囊泡谷氨酸转运体在阿尔兹海默病发病机制中的作用

阿尔兹海默病（Alzheimer’s disease,AD）是一种多因素复杂性神经退行性疾病,β淀粉样蛋白（pamyloid,AB）级联假说和谷氨酸兴奋性毒性是其重要的发病机制。囊泡谷氨酸转运体（vesicularglutamate transporters,VGLUTs）可特异性地将神经元内的谷氨酸转移入突触囊泡,且一个独立功能单位的VGLUT对于完成一个囊泡的填充是必要和充分的,没有VGLUT的突触囊泡中就没有谷氨酸（glutamate,Glul,VGLUT在一定程度上决定了释放进突触间隙Glu的量,是谷氨酸能突触传递的关键因子。在AD中Aβ增多聚集,VGLUTs表达减低,且VGLUTs转运Glu和Glu的囊泡释放与淀粉样前体蛋白（amyloid precursor protein,APP）代谢和A13的释放在突触囊泡的循环中存在行为平行性和共定位。胞外AB的增加可增强囊泡的释放几率,而Glu引起的突触活性增加亦可增加胞外A[3的浓度。APP／Aβ与谷氨酸能系统之间相互影响导致AD的发生,VGLUTs可能在其中发挥重要作用,被认为是治疗AD的潜在的药物靶点和预警标志物。     

谷氨酸转运体GLT-1的剪接变异体研究进展

随着对谷氨酸转运体1(glial glutamate transporter 1, GLT-1)研究的深入,发现GLT-1存在多种剪接变异体.这些变异体除在中枢神经系统大量分布外,在受试动物的其它部位也有分布.中枢神经系统内的GLT-1剪接变异体在星形胶质细胞和神经元上均有表达,且这些剪接变异体在中枢神经系统的分布还呈现出区域定位及亚细胞定位不均衡的特性.     

Ⅱ型囊泡膜谷氨酸转运体阳性终末与γ-氨基丁酸阳性神经元在小鼠腰髓背角的分布及联系

目的 研究Ⅱ型囊泡膜谷氨酸转运体(vesicular glutamate transporter 2,VgluT2)阳性终末与γ-氨基丁酸(γ-aminobutyric acid,GABA)阳性神经元在小鼠腰髓背角的分布和联系。方法采用免疫组织化学方法研究VgluT2阳性终末与GABA阳性神经元在小鼠腰髓背角的分布;采用免疫荧光组织化学双重标记方法研究VgluT2阳性终末与GABA阳性神经元在小鼠腰髓背角的联系。结果VgluT2阳性终末与GABA阳性神经元在小鼠腰髓背角各层均有分布,特别是在Ⅱ层内侧部二分布都较为密集,免疫荧光双重标记后在激光共聚焦显微镜下可见GABA阳性神经元周围有许多VGluT2阳性终末与其胞体或突起密切接触。结论小鼠腰髓背角Ⅱ层内侧部GABA阳性神经元直接接受兴奋性传入。     

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。     

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。     

囊泡膜谷氨酸转运体阳性纤维在大鼠三叉神经运动核内的分布

目的观察I、Ⅱ型囊泡膜谷氨酸转运体阳性纤维在大鼠三叉神经运动核内的分布。方法首先采用免疫荧光三重标记I、Ⅱ型囊泡膜谷氨酸转运体和神经元核蛋白以观察I、Ⅱ型囊泡膜谷氨酸转运体阳性纤维在大鼠三叉神经运动核内的分布;接着注射四甲基罗达明人下颌舌骨肌神经逆行标记三叉神经运动核开口神经元,再采用免疫荧光双重标记I型囊泡膜谷氨酸转运体和神经元核蛋白以观察I、Ⅱ型囊泡膜谷氨酸转运体阳性纤维在大鼠三叉神经运动核开口神经元区和闭口神经元区内的分布差异。结果I型囊泡膜谷氨酸转运体阳性纤维仅在三叉神经运动核背外侧部分布,而Ⅱ型囊泡膜谷氨酸转运体阳性纤维在整个三叉神经运动核内分布;开口神经元区未观察到I型囊泡膜谷氨酸转运体阳性终末。结论闭口神经元接受I、Ⅱ型囊泡膜谷氨酸转运体阳性纤维支配,开口神经元仅仅接受Ⅱ型囊泡膜谷氨酸转运体阳性纤维支配。     

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.     

