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.
The dopamine D1 receptor is centrally involved in mediating the effects of cocaine and is essential for cocaine-induced locomotor sensitization. Changes in D1 receptor expression has been reported in various models of cocaine addiction; however, the mechanisms that mediate these changes in D1 receptor expression are not well understood. Using preadolescent drd1a-EGFP mice and a binge cocaine treatment protocol we demonstrate that the D1 receptor is post-transcriptionally regulated in the caudate-putamen of cocaine-sensitized animal. While cocaine-sensitized mice express high levels of steady state D1 receptor mRNA, the expression of D1 receptor protein is not elevated. We determined that the post-transcriptional regulation of D1 receptor mRNA is rapidly attenuated and D1 receptor protein levels increase within thirty minutes when the sensitized mice are challenged with cocaine. The rapid increase in D1 receptor protein levels requires de novo protein synthesis and correlates with the cocaine-induced hyperlocomotor activity in the cocaine-sensitized mice. The increase in D1 receptor protein levels in the caudate-putamen inversely correlated to the levels of microRNA 142-3p and 382, both of which regulate D1 receptor protein expression. The levels of these two microRNAs decreased significantly within five minutes of cocaine challenge in sensitized mice. The results provide novel insights into the previously unknown rapid kinetics of D1 receptor protein expression which occurs in a time scale that is comparable to the expression of immediate early genes. Furthermore, the results suggests a potential novel role for inherently labile microRNAs in regulating the rapid expression of D1 receptor protein in cocaine-sensitized animals.
The D1 dopamine receptor has been implicated in various addictive behaviors. We have demonstrated that this receptor is post‐transcriptionally regulated (PTR) both in vivo and in vitro. The objective of our study is to determine the molecular mechanisms that mediate D1 receptor PTR. The CAD catecholaminergic cell line expresses endogenous D1 receptor. Upon serum deprivation‐induced differentiation, there is an increase in D1 receptor mRNA expression but not protein. Here we show that the 3′ untranslated region (UTR) is necessary and sufficient for D1 receptor PTR. Deletion and competition studies suggest that the PTR is mediated by a trans‐acting factor interacting within the first 1277 bp of the D1 3'UTR. Dicer knockdown studies indicated a role for micro‐RNAs (miRs). To identity the miRs involved in D1 receptor PTR we compared the global expression profile of all known miRs in non‐differentiated and differentiated CAD cells. Funded by The FM Kirby Foundation and NIH (R03 DA026030) grants to EVK and a PhRMA Foundation Pre‐Doctoral Fellowship to KT.
It is well established that the use of atypical antipsychotics results in weight gain and metabolic diseases such as obesity, type‐2 diabetes and metabolic syndrome. However, the underlying mechanisms for weight gain are not clear. Recent studies have shown that various dopamine receptor subtypes are expressed in the enteric system; however, the physiological role as well the effect of antipsychotics on the enteric dopamine receptors is not well understood. We hypothesized that the dopamine receptors might be involved in the modulation of nutrient transport in the intestine and alteration of this modulation by chronic antipsychotic treatment might contribute towards weight gain. In particular, excessive and chronic intake of fructose is known to adversely affect long‐term health and lead to obesity, type‐2 diabetes and metabolic syndrome. The intestinal fructose transporter GLUT5 is considered as a biomarker of excessive fructose consumption. In this project, we test the hypothesis that antipsychotics acting at D2‐like dopamine receptors modulate the expression or function of intestinal GLUT5 transporters, resulting in weight gain. To test this hypothesis, we compared the ability of clozapine (CLZ) to induce weight gain in wild type C57BL/6 mice and GLUT5‐null mice. Preliminary results showed that CLZ dose‐dependently induced weight gain in the wild type C57BL/6 mice but not in the GLUT5‐null mice. In addition, there was a dose‐dependent increase in fructose uptake in the C57BL/6 wild type mice treated with CLZ. The expression of GLUT5 mRNA was not altered by CLZ treatment suggesting that the increase in fructose uptake induced by CLZ might be mediated by an increase in GLUT5 activity. The results suggest that modulation of intestinal nutrient transport by antipsychotics might contribute to weight gain.
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