Clusterin glycosylation as diagnostic and prognostic biomarker for Alzheimer's disease

项目总结 聚集素是晚发性阿尔茨海默病(AD)的第三大遗传风险因素。集群 促进可溶性淀粉样蛋白b(Ab)通过血脑屏障的运输。聚集素结合可溶性形式的 AB，防止它们聚集成淀粉样纤维。然而，血浆和血液中的 脑脊液聚集素浓度水平与阿尔茨海默病的关系，尽管有大量证据表明聚集素表达上调 并与抗体斑块共定位。聚集素高度糖基化，约占其质量的25% 由N-连接的多聚糖组成。聚集素的糖基化在空间和时间上都是受调节的，它是很重要的 因为它具有细胞外伴侣的功能。聚集素的糖基化可能在血管紧张素转换过程中起重要作用 与抗体的相互作用及其清除。 在这个第一阶段的项目中，我们建议开发、优化和验证一种快速且经济高效的质谱学 (MS)-免疫分析法检测完整的糖基化聚集素及其糖化产物 组成糖链结构。我们将首先优化抗体含量、样本量等检测参数 和稀释，捕获后漂洗，酶促释放唾液酸和含唾液酸酶的完整多糖和 PNGaseF酶和MS采集参数。检测性能将通过测试其 稳健性、准确性、特异性和重复性。我们将对一组匹配的脑脊液和 血浆样本(n=106)来自轻度受损和有AD风险的健康个体。原封不动形式的数据 聚集素在血浆和脑脊液之间的质量峰移动，唾液酸酶和 PNGaseF治疗，簇蛋白糖型的相对比率，以及释放的糖链的ID和比率，将 在队列中进行比较，并与载脂蛋白E基因型、载脂蛋白E异构体特异性糖基化、Aβ42、 将对tau和ptau进行检查。 第一个队列的结果将与第二个队列(n=100)进行验证。 我们的价值主张是一种快速且经济高效的分析聚集素糖形式和多糖的方法，它可以 用来评估聚集素糖基化在AD发病机制中的作用。聚集素糖基化可以 作为评估AD风险的生物标记物，或作为改变其治疗方法的开发的目标 糖基化。

PROJECT SUMMARY Clusterin is the third most predominant genetic risk factor for late onset Alzheimer’s disease (AD). Clusterin facilitates the transport of soluble amyloid-b (Ab) across the blood-brain barrier. Clusterin binds soluble forms of Ab, preventing their aggregation into amyloid fibrils. However, there is no clear correlation between plasma or CSF clusterin concentration levels and AD, even though there is substantial evidence for upregulation of clusterin in the AD brain and colocalization with Ab plaques. Clusterin is heavily glycosylated, with ~25% of its mass comprised of N-linked glycans. Glycosylation of clusterin is spatially and temporally regulated, and it is important for its function as an extracellular chaperone. Glycosylation of clusterin may play an important role in the interaction with Ab and its clearance. In this Phase I project we propose to develop, optimize and validate a fast and cost-efficient mass spectrometry (MS)-immunoassay for detection of clusterin glycoforms that reveals both intact glycosylated clusterin and its component glycan structures. We will first optimize assay parameters such as antibody amount, sample volumes and dilutions, post-capture rinses, enzymatic release of sialic acids and the full glycans with sialidase and PNGaseF enzymes, and MS acquisition parameters. The assay performance will be validated by testing its robustness, accuracy, specificity, and reproducibility. We will use the assay on a cohort of matched CSF and plasma samples (n=106) from mildly impaired and healthy individuals at risk of AD. Data in the form of intact clusterin mass peaks shifts between plasma and CSF, mass shifts within samples following sialidase and PNGaseF treatment, the relative ratios of clusterin glycoforms, and IDs and ratios of the released glycans, will be compared across the cohort, and correlations with apoE genotype, apoE isoform-specific glycosylation, Aβ42, Tau and pTau will be examined. The findings from the first cohort will be validated with a second cohort (n=100). Our value proposition is a fast and cost-effective assay for analyzing clusterin glycoforms and glycans that could be utilized for evaluating the role of clusterin glycosylation in AD pathogenesis. Clusterin glycosylation could serve as a biomarker for assessing AD risk, or as a target for the development of therapeutics that modify its glycosylation.