GBE对大鼠急性分离海马神经元NMDA-激活电流的调制作用

目的:观察不同制型的银杏叶提取物(GBE)对N-甲基-D-天门冬氨酸(NMDA)受体激活电流的影响,并比较其作用。方法:应用全细胞膜片钳记录技术记录急性分离大鼠海马神经NMDA激活电流,比较加药前后电流幅度的变化。结果:大部分受检细胞(81.8%,90/110)对外加NMDA敏感,引起一去敏感的内向电流(INMDA)。此电流可被NMDA受体特异阻断剂(MK-801)所阻断。预加不同制型的GBE均能明显抑制NMDA激活电流(P<0.01),但制型不同抑制效应不一,GBE纳米制剂(nGBE)对INMDA的抑制作用明显优于微米型(mGBE组),抑制率分别为64%±15%,40%±17%(n=8),两组比较差异具有统计学意义(P<0.05)。结论:预加GBE能抑制NM-DA-激活电流,从而对抗海马神经元兴奋毒性脑损伤,起神经保护作用。nGBE对NMDA受体的调控作用优于mGBE制剂。     

免疫调节剂对戊四氮致痫大鼠脑内NMDAR1和NF-κB p65表达的影响

目的探讨免疫调节在癫痫发病机制中的作用。方法采用戊四氮(PTZ)致痫,并分别以免疫增强剂左旋咪唑(LMZ)或抑制剂地塞米松(DXM)进行干预。将实验大鼠随机分为4组:即生理盐水对照组(NS组)、戊四氮组(PTZ组)、左旋咪唑 戊四氮组(LMS PTZ组)、地塞米松 戊四氮组(DXM PTZ组)。各组大鼠给予相应试剂处理后,观察行为学表现并用免疫组织化学方法检测大鼠大脑皮质及海马NMDAR1和NF-κB表达情况。结果动物行为学观察,NS组无癫痫发作,PTZ组发作程度为Ⅲ-Ⅵ级,LMS PTZ组为Ⅴ级,DXM PTZ组为Ⅰ-Ⅱ级。NMDAR1和NF-κB免疫组织化学染色显示,在大鼠大脑皮质及海马,NMDAR1和NF-κB在4组均有表达,且二项指标变化趋势一致,其中PTZ组较NS组表达增强,LMS PTZ组进一步增强,DXM PTZ组较PTZ组和LMS PTZ组明显减弱,其差异均具有显著性意义。结论免疫增强剂或抑制剂可分别上调或下调NMDAR1的表达和NF-κB的活化而干预癫痫的发生、发展过程。     

NMDA receptors‐dependent plasticity in the phototaxis preference behavior induced by visual deprivation in young and adult flies

Adult mammals have experience‐dependent plasticity in visual system, but it is unclear whether adult insects also have this plasticity after the critical period of visual development. Here, we have established a modified Y‐maze apparatus for investigating experience‐dependent plasticity in Drosophila. Using this setup we demonstrate that flies after the critical period have bidirectional modifications of the phototaxis preference behavior (PPB) induced by visual deprivation and experience: Visual deprivation decreases the preference of flies for visible light, while visual experience exerts the opposite effect. We also found an age‐dependent PPB plasticity induced by visual deprivation. Molecular and cellular studies suggest that the N‐methyl‐ d ‐aspartate receptors (NMDARs) mediate ocular dominance plasticity in visual cortex in mammals, but direct behavioral evidence is lacking. Here, we used the genetic approaches to demonstrate that NMDAR1, which is NMDARs subunit in Drosophila, can mediate PPB plasticity in young and adult flies. These findings provide direct behavioral evidence that NMDAR1 mediates PPB plasticity in Drosophila. Our results suggest that mammals and insects have analogous mechanisms for experience‐dependent plasticity and its regulation by NMDAR signaling.     

Dynamic Regulation of N-Methyl-d-aspartate (NMDA) and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors by Posttranslational Modifications

Many molecular mechanisms underlie the changes in synaptic glutamate receptor content that are required by neuronal networks to generate cellular correlates of learning and memory. During the last decade, posttranslational modifications have emerged as critical regulators of synaptic transmission and plasticity. Notably, phosphorylation, ubiquitination, and palmitoylation control the stability, trafficking, and synaptic expression of glutamate receptors in the central nervous system. In the current review, we will summarize some of the progress made by the neuroscience community regarding our understanding of phosphorylation, ubiquitination, and palmitoylation of the NMDA and AMPA subtypes of glutamate receptors.     

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca²⁺ influx and mitochondrial Ca²⁺ overload. A substantial change in intracellular Ca²⁺ homeostasis or remodeling of intracellular Ca²⁺ homeostasis may favor neuron damage in old neurons. For investigating Ca²⁺ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca²⁺ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca²⁺ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca²⁺ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca²⁺ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.     