Ⅰ型囊泡膜谷氨酸转运体在生后发育大鼠三叉神经运动核中的表达

目的观察Ⅰ型囊泡膜谷氨酸转运体在大鼠三叉神经运动核生后发育过程中的表达变化。方法取生后不同发育阶段大鼠脑干,行冰冻切片和Ⅰ型囊泡膜谷氨酸转运体免疫染色,光镜观察。结果刚出生大鼠三叉神经运动核背外侧部即可以观察到Ⅰ型囊泡膜谷氨酸转运体免疫染色阳性结构,随着发育进展,免疫染色阳性逐渐增加,出生7天后Ⅰ型囊泡膜谷氨酸转运体免疫染色模式接近成年水平。结论大鼠三叉神经运动核内Ⅰ型囊泡膜谷氨酸转运体阳性终末在出生后1周内较快地成熟。     

谷氨酸转运体GLAST mRNA在大鼠脑内表达的细胞定位

实验采用荧光双标技术研究谷氨酸转运体ＧＬＡＳＴ ｍ ＲＮＡ 在大鼠脑内表达的细胞定位, 研究表明, 在星形神经胶质细胞和神经元, ＧＬＡＳＴｍ ＲＮＡ 分别与神经胶质纤维蛋白（ＧＦＡＰ） 和神经元特异性烯醇化酶 （ＮＳＥ） 有表达共存, 提示ＧＬＡＳＴ ｍ ＲＮＡ在星形神经胶质细胞和神经元上都有表达。     

Dopamine neurons in culture express VGLUT2 explaining their capacity to release glutamate at synapses in addition to dopamine

Dopamine neurons have been suggested to use glutamate as a cotransmitter. To identify the basis of such a phenotype, we have examined the expression of the three recently identified vesicular glutamate transporters (VGLUT1-3) in postnatal rat dopamine neurons in culture. We found that the majority of isolated dopamine neurons express VGLUT2, but not VGLUT1 or 3. In comparison, serotonin neurons express only VGLUT3. Single-cell RT-PCR experiments confirmed the presence of VGLUT2 mRNA in dopamine neurons. Arguing for phenotypic heterogeneity among axon terminals, we find that only a proportion of terminals established by dopamine neurons are VGLUT2-positive. Taken together, our results provide a basis for the ability of dopamine neurons to release glutamate as a cotransmitter. A detailed analysis of the conditions under which DA neurons gain or loose a glutamatergic phenotype may provide novel insight into pathophysiological processes that underlie diseases such as schizophrenia, Parkinson's disease and drug dependence.     

Function of GABAergic and glutamatergic neurons in the stomach

-Aminobutyric acid (GABA) and L-glutamic acid (L-Glu) are transmitters of GABAergic and glutamatergic neurons in the enteric interneurons, targeting excitatory or inhibitory GABA receptors or glutamate receptors that modulate gastric motility and mucosal function. GABAergic and glutamatergic neuron immunoreactivity have been found in cholinergic enteric neurons in the stomach. GABA and L-Glu may also subserve hormonal and paracrine signaling. Disruption in gastrointestinal function following perturbation of enteric GABA receptors and glutamate receptors presents potential new target sites for drug development.     

Modulatory Effect of Dopamine on High-Affinity Glutamate Uptake in the Rat Striatum

In vivo electrical stimulation of the frontal cortical areas was found to enhance sodium-dependent high-affinity glutamate uptake (HAGU) measured in rat striatal homogenates. This activating effect was counteracted by in vivo administration of apomorphine and by in vitro addition of dopamine (DA; 10(-8) M) in the incubation medium, and potentiated by in vivo haloperidol administration. At the doses used, the dopaminergic compounds had no effect on basal HAGU. alpha-Methylparatyrosine pretreatment was found to enhance slightly basal HAGU as well as the activating effects of cortical stimulation. Interestingly enough, lesion of dopaminergic neurons by substantia nigra injection of 6-hydroxydopamine (6-OHDA) did not cause any significant change either in basal HAGU or in the effect of cortical stimulation. Measurement of DA effects in vitro in experiments combined with in vivo manipulations of the dopaminergic nigrostriatal and corticostriatal systems showed that the capacity of DA to inhibit striatal HAGU depends directly on the level of the uptake activation reached over basal value. These results suggest that under physiological conditions, the dopaminergic nigrostriatal pathway exerts a modulatory presynaptic action on corticostriatal glutamatergic transmission, counteracting increasing glutamatergic activity. In the case of chronic DA depletion induced by 6-OHDA, striatal adaptations may occur modifying the mechanisms acting at corticostriatal nerve terminal level.     

