Regulation of APP Pathway Gene Promoters in Alzheimer's

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。淀粉样蛋白β肽（Abeta）的过量产生导致一系列神经退行性步骤，导致斑块形成和神经元丢失，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）过量生产。β -产生的增加可能是由于APP表达的增加，或者是由于限制β -APP切割酶（BACE）对其进行蛋白水解处理。本提案的目标是研究i) APP的转录调控，因为APP（因此，AP）的生物发生始于转录水平，ii) BACE基因，因为β过量生产可能是由于BACE基因上调导致BACE水平升高。具体目的是：1)研究APP启动子的功能域及不同药物对其活性的影响。我们将对7.9 kb的APP启动子进行功能表征，并研究内在（细胞因子）和外在（金属）因子如何调节启动子活性。启动子将在不同细胞类型和原代神经元培养中通过序列缺失、诱变和转染实验进行研究。2)确定APP和BACE基因调控共有的特定因子和因子的作用。我们将描述il -1 α、tnf - α和CREB转录因子（TF）在4.1kb BACE启动子活性中的作用。3)鉴定细胞类型特异性核因子。APP启动子的一个30 bp的新区域（-76-47）包含一个与至少两种蛋白PuF和SkiP相互作用的调控结构域。我们将测试i)控制APP启动子活性的候选TF和ii)这些TF在正常和AD脑组织中的状态，使用凝胶移位法和dna亲和色谱法。4)研究APP基因多态性对晚发型阿尔茨海默病发病风险的影响。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)进行启动子变异的功能和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区域。APP的表达也通过5'-未翻译区（UTR）进行调控。我们将测试APP5'-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。将分析具有特征FAD家族的细胞系中APP和BACE基因的差异表达。研究APP和BACE基因的调控是了解APP产生导致Aa产生的关键。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。

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.