A Peripheral Blood Biomarker for Alzheimer's Disease

阿尔茨海默病的外周血生物标志物

• 批准号: 10225621
• 负责人: Mark Muller
• 金额: $ 35.58万
• 依托单位: TOPOGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10225621
• 关键词: Acute; Acute Disease; Age; Aging; Alzheimer disease screening; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease diagnosis; Alzheimer' s disease pathology; Alzheimer' s disease patient; Alzheimer’s disease biomarker; Amyloid beta-Protein; Archives; Basic Science; Biological; Biological Assay; Biological Markers; Biological Response Modifiers; Blood; Blood Cells; Blood Platelets; Brain; Cell Line; Cell Lineage; Cells; Chronic Disease; Clinical; Clinical Trials; DNA; DNA Damage; DNA Methylation; DNA Modification Methylases; DNA Repair; Data; Data Analyses; Development; Diabetes Mellitus; Diagnosis; Diagnostic; Disease; Disease Management; Disease Progression; Distal; Drug Screening; Drug Targeting; Early Diagnosis; Elderly; Engineering; Enhancers; Epigenetic Process; Etiology; Event; Fibroblasts; Foundations; Gap Junctions; Gender; Gene Expression; Gene Silencing; Genes; Genetic; Genome; Ginkgo biloba; Goals; Harvest; Heritability; Human; Impaired cognition; Impairment; Insulin; Knowledge; Lead; Leukocytes; Link; Mediating; Mediator of activation protein; Memory; Metabolism; Metformin; Methods; Methylation; MicroRNAs; Mitochondria; Natural Products; Neurofibrillary Tangles; Neuroglia; Neurologic; Neurons; Organ; Oxidative Stress; Paper; Pathologic; Pathway interactions; Patients; Pattern; Peripheral; Pharmaceutical Preparations; Pharmacology; Physiological; Pilot Projects; Positioning Attribute; Presenile Alzheimer Dementia; Publishing; Race; Reactive Oxygen Species; Reporter; Reporter Genes; Reporting; Risk; Sampling; Sensitivity and Specificity; Serum; Smoker; Solid; Somatic Cell; Specificity; Testing; Therapeutic; Time; Tissues; Validation; Work; base; biomarker discovery; brain tissue; cellular imaging; cohort; cost effective; design; diagnostic biomarker; early detection biomarkers; epigenome; epigenome-wide association studies; exosome; human disease; innovation; man; methylation pattern; methylome; nervous system disorder; neuron loss; neuropathology; non-smoker; novel; operation; oxidative DNA damage; patient subsets; peripheral blood; predictive modeling; predictive test; pressure; repaired; response; sample archive; screening; therapeutic target; tool

DNA methylation is a central epigenetic process. All diseases have genetic and/or epigenetic foundations; however only epigenetics can be altered pharmacologically. Thus, epigenetic pathway components are not only excellent epi-therapeutic drug targets, they are also established biomarkers in many acute and chronic diseases. The information stored in 5-methyl-C (mC) is passed down in a heritable and stable fashion; yet, we know that DNA methylation profiles are frequently altered in a number of diseases including Alzheimer’s Disease (AD) where differentially methylated regions (DMRs) have been identified in blood that are similarly found in brain. This means an AD specific pathway or mechanism exists that is mediating alterations in the methylome specifically in peripheral leucocytes, brain and presumably other tissues. Over the past 10y the PI has been working on mC revision pathways and has published numerous papers on the topic. Despite convincing evidence for DMRs in peripheral blood of AD patients, the mechanism of mC re-programming in is unknown. We describe new tools to interrogate mC revisions in neurologically derived cell lines. We devised a screen that uses a neutral GFP gene (i.e., one with no selective pressure) to track changes in DNA methylation in a cellular context. Any forward (more DNA methylation) or backward (less methylation) alters gene expression in a permanent fashion. Cytofluorimetry and cell imaging using engineered reporters, quantifies GFP expression as an indirect readout for mC changes. The method was developed as a screen to identify epi-therapeutics and has been used successfully in this capacity. It is cost effective, simple, robust and will detect biological effectors that alter DNA methylation. We call this the ADEE (AD Epigenetic Effector) screen since it detects epigenetic bio-effectors in AD sera that revise DNA methylation patterns. Also, the ADEE screen works in a lineage specific cell context giving high physiological relevance. Most agree that there is an acute need for peripheral blood biomarkers in AD. We demonstrate that small amounts of serum (5µl corresponding to 5%) induced a strong uptick in GFP expression of a heavily methylated reporter gene, revealing the presence of a potent hypomethylating activity in the peripheral blood of AD patients but not in age-match controls. Since ADEEs appear highly active in AD serum, they may represent early tractable biomarkers for early AD onset, disease progression and management. In support of this, epigenome-wide association studies clearly show that changes in DNA methylation are an early antecedent event to clinical manifestation and are tightly associated with AD neuropathology. Archived serum samples (>600) from the GEMS (Gingko biloba Memory Study) will be used to test reliability of the ADEE biomarker. A subset of the patients in the GEM study displayed no cognitive impairment at baseline (at the start of the trial). Over the course of the next 8y in trial, some patients were diagnosed with AD and serum samples were harvested and stored; thus we have sera on the same cohort before and after diagnosis in sufficient numbers to derive high-powered statistical validation regarding positive and negative predictive power of ADEEs. In summary, the ADEE screen is important and well justified for two key reasons: (1) Similar DMRs are found distributed across brain tissue and peripheral blood leukocytes, so we suspected that the ADEE causing (or enabling) the DMRs will be a circulating factor or factors. (2) We developed an innovative cell-based assay proven to test for the presence of ADEE’s. Since we have strong evidence that AD patient serum contains epigenetic effectors, we are uniquely positioned to validate ADEE as a blood biomarker that differentiates AD from normal patients for early detection. We also plan initial characterizations of the ADEEs in order to optimize the test, extend our knowledge of how AD progresses and (as a long term goal) to identify new druggable epigenetic targets. Our specific aims are designed to: 1. Rigorously Test Reliability of ADEE Biomarkers: specificity and selectivity. 2. Optimize, validate and characterize live cell screening operations and assay quality. 3. Carry out validation data analyses in the same patient cohort before and after AD.

