APP MRNA DYSREGULATION AND ALZHEIMER'S DISEASE

APP mRNA 失调与阿尔茨海默病

• 批准号: 2051915
• 负责人: James S Malter
• 金额: $ 11.69万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-29 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2051915
• 关键词: Alzheimer's disease; RNA binding protein; SDS polyacrylamide gel electrophoresis; adenine nucleotides; affinity chromatography; amyloid proteins; cell free system; chemical binding; complementary DNA; gel electrophoresis; genetic regulation; genetic transcription; interleukin 1; ionophores; laboratory mouse; laboratory rabbit; messenger RNA; molecular cloning; monoclonal antibody; mutant; neurotrophic factors; nucleic acid sequence; oligonucleotides; phorbols; polymerase chain reaction; posttranscriptional RNA processing; tissue /cell culture; transfection; tumor necrosis factor alpha; uracil nucleotides; western blottings

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder which culminates in senile dementia. The etiology of AD is unknown although a variety of data suggests that excessive extracellular deposition of amyloid beta/A4 protein preceded by intracellular accumulation of its coding mRNA may be important. Nucleotide sequence analysis of amyloid protein precursor (APP) -695, 714, 751 and 770 mRNAs reveals the presence of 4 reiterations of the destabilizing pentanucleotide motif adenosine-uridine- uridine-uridine-adenosine (AUUUA) which targets cytokine and oncogene mRNAs for rapid, cytoplasmic degradation. We have recently identified a novel, cytoplasmic protein which specifically binds to the AUUUA elements of cytokine and oncogene mRNAs. This protein (which we have denoted as the adenosine-uridine binding factor or AUBF) is upregulated through phosphorylation by phorbol ester, ionophore or cytokines and likely participates in the stabilization of cytokine mRNAs by binding to the AUUUA motifs. Cytoplasmic lysates from rodent and human brain and a human neuroblastoma cell line constitutively express AUBF activity as well as three additional, novel, brain-specific AUUUA binding activities (denoted AUBF-B). These activities can be modulated by cytokines and specifically bind to APP mRNAs through the AUUUA motifs. Therefore, we hypothesize that the AUUUA containing APP mRNAs are normally unstable. The binding of AUBF or AUBF-B to APP mRNAs through the AUUUA motifs stabilizes the APP message leading to a relative accumulation of APP mRNA and subsequent APP production in the brain of AD patients. Thus the specific aims of this proposal are to: 1). Establish the turnover rates of the four wild type APP mRNAs (695, 714, 751, 770) as well as mutated APP transcripts with reduced numbers of AUUUA motifs after the stable transfection of their coding cDNAs into a human neuroblastoma cell line (SH-SY-5Y). The effects of IL-1, tumor necrosis factor, nerve growth factor and calcium ionophore on the turnover of wild type and mutant APP mRNAs and the level of AUBF and AUBF-B activities will be determined; 2). Purify AUBF-B activities from human brain by RNA affinity chromatography and generate polyclonal and monoclonal antibodies against these proteins. 3). Employ a cell-free RNA degradation system derived from rabbit reticulocyte lysate to assess the turnover rates of wild-type and mutant APP mRNAs; 4). Establish the function of AUBF-B by adding purified protein(s) the cell-free degradation system followed by measurement of the turnover rate of wild-type and mutant APP mRNAs. Cumulatively these studies will clarify the roles of posttranscriptional gene regulation and AUBF-B in the accumulation of APP mRNAs.

阿尔茨海默氏病（AD）是一种进行性神经退行性疾病， 最终在老年痴呆症中。 AD的病因尚不清楚，尽管 多种数据表明淀粉样蛋白的细胞外沉积过多 β/A4蛋白之前的编码mRNA的细胞内积累 可能很重要。 淀粉样蛋白的核苷酸序列分析 前体（APP）-695、714、751和770 mRNA揭示了4 破坏戊核苷酸基序腺苷 - 尿尿苷的重申 尿苷 - 尿苷 - 腺苷（AUUUA）靶向细胞因子和癌基因mRNA 用于快速的细胞质降解。 我们最近确定了一部小说 细胞质蛋白特异性结合与AUUA元素的结合 细胞因子和癌基因mRNA。 这种蛋白质（我们称为 通过 佛波酯，离子载体或细胞因子磷酸化，可能 通过与AUUUA结合，参与细胞因子mRNA的稳定 主题。 来自啮齿动物和人脑的细胞质裂解物以及人类 神经母细胞瘤细胞系组成型表达AUBF活性以及 另外三种新型的，新颖的大脑特异性AUUUA结合活性（表示 aubf-b）。 这些活动可以通过细胞因子，特别是 通过AUUUA图案结合到应用程序mRNA。 因此，我们假设 包含应用程序mRNA的AUUA通常是不稳定的。 aubf的结合 或通过AUUUA主题稳定应用程序的AUBF-B到应用程序mRNA 导致App mRNA的相对积累和随后的应用 在AD患者的大脑中产生。 因此，具体目的 提案为：1）。建立四个野生型应用程序的周转率 mRNA（695、714、751、770）以及降低的突变应用转录本 稳定转染其编码cDNA后的Auuua图案的数量 进入人类神经母细胞瘤细胞系（SH-SY-5Y）。 IL-1的影响， 肿瘤坏死因子，神经生长因子和钙离子载体在 野生型和突变应用程序mRNA的营业额以及AUBF和AUBF-B的水平 将确定活动； 2）。净化人类的AUBF-B活动 大脑通过RNA亲和力色谱法，产生多克隆和单克隆 针对这些蛋白质的抗体。 3）。使用无细胞的RNA降解 源自兔网状细胞裂解物以评估周转率的系统 野生型和突变的应用mRNA； 4）。通过建立AUBF-B的功能 添加纯化的蛋白质无细胞降解系统，然后 测量野生型和突变体应用程序mRNA的周转率。 累计这些研究将阐明转录后的作用 基因调控和APP mRNA积累中的AUBF-B。