Differential expression of the neuronal CB1 cannabinoid receptor in the hippocampus of male Ts65Dn Down syndrome mouse model.

Down syndrome (DS) or Trisomy 21 is the most common genetic cause of mental retardation with severe learning and memory deficits. DS is due to the complete or partial triplication of human chromosome 21 (HSA21) triggering gene overexpression and protein synthesis alterations responsible for a plethora of mental and physical phenotypes. Among the diverse brain target systems that affect hippocampal-dependent learning and memory deficit impairments in DS, the upregulation of the endocannabinoid system (ECS), and notably the overexpression of the cannabinoid type-1 receptor (CB1), seems to play a major role. Combining various protein and gene expression targeted approaches using western blot, qRT-PCR and FISH techniques, we investigated the expression pattern of ECS components in the hippocampus (HPC) of male Ts65Dn mice. Among all the molecules that constitute the ECS, we found that the expression of the CB1 is altered in the HPC of Ts65Dn mice. CB1 distribution is differentially segregated between the dorsal and ventral part of the HPC and within the different cell populations that compose the HPC. CB1 expression is upregulated in GABAergic neurons of Ts65Dn mice whereas it is downregulated in glutamatergic neurons. These results highlight a complex regulation of the CB1 encoding gene (Cnr1) in Ts65Dn mice that could open new therapeutic solutions for this syndrome.

New perspectives on the role of the neurosteroid pregnenolone as an endogenous regulator of type-1 cannabinoid receptor (CB1R) activity and function.

Pregnenolone is a steroid with specific characteristics, being the first steroid to be synthesised from cholesterol at all sites of steroidogenesis, including the brain. For many years, pregnenolone was defined as an inactive precursor of all steroids because no specific target had been discovered. However, over the last decade, it has become a steroid of interest because it has been recognised as being a biomarker for brain-related disorders through the development of metabolomic approaches and advanced analytical methods. In addition, physiological roles for pregnenolone emerged when specific targets were discovered. In this review, we highlight the discovery of the selective interaction of pregnenolone with the type-1 cannabinoid receptor (CB1R). After describing the specific characteristic of CB1Rs, we discuss the newly discovered mechanisms of their regulation by pregnenolone. In particular, we describe the action of pregnenolone as a negative allosteric modulator and a specific signalling inhibitor of the CB1R. These particular characteristics of pregnenolone provide a great strategic opportunity for therapeutic development in CB1-related disorders. Finally, we outline new perspectives using innovative genetic tools for the discovery of original regulatory mechanisms of pregnenolone on CB1-related functions.

Serotonin2B receptors in the rat dorsal raphe nucleus exert a GABA-mediated tonic inhibitory control on serotonin neurons.

The central serotonin2B receptor (5-HT2BR) is a well-established modulator of dopamine (DA) neuron activity in the rodent brain. Recent studies in rats have shown that the effect of 5-HT2BR antagonists on accumbal and medial prefrontal cortex (mPFC) DA outflow results from a primary action in the dorsal raphe nucleus (DRN), where they activate 5-HT neurons innervating the mPFC. Although the mechanisms underlying this interaction remain largely unknown, data in the literature suggest the involvement of DRN GABAergic interneurons in the control of 5-HT activity. The present study examined this hypothesis using in vivo (intracerebral microdialysis) and in vitro (immunohistochemistry coupled to reverse transcription-polymerase chain reaction) experimental approaches in rats. Intraperitoneal (0.16 mg/kg) or intra-DRN (1 µM) administration of the selective 5-HT2BR antagonist RS 127445 increased 5-HT outflow in both the DRN and the mPFC, these effects being prevented by the intra-DRN perfusion of the GABAA antagonist bicuculline (100 µM), as well as by the subcutaneous (0.16 mg/kg) or the intra-DRN (0.1 µM) administration of the selective 5-HT1AR antagonist WAY 100635. The increase in DRN 5-HT outflow induced by the intra-DRN administration of the selective 5-HT reuptake inhibitor citalopram (0.1 µM) was potentiated by the intra-DRN administration (0.5 µM) of RS 127445 only in the absence of bicuculline perfusion. Finally, in vitro experiments revealed the presence of the 5-HT2BR mRNA on DRN GABAergic interneurons. Altogether, these results show that, in the rat DRN, 5-HT2BRs are located on GABAergic interneurons, and exert a tonic inhibitory control on 5-HT neurons innervating the mPFC.

