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Abstract
Amyloid-beta (Aβ) plaques are a major neuropathological feature of Alzheimer's disease (AD). These plaques are primarily composed of aggregates of Aβ peptides generated via the amyloidogenic processing of the amyloid precursor protein. The two major isoforms of Aβ peptide are Aβ40 and Aβ42, of which the latter is highly prone to aggregation. Increased presence and aggregation of intracellular Aβ42 peptides is an early event in the disease progression of AD. Improved understanding of cellular processes involved in Aβ42 aggregation may have implications for understanding AD progression and development of therapeutic strategies.
Here Aβ42 fused to green fluorescent protein (Aβ42GFP) was expressed in each mutant of the homozygous diploid Saccharomyces cerevisiae genome-wide deletion library (Winzeler et al., 1999) to identify proteins and cellular processes that affect intracellular Aβ42 aggregation by assessing the fluorescence associated with expression of the Aβ42GFP fusion protein. This screening identified 110 mutants exhibiting intense Aβ42GFP-associated fluorescence. Four major cellular processes were over-represented in the data set, including phospholipid homeostasis and mitochondrial function. Global analysis of the S. cerevisiae lipidome by quantitative shotgun mass spectrometry led to the discovery that disruption of phosphatidylcholine, phosphatidylserine and/or phosphatidylinositol metabolism had a major effect on intracellular Aβ42 aggregation and localisation. Distinctive subcellular localisation of Aβ42GFP in the phospholipid mutants was observed. Confocal microscopy indicated that Aβ42GFP in the phospholipid mutants was juxtaposed to the nucleus of the cell, associated with the endoplasmic reticulum.
Another novel outcome of the Aβ42GFP fusion protein has been to identify putative roles for two proteins of previously uncharacterised function. This study further exploited the powerful and flexible platform of applying GFP-derived fluorescence-based assay for the screening of the Library of Pharmacologically Active Compounds (LOPAC) and the SPECTRUM chemical/drug library for compounds with amyloidogenic and anti-amyloidogenic properties. These libraries include FDA-approved drugs and identifying such drugs as affecting intracellular Aβ42 aggregation may significantly reduce costs and the exhaustive process of introducing new drugs onto the market. Compounds identified through this approach may potentially change the way these drugs are currently administered to patients. These data provide the first genome-wide evidence of cellular processes and chemical compounds that affect intracellular Aβ42GFP aggregation and may have important implications for understanding cellular mechanisms that affect intracellular Aβ42 aggregation and ultimately disease progression of AD.