The effect of genotype on receptor expression in human alcoholic brain

Severe long-term alcohol misuse leads to localized brain damage that is prominent in superior frontal cortex but less so in other cortical areas e.g. primary motor. Alcohol dependence is also associated with several genetic markers. GABAA receptor expression differs selectively between alcoholics and controls in a manner that conforms to the pathology, whereas glutamate receptors are much less regionally variable in these subjects. We determined whether genotype differentiated the pharmacology of glutamate-NMDA receptors and the expression GABAA receptor subunits transcripts in a locally appropriate way so as to influence the severity of alcohol-induced brain damage.

Interactions between serotonergic genotype and amino acid transmitter receptor expression in human alcoholics

Serotonin can modulate the activity of neural reward pathways that are strongly implicated in mediating the effects of chronic alcohol misuse, and its treatment, in human subjects. In previous work and as discussed elsewhere at this meeting, we and others have found consistent differences in the parameters of GABA and glutamate receptors, and the expression of their component subunit transcripts and proteins, in areas of the alcoholic brain that are altered by alcoholism. We did not fi nd clear changes in GABA and glutamate transport function in such samples, but a series of microarray analyses showed consistent upregulation of the presynaptic GABA/betaine transporter SLC6A12. Microarray studies showed no signifi cant differences in the expression of transcripts associated with 5HT transmission; however, only a small number of such elements were present on the arrays. Here we partitioned GABAA and NMDA pharmacology, and subunit mRNA and protein expression, measured in samples of frontal and motor cortex obtained at autopsy from alcoholics without comorbid disease, alcoholics with liver cirrhosis, and controls, according to 5HTTLPR (SLC6A4) and 5HT1B (HTR1B) polymorphisms. We found no effect of these genotypes on the expression of GABAA receptor gene products, but there was a signifi cant mRNA Transcript X Area X Group X 5HTTLPR Interaction with NMDA subunit isoform expression measured by Real Time PCR with GAPDH normalization. Further analysis showed the effect to be selective for alcoholics with cirrhosis, to be most marked in the pathologically vulnerable frontal cortex, and to vary with subunit transcript (F2,76 = 6.545, P = 0.002). NR1 expression was most affected, followed by NR2A, with NR2B expression least altered. Pilot data suggest 5HT1B genotype may also modulate NMDA subunit expression. Interactions between amino acid and serotonin transmission may infl uence susceptibility to alcohol dependence or pathogenesis

Down-regulation of central glucocorticoid receptor expression using antisense oligonucleotide technology

Endogenous glucocorticoids and serotonin have been implicated in the pathophysiology of depression, anxiety and schizophrenia. This thesis investigates the potential of downregulating expression of central Type II glucocorticoid receptors (GR) both in vitro and in vivo, with empirically-designed antisense oligodeoxynucleotides (ODN), to characterise GR modulation of 5-HT2A receptor expression using quantitative RT-PCR, Western blot analysis and radioligand binding. The functional consequence of GR downregulation is also determined by measuring 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane hydrochloride (DOI) mediated 5-HT2A receptor specific headshakes. Using a library of random antisense ODN probes, RNAse H accessibility mapping of T7-primed, in vitro transcribed GR mRNA revealed several potential cleavage sites and identified an optimally effect GR antisense ODN sequence of 21-mer length (GRAS5). In vitro efficacy studies using rat C6 glioma cells showed a 56% downregulation in GR mRNA levels and 80% downregulation in GR protein levels. In the same cells a 29% upregulation in 5-HT2A mRNA levels and 32% upregulation in 5-HT2A protein levels was revealed. This confirmed the optimal nature of the GRAS5 sequence to produce marked inhibition of GR gene expression, and also revealed GR modulation of the 50-HT2A receptor subtype in C6 glioma cells to be a tonic repression of receptor expression. The distribution of a fluorescently-labelled GRAS5 ODN was detected in diverse areas of the rat brain after single ICV administration, although this fluorescence signal was not sustained over a period of 5 days. However, fluorescently-labelled GRAS5 ODN, when formulated in polymer microspheres, showed diverse distribution in the brain which was maintained for 5 days following a single ICV administration. This produced no apparent neurotoxic effects on rat behaviour and hypothalamic-pituitary-adrenal (HPA) axis homeostasis. Furthermore, a single polymer microsphere injection ICV proved to be an effective means of delivering antisense ODNs and this was adopted for the in vivo efficacy studies. In vivo characterisation of GRAS5 revealed marked downregulation of GR mRNA in rat brain regions such as the frontal cortex (26%), hippocampus (35%), and hypothalamus (39%). Downregulation of GR protein was also revealed in frontal cortex (67%), hippocampus (76%), and hypothalamus (80%). In the same animals upregulation of 5-HT2A mRNA levels was shown in frontal cortex (13%), hippocampus (7%), and hypothalamus (5%) while upregulation in 5-HT2A protein levels was shown in frontal cortex (21 %). This upregulation in 5-HT2A receptor density as a result of antisense-mediated inhibition of GR was further confirmed by a 55% increase in DOl-mediated 5-HT2A receptor specific headshakes. These results demonstrate that GR is involved in tonic inhibitory regulation of 5-HT2A receptor expression and function in vivo, thus providing the potential to control 5-HT2A-linked disorders through corticosteroid manipulation. These experiments have therefore established an antisense approach which can be used to investigate pharmacological characteristics of receptors.