Regulation of RNA polymerase III transcription during transformation of human IMR90 fibroblasts with defined genetic elements.

RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain.

Chronic pain states are characterized by long-term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up-regulation of Cav1.2-comprising L-type calcium channel (Cav1.2-LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR-103, simultaneously regulates the expression of the three subunits forming Cav1.2-LTC in a novel integrative regulation. This regulation is bidirectional since knocking-down or over-expressing miR-103, respectively, up- or down-regulate the level of Cav1.2-LTC translation. Functionally, we show that miR-103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR-103 is down-regulated in neuropathic animals and that miR-103 intrathecal applications successfully relieve pain, identifying miR-103 as a novel possible therapeutic target in neuropathic chronic pain.

Tissue microarray cytometry reveals positive impact of homeodomain interacting protein kinase 2 in colon cancer survival irrespective of p53 function.

The human p53 gene is a tumor suppressor mutated in half of colon cancers. Although p53 function appears important for proliferation arrest and apoptosis induced by cancer therapeutics, the prognostic significance of p53 mutations remains elusive. This suggests that p53 function is modulated at a posttranslational level and that dysfunctions affecting its modulators can have a prognostic impact. Among p53 modulators, homeodomain interacting protein kinase (HIPK) 2 emerges as a candidate "switch" governing p53 transition from a cytostatic to a proapoptotic function. Thus, we investigated the possible prognostic role of HIPK2 on a retrospective series of 80 colon cancer cases by setting up a multiplexed cytometric approach capable of exploring correlative protein expression at the single tumor cell level on TMA. Crossing the data with quantitative PCR and p53 gene sequencing and p53 functional assays, we observed the following: despite a strong impact on p21 transcription, the presence of disabling p53 mutations has no prognostic value, and the increased expression of the HIPK2 protein in tumor cells compared with paired normal tissue cells has a strong impact on survival. Unexpectedly, HIPK2 effect does not appear to be mediated by p53 function because it is also observed in p53-disabling mutated backgrounds. Thus, our results point to a prominent and p53-independent role of HIPK2 in colon cancer survival.

Hormonal, hypothalamic and striatal responses to reduced body weight gain are attenuated in anorectic rats bearing small tumors.

Lack of compensatory or even reduced food intake is frequently observed in weight-losing cancer patients and contributes to increased morbidity and mortality. Our previous work has shown increased transcription factor expression in the hypothalamus and ventral striatum of anorectic rats bearing small tumors. mRNA expression of molecules known to be involved in pathways regulating appetite in these structures was therefore assessed in this study. Given that pain, pro-inflammatory cytokines and metabolic hormones can modify food intake, spinal cord cellular activation patterns and plasma concentrations of cytokines and hormones were also studied. Morris hepatoma 7777 cells injected subcutaneously in Buffalo rats provoked a 10% lower body weight and 15% reduction in food intake compared to free-feeding tumor-free animals 4 weeks later when the tumor represented 1-2% of body mass. No differences in spinal cord activation patterns or plasma concentration of pro-inflammatory cytokines were observed between groups. However, the changes in plasma ghrelin and leptin concentrations found in food-restricted weight-matched rats in comparison to ad libitum-fed animals did not occur in anorectic tumor-bearing animals. Real-time PCR showed that tumor-bearing rats did not display the increase in hypothalamic agouti-related peptide mRNA observed in food-restricted weight-matched animals. In addition, microarray analysis and real-time PCR revealed increased ventral striatal prostaglandin D synthase expression in food-restricted animals compared to anorectic tumor-bearing rats. These findings indicate that blunted hypothalamic AgRP mRNA expression, probably as a consequence of relatively high leptin and low ghrelin concentrations, and reduced ventral striatal prostaglandin D synthesis play a role in maintaining cancer-associated anorexia.

p190B RhoGAP regulates endothelial-cell-associated proteolysis through MT1-MMP and MMP2.

