Use of Giant Plasma Membrane Vesicles (GPMV) to Examine the Lo/Ld Phase Preference of the C99 Domain of the Amyloid Precursor Protein

Abstract

Alzheimer's disease (AD) is the most common form of dementia at the moment cannot be treated, prevented, or even delayed. Despite decades of intensive research efforts, there are still many mysteries regarding the etiology of AD. The predominant hypothesis for AD is that disease pathology begins decades before clinically-detectable symptoms due to the formation of toxic oligomers and aggregates composed mainly of amyloid-β (Aβ) peptides. Amyloid-β is generated when the amyloid precursor protein (APP) transmembrane C-terminal fragment, C99, is cleaved in the membrane by γ-secretase. Cholesterol has long been implicated in AD and shown to increase the generation of Aβ peptides, but it is not known how cholesterol or cholesterol-rich environments impact C99 membrane localization. We are examining the membrane phase preference of C99 localization in membranes using giant plasma membrane-derived vesicles (GPMVs). These cell-derived giant vesicles exhibit phase separation into cholesterol-rich Lo and Ld-like domains that can be directly observed using confocal microscopy. We compare and contrast various methods for preparing GPMVs and report the phase partitioning behavior of both WT C99 and cholesterol binding site-disrupted mutant forms.