DNA甲基化是一个重要的表观遗传过程。所有疾病都有遗传和/或表观遗传 基础;然而，只有表观遗传学可以改变。因此，表观遗传途径 成分不仅是极好的表观治疗药物靶点，它们也是确定的生物标志物， 许多急性和慢性疾病。存储在5-甲基-C（mC）中的信息以一种 遗传和稳定的方式;然而，我们知道，DNA甲基化谱经常改变， 许多疾病，包括阿尔茨海默病（AD），其中差异甲基化区域（DMR） 在血液中发现了类似于在大脑中发现的物质。这意味着AD特异性途径或 存在介导特异性外周白细胞中甲基化组改变的机制， 大脑和其他组织在过去的10年里，PI一直致力于mC翻修 他还发表了许多关于这一主题的论文。尽管有令人信服的证据表明， 在AD患者的外周血中，mC重编程的机制尚不清楚。我们描述了新 在神经学衍生的细胞系中询问mC修订的工具。我们设计了一个屏幕， 中性GFP基因（即，一个没有选择压力）来跟踪细胞中DNA甲基化的变化。 上下文任何正向（更多的DNA甲基化）或反向（更少的甲基化）都会改变基因表达。 永久的时尚。使用工程化报告基因的细胞荧光测定法和细胞成像，定量GFP 表达作为mC变化的间接读数。该方法被开发作为一种筛选来鉴定 并已成功地用于这一能力。它具有成本效益，简单，坚固， 将检测改变DNA甲基化的生物效应物。我们称之为ADEE（AD表观遗传学 效应子）筛选，因为它检测AD血清中修正DNA甲基化模式的表观遗传生物效应子。 此外，ADEE筛选在谱系特异性细胞环境中起作用，从而提供高生理相关性。 大多数人认为，AD患者迫切需要外周血生物标志物。我们证明了 少量的血清（5μl，相当于5%）诱导GFP表达的强烈上升， 高度甲基化的报告基因，揭示了在细胞中存在有效的低甲基化活性。 AD患者的外周血中，但不是在年龄匹配的对照。由于ADEE在AD中表现出高度活性， 血清中，它们可能代表早期AD发作、疾病进展和 管理为了支持这一点，表观基因组关联研究清楚地表明，DNA的变化 甲基化是临床表现的早期先行事件，与AD密切相关 神经病理学 来自GEMS（银杏记忆研究）的存档血清样本（>600）将用于测试 ADEE生物标志物的可靠性。GEM研究中的一部分患者没有表现出认知障碍， 基线时（试验开始时）的损伤。在接下来的8年试验中，一些患者 被诊断患有AD，收集并储存血清样品;因此，我们在 诊断前后的同一队列中有足够数量的患者，以获得高效力的统计学验证 关于ADEE的阳性和阴性预测能力。 总而言之，ADEE屏幕很重要，有两个关键原因： (1)发现类似的DMR分布在脑组织和外周血白细胞中，所以我们 怀疑导致（或启用）DMR的ADEE将是一个或多个循环因素。 (2)我们开发了一种创新的基于细胞的检测方法，证明可以检测ADEE的存在。既然我们 有强有力的证据表明，AD患者血清含有表观遗传效应子，我们处于独特的地位， 验证ADEE作为区分AD与正常患者的早期检测的血液生物标志物。我们 还计划ADEE的初始表征，以优化测试，扩展我们对 如何AD进展和（作为长期目标），以确定新的药物表观遗传目标。 我们的具体目标是： 1.严格测试ADEE生物标志物的可靠性：特异性和选择性。 2.优化、验证和表征活细胞筛选操作和检测质量。 3.在AD前后对同一患者队列进行验证数据分析。