阻断NMDA受体可增强皮质酮对海马脑源性神经营养因子表达的抑制:cAMP反应元件结合蛋白的作用

高浓度的皮质酮可引起海马形态与功能的损伤,其中脑源性神经营养因子(brain-derived neurotrophic factor,BDNF) 表达的改变在海马形态与功能损伤中扮演重要角色。本实验的目的是观察单次皮下注射皮质酮后海马内BDNF-mRNA、前 体蛋白及成熟型蛋白表达的改变,并观察N-甲基-D-天冬氨酸(N-methyl-D-aspartate NMDA)受体阻滞剂MK801对皮质酮 作用的影响。实验结果显示,单次皮下注射皮质酮2 mg／kg,3 h后海马内BDNF mRNA、前体蛋白及成熟型蛋白的表达 均降低;MK801(0．1 mg／kg)对皮质酮的这一作用有增强效果。单独给予皮质酮或注射MK801 30 min后再给予皮质酮, 均能明显降低海马中cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)的磷酸化水平,MK801与 皮质酮联用时CREB的磷酸化水平降低更为显著(与单独给予皮质酮相比,P<0．05)。实验结果提示,CREB磷酸化水平降 低可能是皮质酮引起海马BDNF表达减少的重要中间环节,阻断NMDA受体可加强皮质酮降低BDNF表达的效应。     

芳香烃受体核转位蛋白的结构及相关功能

芳香烃受体核转位蛋白（aryl hydrocarbon receptor nuclear translocator,ARNT）是碱性螺旋-环．螺旋转录因子超家族中新发现的PAS亚家族的成员之一。它是体内许多bHLH-PAS蛋白共同的专性配偶体,可以与芳香烃受体、低氧诱导因子、果蝇SIM蛋白等形成异二聚体并介导许多信号转导过程,从而使个体对环境污染物（如二恶英）、低氧状态等外界因素的改变产生相应的生物学效应,本文就ARNT的基本结构及其在体内的主要生理功能等方面作一综述。     

Recombinant expression of aryl hydrocarbon receptor for quantitative ligand-binding analysis

Recombinant expression of the aryl hydrocarbon receptor (AhR) yields small amounts of ligand-binding-competent AhR. Therefore, Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high-level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding-competent AhR was sensitive to the protein concentration of Sf9 extract, and coexpression of the chaperone p23 failed to affect the yield of functional ligand-binding AhR. The expression system yielded high levels of functional protein, with the ligand-binding capacity (B_max) typically 20-fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity of human and rat AhR were obtained; the K_d for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding-competent AhR, and sufficient AhR for quantitative analysis of ligand binding.     

Prevention of NMDA-induced death of cortical neurons by inhibition of protein kinase Czeta

Excitotoxicity through stimulation of N-methyl-d-aspartate (NMDA) receptors contributes to neuronal death in brain injuries, including stroke. Several lines of evidence suggest a role for protein kinase C (PKC) isoforms in NMDA excitotoxicity. We have used specific peptide inhibitors of classical PKCs (alpha, beta, and gamma), novel PKCs delta and epsilon, and an atypical PKCzeta in order to delineate which subspecies are involved in NMDA-induced cell death. Neuronal cell cultures were prepared from 15-day-old mouse embryos and plated onto the astrocytic monolayer. After 2 weeks in vitro the neurons were exposed to 100 micro m NMDA for 5 min, and 24 h later the cell viability was examined by measuring the lactate dehydrogenase release and bis-benzimide staining. While inhibitors directed to classical (alpha, beta, and gamma) or novel PKCs (delta or epsilon) had no effect, the PKCzeta inhibitor completely prevented the NMDA-induced necrotic neuronal death. Confocal microscopy confirmed that NMDA induced PKCzeta translocation, which was blocked by the PKCzeta inhibitor. The NMDA-induced changes in intracellular free Ca2+ were not affected by the peptides. In situ hybridization experiments demonstrated that PKCzeta mRNA is induced in the cortex after focal brain ischemia. Altogether, the results indicate that PKCzeta activation is a downstream signal in NMDA-induced death of cortical neurons.     

The aryl hydrocarbon receptor, more than a xenobiotic-interacting protein

The aryl hydrocarbon (dioxin) receptor (AhR) has been studied for several decades largely because of its critical role in xenobiotic-induced toxicity and carcinogenesis. Albeit this is a major issue in basic and clinical research, an increasing number of investigators are turning their efforts to try to understand the physiology of the AhR under normal cellular conditions. This is an exciting area that covers cell proliferation and differentiation, endogenous mechanisms of activation, gene regulation, tumor development and cell motility and migration, among others. In this review, we will attempt to summarize the studies supporting the implication of the AhR in those endogenous cellular processes.     