The two isoforms of p190 RhoGAP (p190A and p190B) are important regulators of RhoGTPase activity in mammalian cells. Both proteins are ubiquitously expressed, are involved in the same signalling pathways and interact with the same identified binding partners. In search of isoform functional specificity, we knocked down the expression of each p190 protein using siRNA and examined the resulting phenotypic changes in human umbilical vein endothelial cells (HUVECs). We provide evidence that p190B plays a crucial role in the regulation of MT1-MMP expression and cell-surface presentation, as well as subsequent MMP2 activation. p190B is involved in both local extracellular matrix degradation at podosomes and endothelial cell assembly into tube-like structures in Matrigel. In addition, whereas p190B knockdown does not affect podosome formation, p190A knockdown increases the number of cells showing podosome structures in HUVECs. We conclude that the two p190 RhoGAP isoforms play distinct roles in endothelial cells. In addition, our data reveal an unsuspected role for p190B in the expression of the two collaborative proteases MT1-MMP and MMP2, thereby affecting matrix remodelling and angiogenesis.

Tyrosine hydroxylase and dopamine transporter expression in lactotrophs from postlactating rats: involvement in dopamine-induced apoptosis.

Cessation of lactation causes a massive loss of surplus lactotrophs in the rat pituitary gland. The factors and mechanisms involved in this phenomenon have not yet been elucidated. Besides its inhibitory control on prolactin secretion and lactotroph proliferation, evidence suggests that dopamine (DA) may be a proapoptotic factor for lactotrophs. We therefore tested the proapoptotic effect of DA on pituitary glands from virgin, lactating, and postlactating rats. By measuring mitochondrial membrane potential loss, caspase-3 activation, and nuclear fragmentation, we show that DA induces apoptosis specifically in lactotrophs from postlactating rats. We then determined that this effect was partly mediated by the DA transporter (DAT) rather than the D(2) receptor, as corroborated by the detection of DAT expression exclusively in lactotrophs from postlactating rats. We also observed tyrosine hydroxylase (TH) expression in postlactating lactotrophs that was accompanied by an increase in DA content in the anterior pituitary gland of postlactating compared with virgin rats. Finally, we observed that cells expressing TH coexpressed DAT and cleaved caspase-3. These findings show that DA may play a role in lactotroph regression during the postlactation period by inducing apoptosis. The fact that this process requires DAT and TH expression by lactotrophs themselves suggests that it may be "autocrine" in nature.

Discovery of naturally occurring splice variants of the rat histamine H3 receptor that act as dominant-negative isoforms.

We described previously the cDNA cloning of three functional rat histamine H3 receptor (rH3R) isoforms as well as the differential brain expression patterns of their corresponding mRNAs and signaling properties of the resulting rH3A, rH3B, and rH3C receptor isoforms (Mol Pharmacol 59:1-8). In the current report, we describe the cDNA cloning, mRNA localization in the rat central nervous system, and pharmacological characterization of three additional rH3R splice variants (rH3D, rH3E, and rH3F) that differ from the previously published isoforms in that they result from an additional alternative-splicing event. These new H3R isoforms lack the seventh transmembrane (TM) helix and contain an alternative, putatively extracellular, C terminus (6TM-rH3 isoforms). After heterologous expression in COS-7 cells, radioligand binding or functional responses upon the application of various H3R ligands could not be detected for the 6TM-rH3 isoforms. In contrast to the rH3A receptor (rH3AR), detection of the rH3D isoform using hemagglutinin antibodies revealed that the rH3D isoform remains mainly intracellular. The expression of the rH3D-F splice variants, however, modulates the cell surface expression-levels and subsequent functional responses of the 7TM H3R isoforms. Coexpression of the rH3AR and the rH3D isoforms resulted in the intracellular retention of the rH3AR and reduced rH3AR functionality. Finally, we show that in rat brain, the H3R mRNA expression levels are modulated upon treatment with the convulsant pentylenetetrazole, suggesting that the rH3R isoforms described herein thus represent a novel physiological mechanism for controlling the activity of the histaminergic system.