Co-expression of vesicular glutamate transporters (VGLUT1 and VGLUT2) and their association with synaptic-like microvesicles in rat pinealocytes

A vesicular glutamate transporter (VGLUT) is responsible for the accumulation of l-glutamate in synaptic vesicles in glutamatergic neurons. Two isoforms, VGLUT1 and VGLUT2, have been identified, which are complementarily expressed in these neurons. Mammalian pinealocytes, endocrine cells for melatonin, are also glutamatergic in nature, accumulate l-glutamate in synaptic-like microvesicles (SLMVs), and secrete it through exocytosis. Although the storage of l-glutamate in SLMVs is mediated through a VGLUT, the molecular nature of the transporter is less understood. We recently observed that VGLUT2 is expressed in pinealocytes. In the present study, we show that pinealocytes also express VGLUT1. RT-PCR and northern blot analyses indicated expression of the VGLUT1 gene in pineal gland. Western blotting with specific antibodies against VGLUT1 indicated the presence of VGLUT1 in pineal gland. Indirect immunofluorescence microscopy with a section of pineal gland and cultured cells indicated that VGLUT1 and VGLUT2 are co-localized with process terminal regions of pinealocytes. Furthermore, immunoelectronmicroscopy as well as subcellular fractionation studies revealed that both VGLUT1 and VGLUT2 are specifically associated with SLMVs. These results indicate that both VGLUTs are responsible for storage of l-glutamate in SLMVs in pinealocytes. Pinealocytes are the first exception as to complementary expression of VGLUT1 and VGLUT2.     

中枢神经系统内神经元信号处理(英文)

一种新颖的轴突断端(axon bleb)膜片钳记录方法大力促进了中枢神经系统轴突功能的研究。我们的工作应用这一方法揭示了大脑皮层锥体神经元的数码信号(具全或无特性的动作电位)的爆发和传播机制。在轴突始段(axon initial segment,AIS)远端高密度聚集的低阈值Na+通道亚型Nav1.6决定动作电位的爆发;而在AIS近端高密度聚集的高阈值Na+通道亚型Nav1.2促进动作电位向胞体和树突的反向传播。应用胞体和轴突的同时记录,我们发现胞体阈下膜电位的变化可以在轴突上传播较长的距离并可到达那些离胞体较近的突触前终末。进一步的研究证明了胞体膜电位的变化调控动作电位触发的突触传递,该膜电位依赖的突触传递是一种模拟式的信号传递。轴突上一类特殊K+通道(Kv1)的活动调制动作电位的波形,特别是其波宽,从而调控各种突触前膜电位水平下突触强度的变化。突触前终末的背景Ca2+浓度也可能参与模拟信号的传递。这些发现深化了我们对中枢神经系统内神经信号处理基本原理的认识,进而帮助我们理解脑如何工作。     

Glutamate receptor binding to cat central nervous system membranes

L-[3H]Glutamate exhibited specific binding to fresh membranes of cat CNS under physiological conditions of pH and temperature. This binding occurred in the absence of sodium ions. Kinetic analysis of the data for cerebellum suggested the presence of two distinct binding sites: a high-affinity process (Kd = 0.33 microM) with a capacity of 15 pmol/mg protein and a low-affinity process (Kd = 1.8 microM) which had a capacity of 65 pmol/mg protein. Several structural analogues of glutamic acid were able to appreciably inhibit the binding of [3H]glutamate. The distribution of glutamate binding between 12 regions of the CNS was measured. The amygdaloid complex exhibited the highest binding followed by hippocampus > hypothalamus identical to visual cortex identical to thalamus identical to caudate nucleus > olfactory bulb identical to tectum identical to cerebellum > dorsal pons identical to medulla > cervical spinal cord. These findings are consistent with the binding of [3H]glutamate being to its receptor.     

Function of thyroid hormone transporters in the central nervous system

Background

Iodothyronines are charged amino acid derivatives that cannot passively cross a phospholipid bilayer. Transport of thyroid hormones across plasma membranes is mediated by integral membrane proteins belonging to several gene families. These transporters therefore allow or limit access of thyroid hormones into brain. Since thyroid hormones are essential for brain development and cell differentiation, it is expected that genetic deficiency of such transporters would result in neurodevelopmental derangements.

Scope of review

We introduce concepts of thyroid hormone transport into the brain and into brain cells. Important thyroid hormone transmembrane transporters are presented along with their expression patterns in different brain cell types. A focus is placed on monocarboxylate transporter 8 (MCT8) which has been identified as an essential thyroid hormone transporter in humans. Mutations in MCT8 underlie one of the first described X-linked mental retardation syndromes, the Allan–Herndon–Dudley syndrome.

Major conclusions

Thyroid hormone transporter molecules are expressed in a developmental and cell type-specific pattern. Any thyroid hormone molecule has to cross consecutively the luminal and abluminal membranes of the capillary endothelium, enter astrocytic foot processes, and leave the astrocyte through the plasma membrane to finally cross another plasma membrane on its way towards its target nucleus.

General significance

We can expect more transporters being involved in or contributing to in neurodevelopmental or neuropsychiatric disease. Due to their expression in cellular components regulating the hypothalamus–pituitary–thyroid axis, mutations and polymorphisms are expected to impact on negative feedback regulation and hormonal setpoints. This article is part of a Special Issue entitled Thyroid hormone signalling.