MECHANISMS FOR GLUTAMATE TRANSPORTER ALTERATIONS IN AD

描述(摘自申请者的摘要)：谷氨酸是哺乳动物中枢神经系统中主要的兴奋性神经递质。谷氨酸通常通过位于神经元和神经胶质细胞中的高亲和力、钠依赖的谷氨酸转运体从突触裂隙中清除。谷氨酸转运障碍可导致谷氨酸在突触中过量积聚，从而导致神经毒性。EAAT2是一种星形胶质谷氨酸转运体，是主要的谷氨酸转运体。在阿尔茨海默病(AD)的受累区域以及肌萎缩侧索硬化症(ALS)中发现了这种蛋白的丢失。反义基因敲除研究以及EAAT2基因缺失的小鼠已经证明，EAAT2蛋白的丢失可以导致兴奋性毒性神经元变性。在这些神经退行性疾病中，EAAT2蛋白选择性丢失的原因是什么？这位研究人员最近在ALS中的研究表明，EAAT2的丢失是由于mRNAs的异常，可能是异常剪接的结果。在这项研究中，本研究旨在验证异常的mRNAs可能解释AD中EAAT2的丢失。初步结果表明，AD标本中存在EAAT2mRNAs异常。研究人员将确定异常EAAT2mRNA物种的丰度，评估异常EAAT2mRNAs在AD患者中的患病率，以及它们是否与EAAT2蛋白丢失相关。这位研究人员还建议调查在带有APP(淀粉样前体蛋白)突变的转基因小鼠中是否存在EAAT2 mRNAs的异常。在体内，EAAT2基因的异常是否会导致EAAT2蛋白的丢失并导致神经变性？为了回答这个问题，他们将产生转基因小鼠，表现出人类野生型和异常EAAT2mRNAs的星形胶质细胞特异性表达。是什么机制导致了这些异常的剪接缺陷？他们将确定EAAT2基因是否存在获得性体细胞突变。这位研究人员建议从AD脑中培养星形胶质细胞，并确定是否可以在体外产生异常的EAAT2 mRNAs。

DESCRIPTION (Adapted from applicant's abstract):.Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system. Glutamate is normally cleared from the synaptic cleft by high-affinity, sodium-dependent glutamate transporters located in both neurons and glia. Glutamate transport malfunction can lead to the accumulation of excessive glutamate in the synapse, with subsequent neurotoxicity. EAAT2 is an astroglial glutamate transporter and is the predominant glutamate transporter. A loss of this protein has been found in the affected areas of Alzheimer's disease (AD) as well as in amyotrophic lateral sclerosis (ALS). Antisense knockdown studies as well as EAAT2 null mice have demonstrated that loss of EAAT2 protein can lead to excitotoxic neuronal degeneration. What could account for the selective loss of EAAT2 protein in these neurodegenerative diseases? The investigator's recent studies in ALS have demonstrated that the loss of EAAT2 is due to aberrant mRNAs, possibly as a consequence of abnormal splicing. In this study it is proposed to test the possibility that the aberrant mRNAs could account for the loss of EAAT2 in AD. Preliminary results demonstrate that aberrant EAAT2 mRNAs are present in AD specimens. The investigator will determine the abundance of the aberrant EAAT2 mRNA species and evaluate the prevalence of the aberrant EAAT2 mRNAs in AD patients and whether they are correlated with loss of EAAT2 protein. The investigator also proposes to investigate whether there are aberrant EAAT2 mRNAs in transgenic mice with APP (amyloid precursor protein) mutations. Can aberrant EAAT2 mRNAs lead to loss of EAAT2 protein and contribute to neurodegeneration in vivo? To answer this they will generate transgenic mice manifesting astrocyte-specific expression of the human wild-type and aberrant EAAT2 mRNAs. What mechanism accounts for these aberrant splicing defects? They will determine whether there are acquired somatic mutations in the EAAT2 gene. The investigator proposes to develop astrocyte cultures from AD brain and determine if the aberrant EAAT2 mRNAs can be produced in vitro.