Cholecystokinin (CCK) and CCK receptor expression by human gliomas:evidence for an autocrine/paracrine stimulatory loop

Cholecystokinin (CCK) is a gut-brain peptide has been described to be able to induce mitosis according to recent studies. Additionally, conflicting data has been published on whether tumours of the central and peripheral nervous system in general, and gliomas in particular, express CCK receptors. In the present in vitro study we employed reverse transcription followed by the polymerase chain reaction (RT-PCR) to investigate whether mRNA for CCK-A and CCK-B receptors as well as CCK peptide itself is present in primary human gliomas and the U-87 MG GBM cell line. The data show that 14/14 (100%) of the primary gliomas exhibited mRNA expression for the CCK peptide gene and the B receptor including the U-87 MG cells, whereas, only 2/14 (14%) showed presence of the CCK-A receptor. The presence of CCK receptors together with CCK peptide expression itself suggests presence of an autocrine loop controlling glioma cell growth. In support of this conclusion, a neutralizing antibody against the CCK peptide exhibited a dose dependent inhibition of cell growth whereas, antagonists to CCK caused a dose depend inhibition of exogenous stimulated glioma cell growth in vitro, via the CCK-B receptor which is PKC activated. Assessment of apoptosis and proteasome activity were undertaken and we report that treatment with CCK antagonists decreased proteasome and increased caspase-3 activity. These data indicate that CCK peptide and CCK-B are abundant in human gliomas and they act to stimulate cell growth in an autocrine manner, primarily via the high affinity CCK-B receptor, which was blocked by antagonists to CCK, perhaps via apoptosis.

Resistin down-regulates insulin receptor expression, and modulates cell viability in rodent pancreatic beta-cells

The adipokine resistin is known to induce insulin resistance in rodent tissues. Increases in adipose tissue mass are known to have a negative effect on pancreatic beta-cell function, although the mechanisms are poorly understood. This study investigated the effects of resistin on insulin secretion, insulin receptor expression and cell viability in pancreatic beta-cells. BTC-6 or BRIN-BD11 cells were treated for 24h with resistin, and insulin receptor expression, insulin secretion and cell viability were measured. Incubation with 40ng/ml resistin caused significant decreases in insulin receptor mRNA and protein expression, but did not affect insulin secretion. At low concentrations, resistin caused significant increases in cell viability. These data implicate resistin as a factor that may regulate beta-cell function/viability, and suggests a potential mechanism by which increased adiposity causes beta-cell dysfunction.

Reduced Androgen Receptor Expression Accelerates the Onset of ERBB2 Induced Breast Tumors in Female Mice

Androgen receptor (AR) is commonly expressed in both the epithelium of normal mammary glands and in breast cancers. AR expression in breast cancers is independent of estrogen receptor alpha (ERα) status and is frequently associated with overexpression of the ERBB2 oncogene. AR signaling effects on breast cancer progression may depend on ERα and ERBB2 status. Up to 30% of human breast cancers are driven by overactive ERBB2 signaling and it is not clear whether AR expression affects any steps of tumor progression in this cohort of patients. To test this, we generated mammary specific Ar depleted mice (MARKO) by combining the floxed allele of Ar with the MMTV-cre transgene on an MMTV-NeuNT background and compared them to littermate MMTV-NeuNT, Arfl/+ control females. Heterozygous MARKO females displayed reduced levels of AR in mammary glands with mosaic AR expression in ductal epithelium. The loss of AR dramatically accelerated the onset of MMTV-NeuNT tumors in female MARKO mice. In this report we show that accelerated MMTV-NeuNT-dependent tumorigenesis is due specifically to the loss of AR, as hormonal levels, estrogen and progesterone receptors expression, and MMTV-NeuNT expression were similar between MARKO and control groups. MMTV-NeuNT induced tumors in both cohorts displayed distinct loss of AR in addition to ERα, PR, and the pioneer factor FOXA1. Erbb3 mRNA levels were significantly elevated in tumors in comparison to normal mammary glands. Thus the loss of AR in mouse mammary epithelium accelerates malignant transformation rather than the rate of tumorigenesis.

