高亲和力谷氨酸转运体结构和功能的研究进展

真核生物高亲和力谷氨酸转运体(excitatory amino acid transporters,EAATs)分为GLAST(EAAT1)、GLT-1(EAAT2)、EAAC1(EAAT3)、EAAT4和EAAT5等5个亚型.高亲和力谷氨酸转运体结构学的研究,揭示了谷氨酸转运体的跨膜拓扑结构、真核和原核生物EAATs结构的差异,以及在底物转运过程中的一些底物和协同转运离子的结合位点.其功能学的研究发现,EAATs在参与突触的传递,避免兴奋性氨基酸的毒性效应中发挥重要作用,同时也参与了对学习、记忆以及运动行为的调控.结合我们既往的工作,就近几年EAATs的结构和功能研究做一综述.     

高亲和力谷氨酸转运体

高亲和力谷氨酸转运体主要位于神经元和胶质细胞的细胞膜上,能逆浓度梯度从胞外向胞内摄取谷氨酸,中止谷氨酸能传递,使胞外谷氨酸浓度保持在较低水平,以保护神经元不受谷氨酸的毒性影响。近年来,随着高亲和力谷氨酸转运体的克隆,有关研究迅速发展。本文从高亲和力谷氨酸转运体的克隆、分子结构特征、表达分布、生理功能、结构－功能关系等方面对近年的进展加以综述。     

脑缺血后大鼠神经元和星形胶质细胞P21cip1表达的研究

目的研究大鼠局灶性脑缺血再灌注损伤后细胞周期蛋白依赖性激酶抑制因子P21cip1在神经元和星形胶质细胞的表达。方法建立大鼠大脑中动脉阻塞(MCAo)再灌注模型,应用流式细胞术检测各组MCAo再灌注后不同时期神经元和星形胶质细胞中的P21cip1的表达。结果缺血侧皮层区星形胶质细胞和神经元中的P21cip1的表达在再灌注3d、7d、14d后表达下调,与假手术组比较有显著性差异(P<0.05);神经元中的P21cip1的表达和星形胶质细胞中的P21cip1的表达无显著性差异(P>0.05)。结论局灶性脑缺血再灌注损伤后,缺血侧皮层区星形胶质细胞和神经元的p21cip1表达下调。     

谷氨酸转运体与疼痛

中枢神经系统谷氨酸生理浓度主要依赖神经细胞和神经胶质细胞上谷氨酸转运体维持,谷氨酸转运体的功能紊乱会导致谷氨酸的累积。谷氨酸转运体在吗啡镇痛及耐受中扮演一定的角色,并在神经病理性痛中发挥重要作用。谷氨酸转运体可能作为治疗疼痛的一个潜在的药物靶点。     

关于脑缺血的分子生物学研究

脑缺血的主要直接后果是脑缺氧。脑缺血或脑缺氧除引起一系列神经化学变化与蛋白质的合成降低外,还引起热休克蛋白（ＨＳＰ）和ｃ－ｆｏｓ蛋白等特殊蛋白质的特殊变化。轻度缺血可引起ＨＳＰ７０基因转录与翻译;中度缺血只引起其转录而不翻译;重度缺血时转录与翻译均终止。轻、中度脑缺血引起ｃ－ｆｏｓｍＲＮＡ剧烈而短暂的表达,ｃ－ｆｏｓ、ＪｕｎＢ、ｃ－ｊｕｎ等基因转录增加;严重缺血可能因神经元死伤,导致ｃ－ｆｏｓ蛋白水平降低,但局部胶质细胞ｃ－ｆｏｓ诱导增强。     

中枢神经系统谷氨酸转运体的研究进展

在中枢神经系统,谷氨酸转运体在谷氨酸一谷氨酰胺循环中发挥着重要作用。谷氨酸转运体有高亲和力转运体,即兴奋性氨基酸转运体（excitatory amino acid transporters,EAATs）和低亲和力转运体,即囊泡谷氨酸转运体（vesicular glutamate transporters,VGLUTs）两种类型。其中,VGLUTs的功能是特异地将突触囊泡外的谷氨酸转运进入突触囊泡内,它包括三个成员,分别是VGLUT1、VGLUT2和VGLUT3。一方面,VGLUT1和VGLUT2标记了所有的谷氨酸能神经元,是谷氦酸能神经元和它们轴突末端高度特异的标志;另一方面,VGLUT1标志着皮质一皮质投射,而VGLUT2则标志着丘脑一皮层投射,VGLUT3则位于抑制性突触末端。     

星形胶质细胞调控的谷氨酸代谢与癫痫的关系探讨

癫痫(Epilepsy)是一种常见的慢性神经系统疾病,长期反复发作会逐渐损害患者的认知功能并且导致多种共患疾病.癫痫发病机制复杂,其中谷氨酸代谢异常与癫痫发病关系密切.谷氨酸-谷氨酰胺循环是调节谷氨酸代谢的主要途径,谷氨酸转运体和星形胶质细胞在其中发挥重要作用.因此,本文主要探讨星形胶质细胞及谷氨酸转运体对癫痫的影响.     

