT2A/C) regulate a subset of Dclk1 isoforms in a brain-region-specific manner. The strongest influence was observed for the mianserin-induced expression of an isoform with intron retention. The drug-activated expression of novel alternative Dclk1 isoforms was validated using qPCR. The drug-regulated isoform contains genetic variants of DCLK1 that have been previously associated with schizophrenia and hyperactivity disorder in humans. We identified a short peptide that might originate from the novel DCLK1 protein product. Moreover, protein domains encoded by the regulated variant indicate their potential involvement in the negative regulation of the canonical DCLK1 protein.ConclusionsIn summary, we identified novel isoforms of the neuroplasticity-related gene Dclk1 that are expressed in the brain in response to psychotropic drug treatments.Electronic supplementary materialThe online version of this article (10.1186/s12868-018-0458-4) contains supplementary material, which is available to authorized users.
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