The Influence of Age, Cardiorespiratory Fitness, Exercise and Sedentary Behaviours on Circulating Angiogenic Cells and Cell Surface Receptor Expression.

Cardiovascular disease (CVD) is the biggest killer of people in western civilisation. Age is a significant risk factor for the development for CVD, and treatments and therapies to address this increased risk are crucial to quality of life and longevity. Exercise is one such intervention which has been shown to reduce CVD risk. Age is also associated with endothelial dysfunction, reduced angiogenic capabilities, and reduced ability to repair the vessel wall. Circulating angiogenic cells (CACs) are a subset of circulating cells which assist in the repair and growth of the vasculature and in the maintenance of endothelial function. Reductions in these cells are observed in those with vascular disease compared to age-matched healthy controls. Exercise may reduce CVD risk by improvements in number and/or function of these CACs. Data was collected from human volunteers of various ages, cardiorespiratory fitness (CRF) levels and latent viral infection history status to investigate the effects of chronological age, CRF, viral serology and other lifestyle factors, such as sedentary behaviours and exercise on CACs. The levels of CACs in these volunteers were measured using four colour flow cytometry using various monoclonal antibodies specific to cell surface markers that are used to identify specific subsets of these CACs. In addition, the response to acute exercise of a specific subset of these CACs, termed ‘angiogenic T-cells’ (TANG) were investigated, in a group of well-trained males aged 20-40 years, using a strenuous submaximal exercise bout. Advancing age was associated with a decline in various subsets of CACs, including bone marrow-derived CD34+ progenitors, putative endothelial progenitor cells (EPCs) and also TANG cells. Individuals with a higher CRF were more likely to have higher circulating numbers of TANG cells, particularly in the CD4+ subset. CRF did not appear to modulate CD34+ progenitors or EPC subsets. Increasing sitting time was associated with reduction in TANG cells, but after correcting for the effects of fitness, sitting time no longer negatively affected the circulating number of these cells. Acute exercise was a powerful stimulus for increasing the number of TANG cells (140% increase), potentially through an SDF-1:CXCR4-dependent mechanism, but more studies are required to investigate this. Latent CMV infection was associated with higher number of TANG cells (CD8+), but only in 18-40 year old individuals, and not in an older age group (41-65 year old). The significance of this has yet to be understood. In conclusion, advancing age may contribute to increased CVD risk partly due to the observed reductions in angiogenic cells circulating in the peripheral compartment. Maintaining a high CRF may attenuate this CVD reduction by modulating TANG cell number, but potentially not CD34+ progenitor or EPC subsets. Acute exercise may offer a short window for vascular adaptation through the mobilisation of TANG cells into the circulation.

Chemokine Receptor Expression on Normal Blood CD56(+) NK-Cells Elucidates Cell Partners That Comigrate during the Innate and Adaptive Immune Responses and Identifies a Transitional NK-Cell Population

Studies of chemokine receptors (CKR) in natural killer- (NK-) cells have already been published, but only a few gave detailed information on its differential expression on blood NK-cell subsets. We report on the expression of the inflammatory and homeostatic CKR on normal blood CD56(+low) CD16(+) and CD56(+high)  CD16(-/+low) NK-cells. Conventional CD56(+low) and CD56(+high) NK-cells present in the normal PB do express CKR for inflammatory cytokines, although with different patterns CD56(+low) NK-cells are mainly CXCR1/CXCR2(+) and CXCR3/CCR5(-/+), whereas mostly CD56(+high) NK-cells are CXCR1/CXCR2(-) and CXCR3/CCR5(+). Both NK-cell subsets have variable CXCR4 expression and are CCR4(-) and CCR6(-). The CKR repertoire of the CD56(+low) NK-cells approaches to that of neutrophils, whereas the CKR repertoire of the CD56(+high) NK-cells mimics that of Th1(+) T cells, suggesting that these cells are prepared to migrate into inflamed tissues at different phases of the immune response. In addition, we describe a subpopulation of NK-cells with intermediate levels of CD56 expression, which we named CD56(+int) NK-cells. These NK-cells are CXCR3/CCR5(+), they have intermediate levels of expression of CD16, CD62L, CD94, and CD122, and they are CD57(-) and CD158a(-). In view of their phenotypic features, we hypothesize that they correspond to a transitional stage, between the well-known CD56(+high) and CD56(+low) NK-cells populations.

