Lectin-affinity chromatography coupled proteomics studies and characterization of oxidative modifications to LDL-receptor related protein 1 in mild cognitive impairment and Alzheimer's disease brain: Implications for disease pathogenesis and progression.

摘要

The projects that comprise this dissertation investigated Alzheimer disease (AD) utilizing both discovery- and hypothesis-driven research. The first project of this dissertation research was a discovery-driven proteomics study that employed lectin-affinity chromatography techniques to fractionate proteins based upon changes in glycosylation and/or hydrophobicity patterns in proteins isolated from the brains of subjects with mild cognitive impairment (MCI) and AD. The second, hypothesis-driven project investigated the neurovascular hypothesis of AD by measuring oxidative post-translational modification to LDL-related receptor protein 1 in brain from subjects with MCI and AD.;Known cellular processes regulated by protein glycosylation, such as protein folding, protein regulation, and targeting of proteins to appropriate organelles, are adversely affected in AD brain. Lectin-affinity chromatography fractionates proteins based upon carbohydrate motifs and/or changes in hydrophobicity. The studies conducted in this dissertation utilized concanavalin A (ConA), a lectin that has an affinity for glycoproteins typical of N-linked glycosylation and hydrophobic proteins, and wheat germ agglutinin (WGA), a lectin that has affinity for glycoproteins typical of O-linked glycosylation, to fractionate proteins based on changes in their lectin-affinity. Upon lectin-affinity fractionation, samples were subjected to proteomics techniques for the identification and quantification of the fractionated proteins. Protein samples used in this study were isolated from the brains of subjects with MCI and AD to provide information about AD progression. Identified proteins belonged to several functional classes including chaperones, metabolic, synaptic maintenance, endoplasmic reticulum (ER) function, and cytoskeletal assembly. The proteomics-identified proteins with altered lectin-affinity are consistent with the biochemistry, pathology, and clinical presentation of AD, and provide potential targets to explore further for aberrant functionality in the investigation of AD pathogenesis and progression.;Amyloid β peptide (Aβ), in the form of toxic oligomers, has been hypothesized to contribute to AD pathogenesis and progression by oxidative stress mechanisms. The neurovascular hypothesis of AD states that decreased clearance of Aβ by the LDL-related receptor protein 1 (LRP1), the primary transporter involved in the efflux of Aβ from the brain to the blood, leads to the increased accumulation of Aβ observed in AD brain. Experiments using immunoprecipitation to isolate LRP1 from MCI and AD brain with subsequent probing for oxidative post-translational modifications found a significant increase in the level of 4-hydoxynonenal (HNE), a marker of lipid peroxidation, bound to LRP1 in AD hippocampus.;This result is consistent with the hypothesis that Aβ oxidizes its primary efflux transporter, thereby increasing the levels of Aβ in the brain and exacerbating the oxidative stress observed in AD. Future studies that test therapeutics targeted against lipid peroxidation may prevent the observed oxidative stress to LRP1 and restore the normal efflux of Aβ.;KEYWORDS: Alzheimer's Disease, Mild Cognitive Impairment, Immunoprecipitation, Lectin-affinity Chromatography, Proteomics.