Lithium protection against glutamate excitotoxicity in rat cerebral cortical neurons: involvement of NMDA receptor inhibition possibly by decreasing NR2B tyrosine phosphorylation

The therapeutic mechanisms of lithium for treating bipolar mood disorder remain poorly understood. Recent studies demonstrate that lithium has neuroprotective actions against a variety of insults. Here, we studied neuroprotective effects of lithium against excitotoxicity in cultured cerebral cortical neurons. Glutamate-induced excitotoxicity in cortical neurons was exclusively mediated by NMDA receptors. Pre-treatment of cortical neurons with LiCl time-dependently suppressed excitotoxicity with maximal protection after 6 days of pre-treatment. Significant protection was observed at the therapeutic and subtherapeutic concentration of 0.2-1.6 mm LiCl with almost complete protection at 1 mM. Neuroprotection was also elicited by valproate, another major mood-stabilizer. The neuroprotective effects of lithium coincided with inhibition of NMDA receptor-mediated calcium influx. Lithium pre-treatment did not alter total protein levels of NR1, NR2A and NR2B subunits of NMDA receptors. However, it did markedly reduce the level of NR2B phosphorylation at Tyr1472 and this was temporally associated with its neuroprotective effect. Because NR2B tyrosine phosphorylation has been positively correlated with NMDA receptor-mediated synaptic activity and excitotoxicity, the suppression of NR2B phosphorylation by lithium is likely to result in the inactivation of NMDA receptors and contributes to neuroprotection against excitotoxicity. This action could also be relevant to its clinical efficacy for bipolar patients.     

Regulated release of BDNF by cortical oligodendrocytes is mediated through metabotropic glutamate receptors and the PLC pathway

A number of studies suggest that OLGs (oligodendrocytes), the myelinating cells of the central nervous system, are also a source of trophic molecules, such as neurotrophins that may influence survival of proximate neurons. What is less clear is how the release of these molecules may be regulated. The present study investigated the effects of BDNF (brain-derived neurotrophic factor) derived from cortical OLGs on proximate neurons, as well as regulatory mechanisms mediating BDNF release. Initial work determined that BDNF derived from cortical OLGs increased the numbers of VGLUT1 (vesicular glutamate transporter 1)-positive glutamatergic cortical neurons. Furthermore, glutamate acting through metabotropic, and not AMPA/kainate or NMDA (N-methyl-d-aspartate), receptors increased BDNF release. The PLC (phospholipase C) pathway is a key mediator of metabotropic actions to release BDNF in astrocytes and neurons. Treatment of OLGs with the PLC activator m-3M3FBS [N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide] induced robust release of BDNF. Moreover, release elicited by the metabotropic receptor agonist ACPD [trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid] was inhibited by the PLC antagonist {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, the IP₃ (inositol triphosphate 3) receptor inhibitor 2-APB (2-aminoethoxydiphenylborane) and the intracellular calcium chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)]. Taken together, these results suggest that OLG lineage cells release BDNF, a molecule trophic for proximate neurons. BDNF release is regulated by glutamate acting through mGluRs (metabotropic glutamate receptors) and the PLC pathway. Thus glutamate and BDNF may be molecules that support neuron–OLG interactions in the cortex.     

Demethylation of the aryl hydrocarbon receptor repressor as a biomarker for nascent smokers

Epigenetic modifications to peripheral white blood cell DNA occur in response to a wide variety of exposures. In prior work, we and others have shown that broad changes in DNA methylation, particularly at the aryl hydrocarbon receptor repressor (AHRR) locus, occur in samples from subjects with long histories of smoking. However, given the large number of epigenetic changes that occur in response to prolonged smoking, the primacy of the response at AHRR and the sensitivity of these changes to low levels of smoking are not known. Therefore, we examined the association of smoking to genome lymphocyte DNA methylation status in a representative sample of 399 African American youths living in the rural South that includes 72 subjects with less than one half-pack year of exposure. Consistent with our prior findings, we found a stepwise effect of smoking on DNA methylation among youth with relatively brief exposure histories at a CpG residue in AHRR (cg05575921) (FDR corrected p values; 3 × 10^?7 and 0.09 in the male and female samples, respectively) that was identified in previous studies and at which the effects of smoking were significant, even in those subjects with less than one half pack year exposure. We conclude that AHRR demethylation at cg05575921 in peripheral cells may serve as an early, sensitive biomarker for even low levels of exposure to tobacco smoke, providing a non-self-report alternative for nascent exposure to tobacco smoke. We also suggest that the AHRR/AHR pathway may be functional in the response of peripheral white blood cells to tobacco smoke exposure.     

