Project 3: Development of multiplex assays for clinical monitoring of disease

摘要 阿尔茨海默病的病理特征是形成的神经原纤维缠结和淀粉样斑块 在大脑皮质的灰质区。错误折叠和聚集的蛋白质驻留在这些 病理特征，这可能是神经元死亡的中心，导致区域性萎缩，痴呆， 最终导致死亡。沐浴中枢神经系统的脑脊液是最近端的液体。 30多年来一直是生物标记物发现的焦点。有两个重要的因素 存在于脑脊液中的基因产物，已被证明可预测疾病活动 出现轻度认知障碍的患者的认知功能下降：(1)淀粉样前体蛋白， 其产生Aβ(1-42)和其他片段，以及(2)具有许多磷酸化位点的tau。这个 淀粉样蛋白聚集体中Aβ(1-42)的沉积似乎导致邻近神经元的死亡。由此产生的 损伤会增加释放到脑脊液中的tau蛋白的数量。来自以前的生物标志物 对Aβ蛋白和Tau的研究，似乎可能是整个疾病过程，从Aβ(1-42)沉积到细胞 死亡，与其他神经退行性疾病的安排不同。因此，它是一个令人信服的 假设脑脊液中存在新的生物标志物，它们可能是疾病的媒介 并可能在目前用于诊断阿尔茨海默病的生物标记物上增加更多信息。 以前已经尝试使用发现蛋白质组学来识别差异蛋白质 阿尔茨海默病患者脑脊液中的表达与对照组相比 很少有人努力以临床有意义的方式来扩展这些研究。然而，其中一些努力 已经确定存在更多的淀粉样前体蛋白片段[即除了Aβ(1-42)之外] 和其他蛋白质，这表明翻译后的变化可能是重要的形成 淀粉样蛋白，神经元突触功能障碍，以及随后的神经元死亡。 在这项题为“质谱学目标的鉴定和多种分析方法的发展”的项目中 疾病管理，我们的目标是确定导致改变的翻译后修饰 脑脊液中Lp/Ev浓度、相对分子质量和稳定性。部分原因是 Aβ(1-42)、tau和磷酸化tau作为脑脊液生物标志物的临床翻译花了几十年的时间 早期没有可重复使用的化验方法。因此，该项目的另一个首要目标 是开发精确的、可转移的、有效的靶向蛋白质组分析来定量 可用于研究疾病机制和预测不良预后的脑脊液。

Abstract The pathological hallmarks of Alzheimer’s disease are the neurofibrillary tangles and amyloid plaques that form in gray matter regions of the cerebral cortex. Misfolded and aggregated proteins reside within those pathological features, which may to be central to the neuronal death that causes regional atrophy, dementia, and ultimately death. The cerebrospinal fluid that bathes the central nervous system is the fluid most proximal to the disease and has been the focus of biomarker discovery for more than 30 years. There are two important gene products that are present in cerebrospinal fluid and have been shown to be predictive of disease activity and cognitive decline in patients that present with mild cognitive impairment: (1) the amyloid precursor protein, which gives rise to Aβ(1-42) and other fragments, and (2) tau, which has many phosphorylation sites. The deposition of Aβ(1-42) as amyloid aggregates appears to lead to the death of adjacent neurons. The resulting injury increases the amount of tau protein released into the cerebrospinal fluid. From previous biomarker studies of Aβ proteins and tau, it seems likely that the entire disease process, from Aβ(1-42) deposition to cell death, is orchestrated differently than in other neurodegenerative diseases. As a result, it is a compelling hypothesis that novel biomarkers, which may be mediators of disease, are present in the cerebrospinal fluid and could add more information over the current biomarkers used in the diagnosis of Alzheimer’s disease. There have been previous attempts to use discovery proteomics to identify proteins that are differentially expressed in cerebrospinal fluid in patients with Alzheimer’s disease compared with controls and there have been few efforts to expand upon these studies in a clinically meaningful way. However, some of these efforts have identified the presence of additional fragments of amyloid precursor protein [i.e., in addition to Aβ(1-42)] and other proteins, which suggests that post-translational changes may be important in the formation of amyloid, the dysfunction of neuronal synapses, and the subsequent death of neurons. In this project entitled, “Identification of mass spectrometric targets and development of multiplex assays for disease management,” our objective is to identify the post-translational modifications that lead to altered LP/EV concentration, molecular weight, and stability in cerebrospinal fluid. Part of the reason that the clinical translation of Aβ(1-42), tau, and phosphorylated tau as cerebrospinal fluid biomarkers took decades was that reproducible assays were not available early on. As a result, another overarching goal of this project is to develop precise, transferable, validated targeted proteomic assays to quantify proteins in cerebrospinal fluid that can be used to investigate disease mechanism and predict poor outcomes.