Effects of sodium arsenite on neurite outgrowth and glutamate AMPA receptor expression in mouse cortical neurons.

There has been broad concern that arsenic in the environment exerts neurotoxicity. To determine the mechanism by which arsenic disrupts neuronal development, primary cultured neurons obtained from the cerebral cortex of mouse embryos were exposed to sodium arsenite (NaAsO2) at concentrations between 0 and 2μM from days 2 to 4 in vitro and cell survival, neurite outgrowth and expression of glutamate AMPA receptor subunits were assessed at day 4 in vitro. Cell survival was significantly decreased by exposure to 2μM NaAsO2, whereas 0.5μM NaAsO2 increased cell survival instead. The assessment of neurite outgrowth showed that total neurite length was significantly suppressed by 1μM and 2μM NaAsO2, indicating that the lower concentration of NaAsO2 impairs neuritogenesis before inducing cell death. Immunoblot analysis of AMPA receptor subunit expression showed that the protein level of GluA1, a specific subunit of the AMPA receptor, was significantly decreased by 1μM and 2μM NaAsO2. When immunocytochemistry was used to confirm this effect by staining for GluA1 expression in neuropeptide Y neurons, most of which contain GluA1, GluA1 expression in neuropeptide Y neurons was found to be significantly suppressed by 1μM and 2μM NaAsO2 but to be increased at the concentration of 0.5μM. Finally, to determine whether neurons could be rescued from the NaAsO2-induced impairment of neuritogenesis by compensatory overexpression of GluA1, we used primary cultures of neurons transfected with a plasmid vector to overexpress either GluA1 or GluA2, and the results showed that GluA1/2 overexpression protected against the deleterious effects of NaAsO2 on neurite outgrowth. These results suggest that the NaAsO2 concentration inducing neurite suppression is lower than the concentration that induces cell death and is the same as the concentration that suppresses GluA1 expression. Consequently, the suppression of GluA1 expression by NaAsO2 seems at least partly responsible for neurite suppression induced by NaAsO2.

Mono-allelic Ly49 NK cell receptor expression.

Each cell is equipped with two copies (alleles) of each autosomal gene. While the vast majority use both alleles, occasional genes are expressed from a single allele. The reason for mono-allelic expression is not always evident and can serve distinct purposes. First, it may facilitate the tight control over the dosage of certain gene products such as some growth factors and their receptors or X-linked genes. Second, the differential usage of the two parental alleles may reflect the mechanisms that ensure mono-specificity, e.g. olfactory receptors, T and B cell receptors. The context of allele-specific expression of the murine Ly49 natural killer (NK) cell receptor genes suggests that their allele-specific expression reflects a process that generates clonal variability.

Consequences of sleep deprivation on neurotransmitter receptor expression and function.

Several pieces of evidence suggest that sleep deprivation causes marked alterations in neurotransmitter receptor function in diverse neuronal cell types. To date, this has been studied mainly in wake- and sleep-promoting areas of the brain and in the hippocampus, which is implicated in learning and memory. This article reviews findings linking sleep deprivation to modifications in neurotransmitter receptor function, including changes in receptor subunit expression, ligand affinity and signal transduction mechanisms. We focus on studies using sleep deprivation procedures that control for side-effects such as stress. We classify the changes with respect to their functional consequences on the activity of wake-promoting and/or sleep-promoting systems. We suggest that elucidation of how sleep deprivation affects neurotransmitter receptor function will provide functional insight into the detrimental effects of sleep loss.

Swimming prevents vulnerable atherosclerotic plaque development in hypertensive 2-kidney, 1-clip mice by modulating angiotensin II type 1 receptor expression independently from hemodynamic changes.

Exercise is known to reduce cardiovascular risk. However, its role on atherosclerotic plaque stabilization is unknown. Apolipoprotein E(-/-) mice with vulnerable (2-kidney, 1-clip: angiotensin [Ang] II-dependent hypertension model) or stable atherosclerotic plaques (1-kidney, 1-clip: Ang II-independent hypertension model and normotensive shams) were used for experiments. Mice swam regularly for 5 weeks and were compared with sedentary controls. Exercised 2-kidney, 1-clip mice developed significantly more stable plaques (thinner fibrous cap, decreased media degeneration, layering, macrophage content, and increased smooth muscle cells) than sedentary controls. Exercise did not affect blood pressure. Conversely, swimming significantly reduced aortic Ang II type 1 receptor mRNA levels, whereas Ang II type 2 receptor expression remained unaffected. Sympathetic tone also significantly diminished in exercised 2-kidney, 1-clip mice compared with sedentary ones; renin and aldosterone levels tended to increase. Ang II type 1 downregulation was not accompanied by improved endothelial function, and no difference in balance among T-helper 1, T-helper 2, and T regulatory cells was observed between sedentary and exercised mice. These results show for the first time, in a mouse model of Ang II-mediated vulnerable plaques, that swimming prevents atherosclerosis progression and plaque vulnerability. This benefit is likely mediated by downregulating aortic Ang II type 1 receptor expression independent from any hemodynamic change. Ang II type 1 downregulation may protect the vessel wall from the Ang II proatherogenic effects. Moreover, data presented herein further emphasize the pivotal and blood pressure-independent role of Ang II in atherogenesis.