Demethylation of the aryl hydrocarbon receptor repressor as a biomarker for nascent smokers

Epigenetic modifications to peripheral white blood cell DNA occur in response to a wide variety of exposures. In prior work, we and others have shown that broad changes in DNA methylation, particularly at the aryl hydrocarbon receptor repressor (AHRR) locus, occur in samples from subjects with long histories of smoking. However, given the large number of epigenetic changes that occur in response to prolonged smoking, the primacy of the response at AHRR and the sensitivity of these changes to low levels of smoking are not known. Therefore, we examined the association of smoking to genome lymphocyte DNA methylation status in a representative sample of 399 African American youths living in the rural South that includes 72 subjects with less than one half-pack year of exposure. Consistent with our prior findings, we found a stepwise effect of smoking on DNA methylation among youth with relatively brief exposure histories at a CpG residue in AHRR (cg05575921) (FDR corrected p values; 3 × 10⁻⁷ and 0.09 in the male and female samples, respectively) that was identified in previous studies and at which the effects of smoking were significant, even in those subjects with less than one half pack year exposure. We conclude that AHRR demethylation at cg05575921 in peripheral cells may serve as an early, sensitive biomarker for even low levels of exposure to tobacco smoke, providing a non-self-report alternative for nascent exposure to tobacco smoke. We also suggest that the AHRR/AHR pathway may be functional in the response of peripheral white blood cells to tobacco smoke exposure.     

Antagonistic effects of TrkB and p75(NTR) on NMDA receptor currents in post-synaptic densities transplanted into Xenopus oocytes

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are essential regulators of synaptic function in the adult CNS. A TrkB-mediated effect at excitatory synapses is enhancement of NMDA receptor (NMDA-R)-mediated currents. Recently, opposing effects of TrkB and the pan-neurotrophin receptor p75(NTR) on long-term synaptic depression and long-term potentiation have been reported in the hippocampus. To further study the regulation of NMDA-Rs by neurotrophin receptors in their native protein environment, we micro-transplanted rat forebrain post-synaptic densities (PSDs) into Xenopus oocytes. One-minute incubations of oocytes with BDNF led to dual effects on NMDA-R currents: either TrkB-dependent potentiation or TrkB-independent inhibition were observed. Pro-nerve growth factor, a ligand for p75(NTR) but not for TrkB, produced a reversible, dose-dependent, TrkB-independent and p75(NTR)-dependent inhibition of NMDA-Rs. Fractionation experiments showed that p75(NTR) is highly enriched in the PSD protein fraction. Immunoprecipitation and pull-down experiments further revealed that p75(NTR) is a core component of the PSD, where it interacts with the PDZ3 domain of the scaffolding protein SAP90/PSD-95. Our data provide striking evidence for a rapid inhibitory effect of p75(NTR) on NMDA-R currents that antagonizes TrkB-mediated NMDA-R potentiation. These opposing mechanisms might be present in a large proportion of forebrain synapses and may contribute importantly to synaptic plasticity.     

Commensal microbiota regulates skin barrier function and repair via signaling through the aryl hydrocarbon receptor

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The effect of postnatal manganese exposure on the NMDA receptor signaling pathway in rat hippocampus

Overexposure to manganese (Mn) is associated with neurological disorders in children. Evidence indicated that N‐methyl‐d ‐aspartate (NMDA) receptor signaling pathway was critical for neurobehavioral function. However, whether NMDA receptor signaling pathway contributes to Mn‐induced neurotoxicity remains unknown. In this study, newborn Sprague–Dawley rats were randomly assigned to four groups exposed to 0, 10, 20, and 30 mg/kg of Mn²⁺ by intraperitoneal injection (n = 10/group: five males and five females). After 3 weeks of Mn exposure, messenger RNA (mRNA) and protein expression of NMDA receptor subunits (NR1, NR2A, and NR2B), cAMP‐response element binding protein (CREB), and brain‐derived neurotrophic factor (BDNF) in hippocampus were measured by real‐time quantitative RT‐PCR and Western blot. In Mn‐exposed rats, decreased mRNA and protein expression of NR1, NR2A, and NR2B, CREB, and BDNF was observed. The results imply that downregulated NMDA receptor signaling pathway may be of vital importance in the neuropathological process of Mn‐induced neurotoxicity.