REGULATION OF GLUTAMINE TRANSPORT IN AGING BRAIN

衰老大脑中谷氨酰胺转运的调节

基本信息

批准号：
2859705
负责人：
NEVILLE BROOKES
金额：
$ 7.43万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-01 至 2002-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2859705
关键词：
Xenopus oocyte aminoacid transport antibody formation antisense nucleic acid astrocytes cell senescence glutamate transporter glutamine hybrid cells immunocytochemistry laboratory mouse laboratory rabbit laboratory rat neural transmission neuroregulation oligonucleotides protein isoforms tissue /cell culture

项目摘要

Astrocytes in the brain take up glutamate released by neurons, metabolize glutamate and ammonia to form glutamine, and export glutamine to the extracellular neuronal microenvironment. Of these processes, the export of glutamine is least well understood. Our preliminary studies of murine astrocytes in primary culture show that a widespread transport activity identified functionally as System ASC is the likely mediator of glutamine export. Three cloned transporters (ASCT1, ASCT2, ATB0), related to the glutamate transporter family, are known to exhibit System ASC-like properties, but the substrate specificities of ASCT1 and ATB0 clearly differ from System ASC-mediated glutamine transport in astrocytes. Other preliminary data from our laboratories show that ASCT2 transcripts are abundant in astrocyte cultures. We therefore hypothesize that the ASCT2 transporter mediates glutamine export from astrocytes. In this role, ASCT2 creates a novel linkage between glutamine export and glutamatergic neurotransmission. This linkage occurs because ASCT2 mediates obligatory exchange between glutamine and extracellular L-alanine, L-serine and D-serine, which are endogenous agonists at the glycine modulatory site on the N-methyl-D-aspartate subtype of glutamate receptors. The rate of glutamine export thus exerts a regulatory effect on extracellular levels of agonists at the glycine modulatory site, and consequently on glutamatergic efficacy, with resultant implications for cognitive function and excitotoxic neurodegenerative processes. We propose in this pilot project to clarify the role of the ASCT2 transporter in glutamine export from murine (or alternatively rat) astrocytes using three approaches: (1) We will determine whether the detailed substrate specificity and exchange properties of System ASC-mediated glutamine transport in astrocyte cultures are consistent with the pattern exhibited by the ASCT2 transporter expressed in Xenopus laevis oocytes; (2) We will measure the effect of hybrid depletion of ASCT2, using antisense oligonucleotides, on the velocity of System ASC-mediated glutamine transport in astrocyte cultures; (3) Because ASCT2 expression in astrocyte cultures may not reflect the level of expression in vivo, we will raise in rabbits polyclonal antibodies directed against the ASCT2 transporter in order to detect ASCT2 expression immonocytochemically in astrocytes dissociated acutely from murine brain slices. The results of this pilot project will make possible further investigation of a) the regulation of glutamine export from astrocytes during proliferation, maturation and senescence, and b) the functional implications for the aging brain.

大脑中的星形胶质细胞吸收神经元释放的谷氨酸，代谢谷氨酸和氨形成谷氨酰胺，并将谷氨酰胺输出到细胞外神经元微环境。在这些过程中，谷氨酰胺的输出是人们最不了解的。我们对原代培养物中的鼠星形胶质细胞的初步研究表明，功能上被识别为系统 ASC 的广泛转运活动可能是谷氨酰胺输出的介质。已知与谷氨酸转运蛋白家族相关的三个克隆转运蛋白（ASCT1、ASCT2、ATB0）表现出类似系统 ASC 的特性，但 ASCT1 和 ATB0 的底物特异性明显不同于星形胶质细胞中系统 ASC 介导的谷氨酰胺转运。我们实验室的其他初步数据表明，星形胶质细胞培养物中 ASCT2 转录物丰富。因此，我们假设 ASCT2 转运蛋白介导星形胶质细胞的谷氨酰胺输出。在这个角色中，ASCT2 在谷氨酰胺输出和谷氨酸能神经传递之间建立了一种新的联系。这种联系的发生是因为 ASCT2 介导谷氨酰胺与细胞外 L-丙氨酸、L-丝氨酸和 D-丝氨酸之间的强制性交换，这些物质是谷氨酸受体 N-甲基-D-天冬氨酸亚型上甘氨酸调节位点的内源性激动剂。因此，谷氨酰胺输出速率对甘氨酸调节位点的激动剂的细胞外水平产生调节作用，从而对谷氨酸能功效产生调节作用，从而对认知功能和兴奋毒性神经退行性过程产生影响。我们建议在这个试点项目中使用三种方法阐明 ASCT2 转运蛋白在小鼠（或大鼠）星形胶质细胞谷氨酰胺输出中的作用：（1）我们将确定系统 ASC 介导的谷氨酰胺转运的详细底物特异性和交换特性是否星形胶质细胞培养物中的ASCT2转运蛋白与非洲爪蟾卵母细胞中表达的ASCT2转运蛋白表现出的模式一致； (2) 我们将使用反义寡核苷酸测量 ASCT2 的杂合耗竭对星形胶质细胞培养物中系统 ASC 介导的谷氨酰胺转运速度的影响； (3)由于星形胶质细胞培养物中ASCT2的表达可能不能反映体内的表达水平，我们将在兔中培养针对ASCT2转运蛋白的多克隆抗体，以便通过免疫细胞化学方法检测从小鼠脑切片中急性分离的星形胶质细胞中ASCT2的表达。该试点项目的结果将使进一步研究a）星形胶质细胞在增殖、成熟和衰老过程中谷氨酰胺输出的调节，以及b）对衰老大脑的功能影响成为可能。