The anorexigenic effects of metformin involve increases in hypothalamic leptin receptor expression.

Metformin demonstrates anorectic effects in vivo and inhibits neuropeptide Y expression in cultured hypothalamic neurons. Here we investigated the mechanisms implicated in the modulation of feeding by metformin in animals rendered obese by long-term high-fat diet (diet-induced obesity [DIO]) and in animals resistant to obesity (diet resistant [DR]). Male Long-Evans rats were kept on normal chow feeding (controls) or on high-fat diet (DIO, DR) for 6 months. Afterward, rats were treated 14 days with metformin (75 mg/kg) or isotonic sodium chloride solution and killed. Energy efficiency, metabolic parameters, and gene expression were analyzed at the end of the high-fat diet period and after 14 days of metformin treatment. At the end of the high-fat diet period, despite higher leptin levels, DIO rats had higher levels of hypothalamic neuropeptide Y expression than DR or control rats, suggesting a central leptin resistance. In DIO but also in DR rats, metformin treatment induced significant reductions of food intake accompanied by decreases in body weight. Interestingly, the weight loss achieved by metformin was correlated with pretreatment plasma leptin levels. This effect was paralleled by a stimulation of the expression of the leptin receptor gene (ObRb) in the arcuate nucleus. These data identify the hypothalamic ObRb as a gene modulated after metformin treatment and suggest that the anorectic effects of the drug are potentially mediated via an increase in the central sensitivity to leptin. Thus, they provide a rationale for novel therapeutic approaches associating leptin and metformin in the treatment of obesity.

Inhibitory Receptor Expression Depends More Dominantly on Differentiation and Activation than "Exhaustion" of Human CD8 T Cells.

Under conditions of chronic antigen stimulation, such as persistent viral infection and cancer, CD8 T cells may diminish effector function, which has been termed "exhaustion." Expression of inhibitory Receptors (iRs) is often regarded as a hallmark of "exhaustion." Here we studied the expression of eight different iRs by CD8 T cells of healthy humans, including CTLA-4, PD1, TIM3, LAG3, 2B4, BTLA, CD160, and KLRG1. We show that many iRs are expressed upon activation, and with progressive differentiation to effector cells, even in absence of long-term ("chronic") antigenic stimulation. In particular, we evaluated the direct relationship between iR expression and functionality in CD8 T cells by using anti-CD3 and anti-CD28 stimulation to stimulate all cells and differentiation subsets. We observed a striking up-regulation of certain iRs following the cytokine production wave, in agreement with the notion that iRs function as a negative feedback mechanism. Intriguingly, we found no major impairment of cytokine production in cells positive for a broad array of iRs, as previously shown for PD1 in healthy donors. Rather, the expression of the various iRs strongly correlated with T cell differentiation or activation states, or both. Furthermore, we analyzed CD8 T cells from lymph nodes (LNs) of melanoma patients. Interestingly, we found altered iR expression and lower cytokine production by T cells from metastatic LNs, but also from non-metastatic LNs, likely due to mechanisms which are not related to exhaustion. Together, our data shows that expression of iRs per se does not mark dysfunctional cells, but is rather tightly linked to activation and differentiation. This study highlights the importance of considering the status of activation and differentiation for the study and the clinical monitoring of CD8 T cells.