Neuroprotective Role of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> Extract in Epilepsy and Effect of Glucose Supplementation During Hypoxia: Glutamate Receptor Gene Expression

The experiments were designed to study the glutamate gene expression during epilepsy in adult and hypoxic insult to brain during the neonatal period and the therapeutic role of neuroprotective supplements. We investigated the role of metabotropic glutamate-8 receptor (mGluR8) gene expression in cerebellum during epilepsy and neuroprotective role of Bacopa monnieri extract in epilepsy. We also studied the effect of NMDA receptor 1 (NMDAR1) gene expression during neonatal hypoxia and therapeutic role of glucose, oxygen and epinephrine supplementation. During epilepsy a significant down-regulation (P < 0.01) of mGluR8 gene expression was observed which was up-regulated (P < 0.05) near control level after B. monnieri treatment which is supported by Morris water maze experiment. In hypoxic neonates we observed up-regulation (P < 0.001) of the NMDAR1 gene expression whereas glucose and glucose + oxygen was able to significantly reverse (P < 0.001) the gene expression to near control level when compared to hypoxia and epinephrine treatment which was supported by open field test. Our results showed that B. monnieri treatment to epileptic rats significantly brought the reversal of the down-regulated mgluR8 gene expression toward control level. In neonatal rats, hypoxia induced expressional and functional changes in the NMDAR1 receptors of neuronal cells which is corrected by supplementation of glucose alone or glucose followed by oxygen during the resuscitation to prevent the glutamate related neuronal damage. Thus, the results suggest the clinical significance of corrective measures for epileptic and hypoxic management.     

NMDAR1蛋白膜外抗原结构域的重组表达、纯化和免疫反应原性鉴定

构建编码NMDAR1蛋白膜外片段的原核表达重组质粒,在大肠杆菌中诱导表达、纯化并鉴定其免疫反应原性。根据人NMDAR1基因序列,利用Phyre 2软件预测蛋白的三级结构并分析其结构域。设计引物用RT-PCR方法扩增编码NMDAR1膜外蛋白不同结构域的核酸片段,并插入原核表达载体pCold-SUMO构建重组质粒。转化DH5α感受态细胞,菌落PCR鉴定,阳性单克隆进行测序验证。鉴定正确的重组体转化大肠杆菌BL21(DE3),IPTG诱导目的蛋白的表达和纯化,Ni-NTA柱亲和层析和凝胶过滤层析纯化蛋白,酶切切除融合蛋白6His-SUMO标签,用AKTA Purifier进行凝胶过滤层析,收集纯化蛋白。利用SDS-PAGE鉴定蛋白纯度,并用Western blotting进行免疫反应性鉴定。克隆获得NMDAR1膜外部分的三段DNA序列,分别是NR1-M1(编码19–393 aa)、NR1-S1(编码394–544 aa)和NR1-S2(编码663–800 aa)。其中NR1-S1和NR1-S2片段之间以G(甘氨酸)和T(苏氨酸)作为接头连接成为复合片段。经菌落PCR筛选和测序鉴定,成功构建了重组质粒p Cold-SUMO-M1和p Cold-SUMO-S1-GT-S2。SDS-PAGE鉴定结果表明重组质粒在大肠杆菌中经诱导可表达可溶性NR1-M1及NR1-S1-GT-S2蛋白。对表达产物进行亲和层析和凝胶过滤层析获得了高纯度的目标蛋白。Western blotting证实纯化的目的蛋白能与相应抗体发生特异性结合反应。本研究成功构建了NMDAR1蛋白膜外抗原结构域的原核表达系统,并获得了具有免疫反应性的NR1-M1及NR1-S1-GT-S2纯化蛋白。该蛋白有望用于NMDAR1蛋白的功能研究及自身抗体的检测。     

AMSH is required to degrade ubiquitinated proteins in the central nervous system

Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. AMSH (associated molecule with the SH3 domain of STAM) is a deubiquitination enzyme that participates in the endosomal sorting of several cell-surface molecules. AMSH impairment results in missorted ubiquitinated cargoes in vitro and severe neurodegeneration in vivo, but it is not known how AMSH deficiency causes neuronal damage in the brain. Here, we demonstrate that AMSH^−/− mice developed ubiquitinated protein accumulations as early as embryonic day 10 (E10), and that severe deposits were present in the brain at postnatal day 8 (P8) and P18. Interestingly, TDP-43 was found to accumulate and colocalize with glial marker-positive cells in the brain. Glutamate receptor and p62 accumulations were also found; these molecules colocalized with ubiquitinated aggregates in the brain. These data suggest that AMSH plays an important role in degrading ubiquitinated proteins and glutamate receptors in vivo. AMSH^−/− mice provide an animal model for neurodegenerative diseases, which are commonly characterized by the generation of proteinaceous aggregates.     

Sustained calcium signalling and caspase-3 activation involve NMDA receptors in thymocytes in contact with dendritic cells

L-glutamate, the major excitatory neurotransmitter, also has a role in non-neuronal tissues and modulates immune responses. Whether NMDA receptor (NMDAR) signalling is involved in T-cell development is unknown. In this study, we show that mouse thymocytes expressed an array of glutamate receptors, including NMDARs subunits. Sustained calcium (Ca²⁺) signals and caspase-3 activation in thymocytes were induced by interaction with antigen-pulsed dendritic cells (DCs) and were inhibited by NMDAR antagonists MK801 and memantine. NMDARs were transiently activated, triggered the sustained Ca²⁺ signal and were corecruited with the PDZ-domain adaptor postsynaptic density (PSD)-95 to thymocyte-DC contact zones. Although T-cell receptor (TCR) activation was sufficient for relocalization of NMDAR and PSD-95 at the contact zone, NMDAR could be activated only in a synaptic context. In these T-DC contacts, thymocyte activation occurred in the absence of exogenous glutamate, indicating that DCs could be a physiological source of glutamate. DCs expressed glutamate, glutamate-specific vesicular glutamate transporters and were capable of fast glutamate release through a Ca²⁺-dependent mechanism. We suggest that glutamate released by DCs could elicit focal responses through NMDAR-signalling in T cells undergoing apoptosis. Thus, synapses between T and DCs could provide a functional platform for coupling TCR activation and NMDAR signalling, which might reflect on T-cell development and modulation of the immune response.