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Abstract
Apolipoprotein D (apoD) is a widely expressed, multifunctional apolipoprotein known for its lipid antioxidant properties in the brain. It is upregulated under oxidative stress conditions including aging and in neurodegenerative diseases. The mechanism of lipid antioxidant function of apoD and reasons for its upregulation in neurodegenerative diseases is unknown. Hence the aim of this thesis was to study the lipid antioxidant mechanism of apoD and its relevance to the most common form of neurodegenerative disease, Alzheimer's disease (AD). To study the mechanism of lipid antioxidant function of apoD, the interaction of recombinant apoD and its methionine mutants (methionine substituted with alanine) with hydroperoxyeicosatetraenoic acids (HpETE) were analysed using HPLC and amino acid analysis techniques. The Met93 residue of apoD was required for the reduction of HpETE to its hydroxyeicosatetraenoic acid derivative (HETE), and this reaction was concomitant with the formation of methionine sulfoxide (MetSO). Western blot analysis revealed that the oxidation of apoD Met residues causes apoD aggregation. This was supported by in-silico simulations which indicated that the oxidation of Met93 is accompanied by destabilisation of a surface-exposed hydrophobic domain that is predicted to increase the propensity for apoD to self-associate. Furthermore, the link between lipid peroxidation, apoD expression and aggregation of apoD was analysed with disease progression in AD brain using High performance liquid chromatography (HPLC), Gas chromatography Mass spectrometry (GC-MS) and Western blot analysis. Lipid conjugated dienes and F2- isoprostanes, measured as markers of lipid peroxidation, were increased in later stages of the disease. The expression of apoD also increased with disease progression and formation of apoD dimers were detected in the insoluble protein fraction of the hippocampus in late stage AD patients. Moreover, the formation of apoD dimers is closely correlated to lipid conjugated diene levels in AD hippocampus. In summary, the Met93 residue of apoD is required for the lipid antioxidant function of apoD. Furthermore, oxidation of methionine residues, particularly Met93, results in self-association of apoD and production of a stable dimer that is observed in vitro and in post-mortem brain derived from late stage AD cases. The increase in apoD expression with disease progression, similar to other antioxidants, suggests that apoD has the potential to reduce lipid peroxidation in AD brain. However, its self-association in late disease stages may prevent it from acting as a lipid antioxidant.