小胶质细胞生理特点及在脑缺血损伤中的作用

小胶质细胞作为中枢神经系统的免疫细胞,监测中枢神经系统微环境的变化,对微环境变化以及损伤性刺激发生快速反应。脑缺血情况下,小胶质细胞快速活化变形产生细胞毒性物质和细胞因子等,并发生一系列生物活性反应。在脑缺血的病理生理中扮演重要角色,为了探究小胶质细胞在脑缺血再灌注损伤中的作用,我们对最近几年的研究进行了综合,为以后的基础研究和临床治疗提供参考。     

大鼠局灶性脑缺血后缺血边缘区NG_2细胞的表达

目的观察局灶性脑缺血后缺血边缘区海马和皮层NG2细胞的动态表达,探讨其在脑缺血神经损伤与修复过程中所起的作用。方法将大鼠随机分为假手术组(sham)和脑缺血再灌注组,脑缺血再灌注组采用线栓法制备大鼠大脑中动脉阻塞再灌流模型(MCAO),假手术组不插入线栓,采用免疫荧光组织化学法结合共聚焦显微镜成像观察sham组及脑缺血后3d,7d,30d不同时间点缺血边缘区的海马CA1区和皮层区NG2的动态表达情况。结果脑缺血再灌注后缺血边缘区海马和皮层NG2胶质细胞表达增加,缺血后7d最明显。结论脑缺血后缺血边缘区存在NG2细胞的增生和形态变化可能与脑缺血后损伤修复密切相关。     

兴奋性氨基酸转运体2在神经退行性变中的作用

谷氨酸是脑内必需的兴奋性神经递质之一,兴奋性氨基酸转运体（Excitatory amino acid transporterEAAT）2是最主要的谷氨酸转运体,负责脑内90%以上的谷氨酸再摄取,调节突触间隙的谷氨酸浓度。EAAT2功能紊乱导致胞外谷氨酸过量积聚,在多种神经退行性疾病的发病过程中起重要作用,如阿尔茨海默病、亨廷顿舞蹈病、肌萎缩侧索硬化等。对于人EAAT2启动子的研究发现,NF-kB在星形胶质细胞中对EAAT2表达起关键作用。通过筛选1 040种FDA批准的化合物,发现多种β-内酰胺类抗生素如头孢曲松钠等是EAAT2的转录激活剂,可以增加EAAT2的蛋白表达水平,产生神经保护作用。     

Neuronal Expression of Splice Variants of “Glial” Glutamate Transporters in Brains Afflicted by Alzheimer’s Disease: Unmasking an Intrinsic Neuronal Property

Anomalies in glutamate homeostasis may contribute to the pathological processes involved in Alzheimer’s disease (AD). Glutamate released from neurons or glial cells is normally rapidly cleared by glutamate transporters, most of which are expressed at the protein level by glial cells. However, in some patho-physiological situations, expression of glutamate transporters that are normally considered to be glial types, appears to be evoked in populations of distressed neurons. This study analysed the expression of exon-skipping forms of the three predominant excitatory amino acid (glutamate) transporters (EAATs1-3) in brains afflicted with AD. We demonstrate by immunocytochemistry in temporal cortex, the expression of these proteins particularly in limited subsets of neurons, some of which appeared to be dys-morphic. Whilst the neuronal expression of the “glial” glutamate transporters EAAT1 and EAAT2 is frequently considered to represent the abnormal and ectopic expression of such transporters, we suggest this may be a misinterpretation, since neurons such as cortical pyramidal cells normally express abundant mRNA for these EAATs (but little if any EAAT protein expression). We hypothesize instead that distressed neurons in the AD brain can turn on the translation of pre-existent mRNA pools, or suppress the degradation of alternately spliced glutamate transporter protein, leading to the “unmasking” of, rather than evoked expression of “glial” glutamate transporters in stressed neurons. Special issue article in honor of Dr. Graham Johnston.     