Regulation of GABA(A) and glutamate receptor expression, synaptic facilitation and long-term potentiation in the hippocampus of prion mutant mice

Background: Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrP(sc)) of the natural cellular prion protein (PrP(c)) encoded by the Prnp gene. Although several roles have been attributed to PrP(c), its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrP(c) studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation. Principal Findings: Here we explore the role of PrP(c) expression in neurotransmission and neural excitability using wild-type, Prnp -/- and PrP(c)-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp -/- mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp -/- and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABA(A) and AMPA-kainate receptors are co-regulated in both Prnp -/- and Tg20 mice. Conclusions/Significance: Present results demonstrate that PrP(c) is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABA(A) and AMPA-Kainate neurotransmission. New PrP(c) functions have recently been described, which point to PrP(c) as a target for putative therapies in Alzheimer's disease. However, our results indicate that a "gain of function" strategy in Alzheimer's disease, or a "loss of function" in prionopathies, may impair PrP(c) function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies.

Developmentally regulated extinction of Ly-49 receptor expression permits maturation and selection of NK1.1+ T cells.

Clonally distributed inhibitory receptors negatively regulate natural killer (NK) cell function via specific interactions with allelic forms of major histocompatibility complex (MHC) class I molecules. In the mouse, the Ly-49 family of inhibitory receptors is found not only on NK cells but also on a minor (NK1.1+) T cell subset. Using Ly-49 transgenic mice, we show here that the development of NK1.1+ T cells, in contrast to NK or conventional T cells, is impaired when their Ly-49 receptors engage self-MHC class I molecules. Impaired NK1.1+ T cell development in transgenic mice is associated with a failure to select the appropriate CD1-reactive T cell receptor repertoire. In normal mice, NK1.1+ T cell maturation is accompanied by extinction of Ly-49 receptor expression. Collectively, our data imply that developmentally regulated extinction of inhibitory MHC-specific receptors is required for normal NK1.1+ T cell maturation and selection.

Nuclear triiodothyronine receptor expression is regulated by axon-Schwann cell contact.

Thyroid hormones, which play an important role in the development and regeneration of the nervous system, require the presence of specific nuclear T3 receptors (NT3R). In this study we provide evidence that NT3R expression by Schwann cells was up-regulated in response to a loss of axonal contact in vitro and in vivo. In dorsal root ganglia explant cultures, Schwann cells which accompanied axons (nerve fibres) were devoid of NT3R. When Schwann cells were orphaned from axon contact by axon transection, all the nuclei of these cells displayed NT3R immunoreactivity. Similar results were obtained in situ; in adult rat sciatic nerve, Schwann cells which ensheathed healthy axons never expressed NT3R immunoreactivity. After sciatic nerve transection in vivo the nuclei of Schwann cells deprived of axonal contact displayed a clear NT3R immunoreaction.

Odorant Receptor (Or) genes: polymorphism and divergence in the D. melanogaster and D. pseudoobscura Lineages

Background: In insects, like in most invertebrates, olfaction is the principal sensory modality, which provides animals with essential information for survival and reproduction. Odorant receptors are involved in this response, mediating interactions between an individual and its environment, as well as between individuals of the same or different species. The adaptive importance of odorant receptors renders them good candidates for having their variation shaped by natural selection. Methodology/Principal Findings: We analyzed nucleotide variation in a subset of eight Or genes located on the 3L chromosomal arm of Drosophila melanogaster in a derived population of this species and also in a population of Drosophila pseudoobscura. Some heterogeneity in the silent polymorphism to divergence ratio was detected in the D. melanogaster/D. simulans comparison, with a single gene (Or67b) contributing ~37% to the test statistic. However, no other signals of a very recent selective event were detected at this gene. In contrast, at the speciation timescale, the MK test uncovered the footprint of positive selection driving the evolution of two of the encoded proteins in both D. melanogaster ¿OR65c and OR67a ¿and D. pseudoobscura ¿OR65b1 and OR67c. Conclusions: The powerful polymorphism/divergence approach provided evidence for adaptive evolution at a rather high proportion of the Or genes studied after relatively recent speciation events. It did not provide, however, clear evidence for very recent selective events in either D. melanogaster or D. pseudoobscura.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma.

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Changes in nuclear 3,5,3'-triiodothyronine receptor expression in rat dorsal root ganglia and sciatic nerve during development: comparison with regeneration.

