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

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.