Glutamate Transporters: Expression and Function in Oligodendrocytes

Glutamate, the main excitatory neurotransmitter of the vertebrate central nervous system (CNS), is well known as a regulator of neuronal plasticity and neurodevelopment. Such glutamate function is thought to be mediated primarily by signaling through glutamate receptors. Thus, it requires a tight regulation of extracellular glutamate levels and a fine-tuned homeostasis that, when dysregulated, has been associated with a wide range of central pathologies including neuropsychiatric, neurodevelopmental, and neurodegenerative disorders. In the mammalian CNS, extracellular glutamate levels are controlled by a family of sodium-dependent glutamate transporters belonging to the solute carrier family 1 (SLC1) that are also referred to as excitatory amino acid transporters (EAATs). The presumed main function of EAATs has been best described in the context of synaptic transmission where EAATs expressed by astrocytes and neurons effectively regulate extracellular glutamate levels so that synapses can function independently. There is, however, increasing evidence that EAATs are expressed by cells other than astrocytes and neurons, and that they exhibit functions beyond glutamate clearance. In this review, we will focus on the expression and functions of EAATs in the myelinating cells of the CNS, oligodendrocytes. More specifically, we will discuss potential roles of oligodendrocyte-expressed EAATs in contributing to extracellular glutamate homeostasis, and in regulating oligodendrocyte maturation and CNS myelination by exerting signaling functions that have traditionally been associated with glutamate receptors. In addition, we will provide some examples for how dysregulation of oligodendrocyte-expressed EAATs may be involved in the pathophysiology of neurologic diseases.

     

Changes in expression of neuronal and glial glutamate transporters in lead-exposed adult rat brain

Excitatory amino acid transporters (EAATs) are membrane-bound proteins localized in glial and neuronal cells which transport glutamate (Glu) in a process essential for terminating its action and protecting neurons from excitotoxic damage. Since Pb-induced neurotoxicity has a glutamatergic component and astrocytes serve as a cellular Pb deposition site, it was of interest to investigate the response of main glutamate transporters to short-term lead exposure in the adult rat brain (25mg/kg b.w. of lead acetate, i.p. for 3 days). We examined the expression of mRNA and protein of GLAST, GLT-1 and EAAC1 in homogenates obtained from cerebellum, hippocampus and forebrain. Molecular evidence is provided which indicates that, of the two glial transporters, GLT-1 is more susceptible than GLAST to the neurotoxic effect arising from Pb. RT-PCR analysis revealed highly decreased expression of GLT-1 mRNA in forebrain and hippocampus. In contrast, GLAST was overexpressed in forebrain and in cerebellum. In the case of EAAC1, the enhanced expression of mRNA and protein of transporter was observed only in forebrain. The results demonstrate regional differences in the expression of glutamate transporters after short-term exposure to Pb. In forebrain, downregulation of GLT-1 is compensated by enhanced expression of GLAST, while in hippocampus, the expression of both is lowered. This observation suggests that under conditions of Pb toxicity in adult rat brain, the hippocampus is most vulnerable to the excitotoxic cell damage arising from impaired clearance of the released glutamate.     

The Role of Excitatory Amino Acid Transporters in Cerebral Ischemia

Glutamate is an excitatory neurotransmitter that plays a major role in the pathogenesis of ischemia brain injury. The regulation of glutamate neurotransmission is carried out by excitatory amino acid transporters (EAATs) that act through reuptake of glutamate into cells. EAATs may also release glutamate into the extracellular space in a calcium-independent manner during ischemia and dysfunction of EAATs is specifically implicated in the pathology of cerebral ischemia. Recent studies show that up-regulation of EAAT2 provides neuroprotection during ischemic insult. This review summarizes current knowledge regarding the role of EAATs in cerebral ischemia.     

Na+-dependent high-affinity glutamate transport in macrophages

Excessive accumulation of glutamate in the CNS leads to excitotoxic neuronal damage. However, glutamate clearance is essentially mediated by astrocytes through Na+-dependent high-affinity glutamate transporters (excitatory amino acid transporters (EAATs)). Nevertheless, EAAT function was recently shown to be developmentally restricted in astrocytes and undetectable in mature astrocytes. This suggests a need for other cell types for clearing glutamate in the brain. As blood monocytes infiltrate the CNS in traumatic or inflammatory conditions, we addressed the question of whether macrophages expressed EAATs and were involved in glutamate clearance. We found that macrophages derived from human blood monocytes express both the cystine/glutamate antiporter and EAATs. Kinetic parameters were similar to those determined for neonatal astrocytes and embryonic neurons. Freshly sorted tissue macrophages did not possess EAATs, whereas cultured human spleen macrophages and cultured neonatal murine microglia did. Moreover, blood monocytes did not transport glutamate, but their stimulation with TNF-alpha led to functional transport. This suggests that the acquisition of these transporters by macrophages could be under the control of inflammatory molecules. Also, monocyte-derived macrophages overcame glutamate toxicity in neuron cultures by clearing this molecule. This suggests that brain-infiltrated macrophages and resident microglia may acquire EAATs and, along with astrocytes, regulate extracellular glutamate concentration. Moreover, we showed that EAATs are involved in the regulation of glutathione synthesis by providing intracellular glutamate. These observations thus offer new insight into the role of macrophages in excitotoxicity and in their response to oxidative stress.     