The action of the thyroid hormones on responsive cells in the peripheral nervous system requires the presence of nuclear triiodothyronine receptors (NT3R). These nuclear receptors, including both the alpha and beta subtypes of NT3R, were visualized by immunocytochemistry with the specific 2B3 monoclonal antibody. In the dorsal root ganglia (DRG) of rat embryos, NT3R immunoreactivity was first discretely revealed in a few neurons at embryonic day 14 (E14), then strongly expressed by all neurons at E17 and during the first postnatal week; all DRG neurons continued to possess clear NT3R immunostaining, which faded slightly with age. The peripheral glial cells in the DRG displayed a short-lived NT3R immunoreaction, starting at E17 and disappearing from the satellite and Schwann cells by postnatal days 3 and 7 respectively. In the developing sciatic nerve, Schwann cells also exhibited transient NT3R immunoreactivity restricted to a short period ranging from E17 to postnatal day 10; the NT3R immunostaining of the Schwann cells vanished proximodistally along the sciatic nerve, so that the Schwann cells rapidly became free of detectable NT3R immunostaining. However, after the transection or crushing of an adult sciatic nerve, the NT3R immunoreactivity reappeared in the Schwann cells adjacent to the lesion by 2 days, then along the distal segment in which the axons were degenerating, and finally disappeared by 45 days, when the regenerating axons were allowed to re-occupy the distal segment.(ABSTRACT TRUNCATED AT 250 WORDS)

Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function.

We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression.

Glucagon-like peptide-1 increases beta-cell glucose competence and proliferation by translational induction of insulin-like growth factor-1 receptor expression.

Glucagon-like peptide-1 (GLP-1) protects beta-cells against apoptosis, increases their glucose competence, and induces their proliferation. We previously demonstrated that the anti-apoptotic effect was mediated by an increase in insulin-like growth factor-1 receptor (IGF-1R) expression and signaling, which was dependent on autocrine secretion of insulin-like growth factor 2 (IGF-2). Here, we further investigated how GLP-1 induces IGF-1R expression and whether the IGF-2/IGF-1R autocrine loop is also involved in mediating GLP-1-increase in glucose competence and proliferation. We show that GLP-1 up-regulated IGF-1R expression by a protein kinase A-dependent translational control mechanism, whereas isobutylmethylxanthine, which led to higher intracellular accumulation of cAMP than GLP-1, increased both IGF-1R transcription and translation. We then demonstrated, using MIN6 cells and primary islets, that the glucose competence of these cells was dependent on the level of IGF-1R expression and on IGF-2 secretion. We showed that GLP-1-induced primary beta-cell proliferation was suppressed by Igf-1r gene inactivation and by IGF-2 immunoneutralization or knockdown. Together our data show that regulation of beta-cell number and function by GLP-1 depends on the cAMP/protein kinase A mediated-induction of IGF-1R expression and the increased activity of an IGF-2/IGF-1R autocrine loop.

Genetic Factors in the Regulation of Striatal and Extrastriatal Dopamine D2 Receptor Expression

Positron emission tomography (PET) studies on healthy individuals have revealed a marked interindividual variability in striatal dopamine D2 receptor density that can be partly accounted for by genetic factors. The examination of the extrastriatal lowdensity D2 receptor populations has been impeded by the lack of suitable tracers. However, the quantification of these D2 receptor populations is now feasible with recently developed PET radioligands. The objective of this thesis was to study brain neurobiological correlates of common functional genetic variants residing in candidate genes relevant for D2 receptor functioning. For this purpose, healthy subjects were studied with PET imaging using [₁₁C]raclopride and [₁₁C]FLB457 as radioligands. The candidate genes examined in this work were the human D2 receptor gene (DRD2) and the catechol-Omethyltransferase gene (COMT). The region-specific genotypic influences were explored by comparing D2 receptor binding properties in the striatum, the cortex and the thalamus. As an additional study objective, the relationship between cortical D2 receptor density and a cognitive phenotype i.e. verbal memory and learning was assessed. The main finding of this study was that DRD2 C957T genotype altered markedly D2 receptor density in the cortex and the thalamus whereas in the striatum the C957T genotype affected D2 receptor affinity, but not density. Furthermore, the A1 allele of the DRD2-related TaqIA polymorphism showed increased cortical and thalamic D2 receptor density, but had the opposite effect on striatal D2 receptor density. The DRD2 –141C Ins/Del or the COMT Val158Met genotypes did not change D2 receptor binding properties. Finally, unlike previously reported, cortical D2 receptor density did not show any significant correlation with verbal memory function. The results of this study suggest that the C957T and the TaqIA genotypes have region-specific neurobiological correlates in brain dopamine D2 receptor availability in vivo. The biological mechanisms underlying these findings are unclear, but they may be related to the region-specific regulation of dopamine neurotranssion, gene/receptor expression and epigenesis. These findings contribute to the understanding of the genetic regulation of dopamine and D2 receptor-related brain functions in vivo in man. In addition, the results provide potentially useful endophenotypes for genetic research on psychiatric and neurological disorders.