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: 7268907
• 负责人: DEBOMOY K LAHIRI
• 金额: $ 29.43万
• 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 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7268907
• 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; Endopeptidases; Enzyme Gene; Enzymes; 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; Rate; 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）：我们的目标是研究阿尔茨海默病（AD）的基因调控，基于阿尔茨海默病的“淀粉样蛋白假说”。淀粉样β-肽（Abeta）的过度产生导致神经退行性步骤的级联反应，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什么因素导致Abeta及其大的Abeta前体蛋白（APP）过度生产。增加的Abeta产生可能是由于APP表达增加，或通过限制性β-APP裂解酶（BACE）进行蛋白水解加工。本提案的目标是研究i）APP的转录调节，因为APP（以及因此AP）生物合成始于转录水平，以及ii）BACE基因，因为Abeta过量产生可能是由于该基因上调导致的BACE水平增加。具体目的是：1）研究APP启动子的功能结构域及不同试剂对其活性的影响。我们将在功能上表征7.9 kb APP启动子，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。启动子将通过在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。2)确定APP和BACE基因调控中共同的特异性因子和细胞因子的作用。我们将描述IL-1 α、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）包含与至少两种蛋白质PuF和SkiP相互作用的调节结构域。我们将测试i）控制APP启动子活性的候选TF和ii）使用凝胶位移测定和DNA亲和色谱法在正常和AD脑组织中这种TF的状态。4)研究APP基因多态性对晚发性阿尔茨海默病发病风险的影响。我们在-3829和-1023位点发现了两个可能与阿尔茨海默病相关的多态性。我们将i）用启动子变体进行功能和DNA-蛋白质结合研究，ii）将启动子研究与APP和Abeta水平相关联。5)研究APP-5 '-UTR区。APP的表达也通过5 '非翻译区（UTR）进行调节。我们将测试APP 5 '-UTR在转录和转录后水平的双重作用，并研究其与细胞因子的相互作用。将分析来自具有特征性FAD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APP产生导致Aa产生至关重要。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。