Physical and functional interaction of NCX1 and EAAC1 transporters leading to glutamate-enhanced ATP production in brain mitochondria

Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na(+)-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.     

Amyloid-beta peptide decreases expression and function of glutamate transporters in nervous system cells

Glutamate is an essential excitatory neurotransmitter that regulates brain functions, and its activity is tightly regulated by glutamate transporters. Excess glutamate in the synaptic cleft and dysfunction of excitatory amino acid transporters have been shown to be involved in development of Alzheimer’s disease, but the precise regulatory mechanism is poorly understood. Using a D-[³H]-aspartic acid uptake assay, we found that Aβ_1-42 oligomers impaired glutamate uptake in astrocytes and neurons. In astrocytes, this process was accompanied by reduced expression of GLT-1 and GLAST as detected by Western blot and immunocytofluorescence. However, mRNA levels of EAATs detected by qPCR in astrocytes and neurons were not altered, which suggests that this process is post-translational. Co-localization analysis using immunocytofluorescence showed that ubiquitylation of GLT-1 significantly increased. Therefore, we hypothesized that Aβ_1-42 oligomers-induced endocytosis of astrocytic GLT-1 may be involved in ubiquitylation. In addition, Aβ_1-42 oligomers enhanced secretion of IL-1β, TNF-α, and IL-6 into culture supernatant, which may be correlated with an inflammatory response and altered EAATs expression or function in Alzheimer’s disease. These findings support the idea that dysregulation of the glutamatergic system may play a significant role in pathogenesis of Alzheimer’s disease. Furthermore, enhancing expression or function of EAATs in astrocytes and neurons might be a new therapeutic approach in treatment of Alzheimer’s disease.     

Modulation of Glutamate Transport and Receptor Binding by Glutamate Receptor Antagonists in EAE Rat Brain

The etiology of multiple sclerosis (MS) is currently unknown. However, one potential mechanism involved in the disease may be excitotoxicity. The elevation of glutamate in cerebrospinal fluid, as well as changes in the expression of glutamate receptors (iGluRs and mGluRs) and excitatory amino acid transporters (EAATs), have been observed in the brains of MS patients and animals subjected to experimental autoimmune encephalomyelitis (EAE), which is the predominant animal model used to investigate the pathophysiology of MS. In the present paper, the effects of glutamatergic receptor antagonists, including amantadine, memantine, LY 367583, and MPEP, on glutamate transport, the expression of mRNA of glutamate transporters (EAATs), the kinetic parameters of ligand binding to N-methyl-D-aspartate (NMDA) receptors, and the morphology of nerve endings in EAE rat brains were investigated. The extracellular level of glutamate in the brain is primarily regulated by astrocytic glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Excess glutamate is taken up from the synaptic space and metabolized by astrocytes. Thus, the extracellular level of glutamate decreases, which protects neurons from excitotoxicity. Our investigations showed changes in the expression of EAAT mRNA, glutamate transport (uptake and release) by synaptosomal and glial plasmalemmal vesicle fractions, and ligand binding to NMDA receptors; these effects were partially reversed after the treatment of EAE rats with the NMDA antagonists amantadine and memantine. The antagonists of group I metabotropic glutamate receptors (mGluRs), including LY 367385 and MPEP, did not exert any effect on the examined parameters. These results suggest that disturbances in these mechanisms may play a role in the processes associated with glutamate excitotoxicity and the progressive brain damage in EAE.     

High-density presynaptic transporters are required for glutamate removal from the first visual synapse

Reliable synaptic transmission depends not only on the release machinery and the postsynaptic response mechanism but also on removal or degradation of transmitter from the synaptic cleft. Accumulating evidence indicates that postsynaptic and glial excitatory amino acid transporters (EAATs) contribute to glutamate removal. However, the role of presynaptic EAATs is unclear. Here, we show in the mouse retina that glutamate is removed from the synaptic cleft at the rod to rod bipolar cell (RBC) synapse by presynaptic EAATs rather than by postsynaptic or glial EAATs. The RBC currents evoked by electrical stimulation of rods decayed slowly after pharmacological blockade of EAATs. Recordings of the evoked RBC currents from EAAT subtype-deficient mice and the EAAT-coupled anion current reveal that functional EAATs are localized to rod terminals. Model simulations suggest that rod EAATs are densely packed near the release site and that rods are equipped with an almost self-sufficient glutamate recollecting system.