Regulation of neuronal NO by apolipoprotein E

载脂蛋白 E 对神经元 NO 的调节

• 批准号: 6479691
• 负责人: CAROL Anne COLTON
• 金额: $ 36.58万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-03-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6479691
• 关键词: Alzheimer's disease; RNase protection assay; aminoacid metabolism; apolipoprotein E; arginine; enzyme activity; genetically modified animals; genotype; human tissue; immunocytochemistry; laboratory mouse; neurons; nitric oxide synthase; nitrogen oxides; oxidative stress; polymerase chain reaction; protein isoforms; protein structure function; synapses; tissue /cell culture; western blottings

DESCRIPTION (Adapted from applicant's abstract): Loss of synaptic contacts, formation of neurofibrillary tangles and neuronal death are characteristic features of Alzheimer's disease. Oxidative stress causes neurodegeneration and affected neurons in AD demonstrate biochemical footprints of the past presence of oxidative stress. We reported that apolipoprotein E (apoE) regulates NO production, a key reactive oxygen/nitrogen species that causes oxidative stress. Our additional new data demonstrate that apolipoprotein E alters the neuronal uptake of L-arginine, the substrate* converted by nitric oxide synthase (NOS) into NO. We hypothesize that apoE regulates the function of cationic amino acid (CAT) transporters in an isoform specific manner, thereby altering the entry of L- arginine into the neuron which is then converted to NO by the action of NOS. We will test this hypothesis by measuring the functional activity of the CAT transporter in murine primary neuronal cultures exposed to human apoE and its isoforms and in primary neuronal cultures from mouse models expressing isoforms of human APOE and its gene products. Using semi-quantitative and quantitative measures, we will determine the effect of apoE and APOE genotype on CAT transporter mRNA and protein expression. Translation to NO production will be measured both directly by determining supernatant nitrite levels produced by NOS -containing neurons and indirectly by measuring indices of past-presence of NO (nitrotyrosine and hemeoxygenase 1). The validity of the mouse model will be tested by determining the apoE isoform dependency of arginine transporters in post-mortem human brain from individuals with and without previous clinical signs of dementia due to Alzheimer's disease.

描述（改编自申请人的摘要）：突触接触丧失， 神经原纤维缠结的形成和神经元死亡是其特征 阿尔茨海默病的特征。氧化应激会导致神经退行性变 AD 中受影响的神经元表现出过去存在的生化足迹 氧化应激。我们报道了载脂蛋白 E (apoE) 调节 NO 产生，一种导致氧化的关键活性氧/氮物种 压力。我们额外的新数据表明载脂蛋白 E 改变了 神经元摄取 L-精氨酸，这是一氧化氮转化的底物* 合酶（NOS）转化为NO。我们假设 apoE 调节以下功能： 阳离子氨基酸（CAT）转运蛋白以异构体特异性方式，从而 改变 L-精氨酸进入神经元，然后转化为 NO 通过NOS的作用。我们将通过测量泛函来检验这个假设 小鼠原代神经元培养物中 CAT 转运蛋白的活性 人类 apoE 及其亚型以及小鼠模型的原代神经元培养物 表达人类 APOE 及其基因产物的亚型。使用 半定量和定量措施，我们将确定的效果 apoE 和 APOE 基因型对 CAT 转运蛋白 mRNA 和蛋白表达的影响。 转化为 NO 产量将通过确定直接测量 含有 NOS 的神经元产生的上清液亚硝酸盐水平，并间接 通过测量NO（硝基酪氨酸和血红素加氧酶）过去存在的指数 1）。通过测定apoE来检验小鼠模型的有效性 死后人脑中精氨酸转运蛋白的异构体依赖性 先前有或没有痴呆症临床症状的个体 阿尔茨海默病。