Regulation of APP Pathway Gene Promoters in Alzheimer's

阿尔茨海默病中 APP 通路基因启动子的调控

• 批准号: 7674686
• 负责人: DEBOMOY K LAHIRI
• 金额: $ 28.76万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7674686
• 关键词: 5' Flanking Region; 5' Untranslated Regions; Abeta synthesis; Accounting; Address; Affect; Affinity Chromatography; Aging; Alleles; Alzheimer disease prevention; Alzheimer' s Disease; Alzheimer' s disease risk; Amyloid; Amyloid Proteins; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Apolipoprotein E; Apolipoproteins; Applications Grants; Biogenesis; Brain; Brain region; C-terminal; CREB1 gene; Cell Line; Cleaved cell; DNA-Binding Proteins; Deletion Mutagenesis; Deposition; Diet; Disease; Drug Delivery Systems; Electrophoretic Mobility Shift Assay; Enzyme Gene; Enzymes; Epidemiology; Etiology; Family; Fatty acid glycerol esters; Gene Expression; Gene Expression Regulation; Gene Proteins; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Transcription; Genomics; Goals; Growth Factor; Inflammatory; Interleukin-1 alpha; Journals; Late Onset Alzheimer Disease; Length; Mediating; Memory; Metals; Molecular Chaperones; Nerve Degeneration; Neurons; Nuclear; Nucleic Acid Regulatory Sequences; Paper; Pathogenesis; Pathway interactions; Peer Review; Peptide Hydrolases; Peptides; Play; Process; Production; Progress Reports; Property; Protein Precursors; Proteins; Proteolytic Processing; Publications; Publishing; Regulation; Regulatory Element; Regulatory Pathway; Research Personnel; Resources; Risk; Role; SP1 gene; Site; Site-Directed Mutagenesis; Testing; Tissues; Transcriptional Regulation; Transfection; Tumor Necrosis Factor-alpha; Up-Regulation; Variant; Work; base; brain tissue; cell type; cognitive function; cytokine; enzyme activity; human TNF protein; interest; microbial alkaline proteinase inhibitor; neuron loss; novel; programs; promoter; protein expression; research study; secretase; transcription factor

DESCRIPTION (provided by applicant): Our goal is to study gene regulation in Alzheimer's disease (AD), based on the "amyloid hypothesis" of Alzheimer's disease. Overproduction of the amyloid beta-peptide (Abeta) causes a cascade of neurodegenerative steps resulting in plaque formation and neuronal loss that characterize Alzheimer's disease. The unresolved key question in the field is what factors cause overproduction of Abeta and its large Abeta precursor protein (APP). Increased Abeta production may result from an increase in APP expression, or in its proteolytic processing by the limiting beta-APP cleaving enzyme (BACE). The goals of this proposal are to investigate the transcriptional regulation of i) APP, because APP (and, hence, AP) biogenesis begins at the level of transcription, and ii) BACE gene, as Abeta overproduction may be due to increased BACE level as a result of upregulation in this gene. Specific Aims are: 1) To study the functional domains of the APP promoter and effects of different agents on its activity. We will functionally characterize the 7.9 kb APP promoter and study how intrinsic (cytokines) and extrinsic (metals) factors regulate promoter activity. Promoter will be studied by serial deletions, mutagenesis and transfection experiments in different cell types and primary neuronal cultures. 2) To identify the effects of specific factors and cvtokines common to both APP and BACE gene regulation. We will characterize the role of IL-1alpha, TNF-alpha and CREB transcription factor (TF) on 4.1kb BACE promoter activity. 3) To identify cell type-specific nuclear factors. A 30 bp novel region (-76-47) of the APP promoter contains a regulatory domain that interacts with at least two proteins, PuF and SkiP. We will test i) the candidate TFs that control APP promoter activity and ii) the status of such TF in normal and AD brain tissues using gel shift assay and DNA-affinity chromatography. 4) To characterize APP gene polymorphisms that influences the risk of late-onset Alzheimer's disease. We discovered two polymorphisms at -3829 and -1023 that may be associated with Alzheimer's disease. We will i) do functional and DNA-protein binding studies with promoter variants and ii) correlate promoter studies with levels of APP and Abeta. 5) To study the APP-5'-UTR region. APP expression is also regulated via the 5'-untranslated region (UTR). We will test a dual role for the APP5'-UTR at both transcriptional and post-transcriptional levels, and study its interaction with cytokines. Cell lines from families with characterized FAD will be analyzed for differential expression of the APP and BACE genes. Studying APP and BACE gene regulation is crucial to understand APP production leading to Aa generation. These studies should help developing suitable drug targets for the treatment of Alzheimer's disease.

描述（由申请人提供）：我们的目标是基于阿尔茨海默病的“淀粉样蛋白假说”，研究阿尔茨海默病（AD）的基因调控。 β-淀粉样肽 (Abeta) 的过量产生会导致一系列神经退行性步骤，导致斑块形成和神经元损失，这是阿尔茨海默病的特征。该领域尚未解决的关键问题是哪些因素导致Abeta及其大的Abeta前体蛋白（APP）过量产生。 Abeta 产量增加可能是由于 APP 表达增加或限制性 β-APP 裂解酶 (BACE) 对其蛋白水解加工造成的。该提案的目标是研究 i) APP 的转录调控，因为 APP（以及 AP）的生物起源始于转录水平，以及 ii) BACE 基因，因为 Abeta 过量生产可能是由于该基因上调导致 BACE 水平增加。具体目标是： 1) 研究APP启动子的功能域以及不同药物对其活性的影响。我们将对 7.9 kb APP 启动子进行功能表征，并研究内在（细胞因子）和外在（金属）因素如何调节启动子活性。将通过不同细胞类型和原代神经元培养物中的连续缺失、诱变和转染实验来研究启动子。 2) 确定 APP 和 BACE 基因调控共有的特定因素和细胞因子的影响。我们将描述 IL-1α、TNF-α 和 CREB ​​转录因子 (TF) 对 4.1kb BACE 启动子活性的作用。 3) 鉴定细胞类型特异性核因子。 APP 启动子的 30 bp 新区域 (-76-47) 包含一个与至少两种蛋白质 PuF 和 SkiP 相互作用的调节结构域。我们将使用凝胶位移测定和 DNA 亲和层析测试 i) 控制 APP 启动子活性的候选 TF，以及 ii) 正常和 AD 脑组织中此类 TF 的状态。 4) 表征影响迟发性阿尔茨海默氏病风险的 APP 基因多态性。我们发现-3829和-1023位点的两个多态性可能与阿尔茨海默病有关。我们将 i) 使用启动子变体进行功能和 DNA 蛋白结合研究，ii) 将启动子研究与 APP 和 Abeta 水平相关联。 5) 研究APP-5'-UTR区域。 APP 表达也通过 5'-非翻译区 (UTR) 进行调节。我们将测试 APP5'-UTR 在转录和转录后水平上的双重作用，并研究其与细胞因子的相互作用。将分析来自具有 FAD 特征的家族的细胞系的 APP 和 BACE 基因的差异表达。研究 APP 和 BACE 基因调控对于了解导致 Aa 生成的 APP 生产至关重要。这些研究应该有助于开发治疗阿尔茨海默病的合适药物靶点。