An Innovative Approach to Study Alzheimer Disease Blood Biomarkers

 描述(申请人提供)：阿尔茨海默病(AD)是21世纪的公共卫生危机。除非采取措施，否则到2050年，AD的成本预计将超过1.1万亿美元，从而给社会带来巨大负担(www.alz.org)。在诊断阶段，AD的病理已经相当先进。因此，迫切需要早期(或病理前)诊断生物标志物。AD的一个特征是大量AD特异性泛素化蛋白(ubi-蛋白，例如ubi-app)在脑组织中的显著积累。该项目将使用一种新发明的方法来研究早期AD相关的ubi蛋白血液生物标记物。制备针对经单体分子翻译后修饰的蛋白质的抗体的方法已经非常成熟，例如磷酸化蛋白质。磷酸化位点特异性抗体已被广泛应用于获取知识、诊断疾病和开发治疗药物等整个生命科学领域。相比之下，目前还没有一种方法可以使抗体以结合位点特定的方式识别聚合蛋白结合物。聚合物蛋白偶联是指两个多肽通过蛋白质泛素化等氨基酸侧链发生共价偶联。人们一直致力于产生泛素-蛋白质结合位点特异性抗体，但没有成功。通过使用最先进的定量蛋白质组学技术，我们最近发现，在转基因AD小鼠模型的早期和晚期，大脑样本中许多ubi蛋白结合位点特异的多肽片段显著增加。因此，我们采用了一种新的方法来制备两种泛素结合位点特异性抗体，用于在转基因AD小鼠模型和动物缺血再灌注中风模型中检测潜在的血液生物标记物。我们发现，这些ubi蛋白结合位点特异性表位在转基因AD小鼠的血液样本中显著增加，但在大脑中动脉闭塞(卒中模型)动物的血液样本中它们的水平很低，基本没有变化。本项目将使用这种新的方法产生ubi蛋白结合位点特异性抗体，以研究来自两个不同转基因小鼠AD模型的脑组织和血液样本中潜在的AD ubi蛋白生物标志物。如果这项计划的目标能够实现，这些创新的方法可以进一步应用于研究脑组织、脑脊液和血液样本中的新的人类AD生物标记物。因此，发展早期人类AD生物标志物的临床检测是AD研究、诊断和药物开发的长期目标。

 DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the public health crisis of the 21st century. Unless something is done, AD is projected to cost over $1.1 trillion by 2050, thus placing a huge burden on society (www.alz.org). At the diagnostic stage, AD pathologies are already quite advanced. Therefore, early (or pre-pathological) diagnostic biomarkers are desperately needed. A hallmark of AD is significant accumulation of an array of AD-specific ubiquitinated proteins (ubi- proteins, e.g., ubi-APP) in brain tissue. This project will use a newl invented method to study early AD-related ubi-protein blood biomarkers. Methods of making antibodies against proteins post-translationally modified by a monomeric molecule, e.g., phosphorylated proteins, are already very well established. Phosphorylation site- specific antibodies have been widely used in acquiring knowledge, diagnosing disease, and developing therapeutic agents throughout the whole spectrum of life sciences. In comparison, there is no method currently available for making antibodies recognizing polymeric protein conjugates in a conjugation site-specific manner. Polymeric protein conjugation is defined as covalent conjugation between two polypeptides via amino acid side chains such as protein ubiquitination. Extensive efforts have been devoted to generating ubiquitin-to-protein conjugation site-specific antibodies, but without success. By using a state-of-the-art quantitative proteomic technology, we recently found that many ubi-protein conjugation site-specific peptide fragments are dramatically increased in brain samples from a transgenic AD mouse model at both early "pre- pathological" and "advanced" stages. We therefore employed a new method to make two ubiquitin conjugation site-specific antibodies for detecting potential blood biomarkers in a transgenic AD mouse model and an animal ischemia-reperfusion stroke model. We found that these ubi-protein conjugation site-specific epitopes were significantly increased in blood samples from the transgenic AD mice, but their levels were very low and basically unchanged in the blood samples from animals after middle cerebral artery occlusion (stroke model). This project will employ this new method to generate ubi-protein conjugation site-specific antibodies to study potential AD ubi- protein biomarkers in brain tissue and blood samples from two different transgenic mouse AD models. If the objectives of this proposal are achieved, these innovative approaches can be further applied to study new human AD biomarkers in brain tissue, CSF and blood samples. Therefore, the long-term objectives are to develop clinical assays of early human AD biomarkers for AD research, diagnosis and drug development.