A Peripheral Blood Biomarker for Alzheimer's Disease

DNA甲基化是一个中心的表观遗传过程。所有疾病都有遗传性和/或表观遗传性 基础；然而，只有表观遗传学才能从药理上改变。因此，表观遗传途径 成分不仅是良好的表观治疗药物靶点，它们也是在 许多急慢性疾病。存储在5-甲基-C(MC)中的信息在 可遗传和稳定的方式；然而，我们知道DNA甲基化特征经常以一种 疾病的数量，包括阿尔茨海默病(AD)的差异甲基化区域(DMR) 已经在血液中发现，在大脑中也有类似的发现。这意味着AD特定的途径或 存在一种机制，即介导外周血白细胞中特定的甲基组的变化， 大脑，大概还有其他组织。在过去的10年里，PI一直致力于MC的修订 路径，并发表了许多关于这一主题的论文。尽管有令人信服的证据证明DMR在 AD患者外周血中MC重编程的机制尚不清楚。我们描述了新的 在神经学来源的细胞系中审问MC修订的工具。我们设计了一个屏幕，它使用 中性GFP基因(即没有选择压力的基因)，用于跟踪细胞中DNA甲基化的变化 背景。任何向前(更多的DNA甲基化)或向后(更少的甲基化)都会改变基因的表达 一种永久的时尚。使用工程记者的细胞荧光测定法和细胞成像，量化绿色荧光蛋白 表达式作为MC更改的间接读数。该方法被开发为一种屏幕，用于识别 计划免疫疗法，并已成功地用于这一能力。它具有成本效益、简单、坚固和 将检测改变DNA甲基化的生物效应物。我们称之为ADIE(AD表观遗传学 效应器)筛选，因为它在AD血清中检测到修改DNA甲基化模式的表观遗传生物效应器。 此外，ADIE屏幕在特定血统的细胞环境中工作，提供高度的生理相关性。 大多数人同意AD患者迫切需要外周血生物标记物。我们证明了 微量血清(5微克L相当于5%)可诱导小鼠成纤维细胞绿色荧光蛋白表达显著上调。 高度甲基化的报告基因，揭示了在体内存在有效的去甲基化活性 阿尔茨海默病患者外周血，而年龄匹配对照组则不是。由于ADEE在AD中看起来非常活跃 血清中，它们可能是早期AD发病、疾病进展和 管理层。为了支持这一点，表观基因组范围的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兆事件，与AD密切相关 神经病理学。 来自宝石(银杏记忆研究)的存档血清样本(&gt；600)将用于测试 ADIE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能力 基线损伤(试验开始时)。在接下来的8年试验中，一些患者 被诊断出患有阿尔茨海默病，并收集和储存血清样本；因此，我们有关于 在诊断前和诊断后有足够数量的相同队列，以获得强大的统计验证 关于ADEE的正负预测能力。 总而言之，ADIE屏幕非常重要，有两个关键原因： (1)相似的DMR分布于脑组织和外周血白细胞，因此我们 怀疑引起(或促成)DMRS的ADE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，被证明可以检测阿迪氏综合症的存在。 有强有力的证据表明AD患者血清含有表观遗传效应，我们处于独特的地位 验证ADIE是区分AD患者和正常患者的血液生物标志物，以便早期发现。我们 还计划ADEE的初始表征，以优化测试，扩展我们的知识 AD的进展和(作为一个长期目标)确定新的可用药的表观遗传学靶点。 我们的具体目标是： 1.严格检验ADIE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前后同一患者队列中进行验证数据分析。

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.