The D1 dopamine receptor subtype is expressed in the brain, kidney and lymphocytes. D1 receptor function has been extensively studied and the receptor has been shown to modulate a wide range of physiological functions and behaviors. The expression of D1 receptor is known to change during development, disease states and chronic treatment; however, the molecular mechanisms that mediate the changes in D1 receptor expression under these circumstances are not well understood. While previous studies have identified extracellular factors and signaling mechanisms regulating the transcription of D1 receptor gene, very little is known about other regulatory mechanisms that modulate the expression of the D1 receptor gene. Here we report that the D1 receptor is post-transcriptionally regulated during postnatal mouse brain development and in the mouse CAD catecholaminergic neuronal cell line. We demonstrate that this post-transcriptional regulation is mediated by a molecular mechanism involving noncoding RNA. We show that the 1277 bp 3′untranslated region of D1 receptor mRNA is necessary and sufficient for mediating the post-transcriptional regulation. Using deletion and site-directed mutagenesis approaches, we show that the D1 receptor post-transcriptional regulation is specifically mediated by microRNA miR-142-3p interacting with a single consensus binding site in the 1277 bp 3′untranslated region of D1 receptor mRNA. Inhibiting endogenous miR-142-3p in CAD cells increased endogenous D1 receptor protein expression levels. The increase in D1 receptor protein levels was biologically significant as it resulted in enhanced D1 receptor-mediated signaling, determined by measuring the activation of both, adenylate cyclase and, the dopamine- and cAMP-regulated phosphoprotein, DARPP-32. We also show that there is an inverse correlation between miR-142-3p levels and D1 receptor protein expression in the mouse brain during postnatal development. This is the first study to demonstrate that the post-transcriptional regulation of D1 receptor expression is mediated by microRNA-induced translational suppression.
A genomics-based approach to identify pharmacodynamic biomarkers was used for a cyclin-dependent kinase inhibitory drug. R547 is a potent cyclin-dependent kinase inhibitor with a potent antiproliferative effect at pharmacologically relevant doses and is currently in phase I clinical trials. Using preclinical data derived from microarray experiments, we identified pharmacodynamic biomarkers to test in blood samples from patients in clinical trials. These candidate biomarkers were chosen based on several criteria: relevance to the mechanism of action of R547, dose responsiveness in preclinical models, and measurable expression in blood samples. We identified 26 potential biomarkers of R547 action and tested their clinical validity in patient blood samples by quantitative real-time PCR analysis. Based on the results, eight genes (FLJ44342, CD86, EGR1, MKI67, CCNB1, JUN, HEXIM1, and PFAAP5) were selected as dose-responsive pharmacodynamic biomarkers for phase II clinical trials.
Sexual aggression can disrupt processes related to learning as females emerge from puberty into young adulthood. To model these experiences in laboratory studies, we developed SCAR, which stands for Sexual Conspecific Aggressive Response. During puberty, a rodent female is paired daily for 30-min with a sexually-experienced adult male. During the SCAR experience, the male tracks the anogenital region of the female as she escapes from pins. Concentrations of the stress hormone corticosterone were significantly elevated during and after the experience. Moreover, females that were exposed to the adult male throughout puberty did not perform well during training with an associative learning task nor did they learn well to express maternal behaviors during maternal sensitization. Most females that were exposed to the adult male did not learn to care for offspring over the course of 17 days. Finally, females that did not express maternal behaviors retained fewer newly-generated cells in their hippocampus whereas those that did express maternal behaviors retained more cells, most of which would differentiate into neurons within weeks. Together these data support SCAR as a useful laboratory model for studying the potential consequences of sexual aggression and trauma for the female brain during puberty and young adulthood.
In adult mice, repeated cocaine administration induces behavioral sensitization measured as increased horizontal locomotor activity. Cocaine-induced locomotor sensitization has been well characterized in adult mice. In adult animals, the D1 dopamine receptor is important for mediating effects of cocaine. The effect of cocaine on D1 receptor expression and function in preadolescent animals is less understood. The recently described drd1-Enhanced Green Fluorescent Protein (drd1-EGFP) reporter mouse is a useful model for performing such mechanistic studies; however, preadolescent drd1-EGFP mice have not been characterized previously. Here we studied cocaine-induced locomotor sensitization in preadolescent drd1-EGFP reporter mice. We administered 15 mg/kg cocaine three times daily at one hour intervals for seven consecutive days beginning on postnatal day 23 to drd1-EGFP reporter mice and the commonly used C57BL/6 mice. Under this regimen, preadolescent mice of both strains exhibited cocaine-induced locomotor sensitization; however, by day 7 the cocaine-induced locomotor activity in the drd1-EGFP mice was maintained for a longer duration compared to the C57BL/6 mice. The preadolescent drd1-EGFP mice also exhibited elevated basal locomotor activity in a novel environment and had higher D1 and D2 dopamine receptor mRNA levels in the caudate nucleus compared to the C57BL/6 mice. The cocaine-induced locomotor sensitization was not retained when the drd1-EGFP mice were maintained cocaine-free for two weeks suggesting that in preadolescent drd1-EGFP mice the cocaine-induced changes do not persist.
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