16α-[18F]-fluoro-17ß-oestradiol ([18F]FES): A biomarker for imaging oestrogen receptor expression with positron emission tomography (PET)

International audience

Expression pattern of the cannabinoid receptor genes in the frontal cortex of mood disorder patients and mice selectively bred for high and low fear

Although the endocannabinoid system (ECS) has been implicated in brain development and various psychiatric disorders, precise mechanisms of the ECS on mood and anxiety disorders remain unclear. Here, we have investigated developmental and disease-related expression pattern of the cannabinoid receptor 1 (CB1) and the cannabinoid receptor 2 (CB2) genes in the dorsolateral prefrontal cortex (PFC) of humans. Using mice selectively bred for high and low fear, we further investigated potential association between fear memory and the cannabinoid receptor expression in the brain. The CB1, not the CB2, mRNA levels in the PFC gradually decrease during postnatal development ranging in age from birth to 50 years (r 2 > 0.6 & adj. p < 0.05). The CB1 levels in the PFC of major depression patients were higher when compared to the age-matched controls (adj. p < 0.05). In mice, the CB1, not the CB2, levels in the PFC were positively correlated with freezing behavior in classical fear conditioning (p < 0.05). These results suggest that the CB1 in the PFC may play a significant role in regulating mood and anxiety symptoms. Our study demonstrates the advantage of utilizing data from postmortem brain tissue and a mouse model of fear to enhance our understanding of the role of the cannabinoid receptors in mood and anxiety disorders

Expression of eph-family receptor tyrosine kinases and ephrins in the tadpole of the frog Xenopus laevis, and possible roles in the development of retinotectal topography

Assembling a nervous system requires exquisite specificity in the construction of neuronal connectivity. One method by which such specificity is implemented is the presence of chemical cues within the tissues, differentiating one region from another, and the presence of receptors for those cues on the surface of neurons and their axons that are navigating within this cellular environment.

Connections from one part of the nervous system to another often take the form of a topographic mapping. One widely studied model system that involves such a mapping is the vertebrate retinotectal projection-the set of connections between the eye and the optic tectum of the midbrain, which is the primary visual center in non-mammals and is homologous to the superior colliculus in mammals. In this projection the two-dimensional surface of the retina is mapped smoothly onto the two-dimensional surface of the tectum, such that light from neighboring points in visual space excites neighboring cells in the brain. This mapping is implemented at least in part via differential chemical cues in different regions of the tectum.

The Eph family of receptor tyrosine kinases and their cell-surface ligands, the ephrins, have been implicated in a wide variety of processes, generally involving cellular movement in response to extracellular cues. In particular, they possess expression patterns-i.e., complementary gradients of receptor in retina and ligand in tectum- and in vitro and in vivo activities and phenotypes-i.e., repulsive guidance of axons and defective mapping in mutants, respectively-consistent with the long-sought retinotectal chemical mapping cues.

The tadpole of Xenopus laevis, the South African clawed frog, is advantageous for in vivo retinotectal studies because of its transparency and manipulability. However, neither the expression patterns nor the retinotectal roles of these proteins have been well characterized in this system. We report here comprehensive descriptions in swimming stage tadpoles of the messenger RNA expression patterns of eleven known Xenopus Eph and ephrin genes, including xephrin-A3, which is novel, and xEphB2, whose expression pattern has not previously been published in detail. We also report the results of in vivo protein injection perturbation studies on Xenopus retinotectal topography, which were negative, and of in vitro axonal guidance assays, which suggest a previously unrecognized attractive activity of ephrins at low concentrations on retinal ganglion cell axons. This raises the possibility that these axons find their correct targets in part by seeking out a preferred concentration of ligands appropriate to their individual receptor expression levels, rather than by being repelled to greater or lesser degrees by the ephrins but attracted by some as-yet-unknown cue(s).

Aurora-A expression, hormone receptor status and clinical outcome in hormone related cancers

Aims: We investigated the correlation between protein expression of Aurora-A with hormone receptor expression and clinicopathological parameters in ovarian, breast and prostate cancer.

Exposure to anthrax toxin alters human leucocyte expression of anthrax toxin receptor 1

Anthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen (PA) with one of three reported cell surface toxin receptors (ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 (TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon (IFN)-γ responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection.