American Society for Cell Biology (2019) Poster presentation .
The source of intracellular cholesterol affects the localization of Amyloid Precursor Protein and BACE in cultured cells.
Michael C. Carinha, Phung Huynh, Anna Tarren, Edward B. Cluett
Ithaca College, Ithaca, NY 14850
Ca2+-independent phospholipase A2 inhibitors (PLAIs) induce the accumulation of cholesterol in the Endocytic Recycling Compartment (ERC), but only when sufficient amounts of LDL are available to cells. Certain proteins that associate with cholesterol also accumulate in the ERC under these conditions, notably Amyloid Precursor Protein (APP). Immunoblotting revealed that the processing of APP and association of APP-derived fragments with detergent-insoluble complexes was altered by PLAI treatment, but whether pathogenic processing occurred could not be determined. Altered processing of APP that leads to the production of the pathogenic form of the protein has been associated with higher levels of cholesterol. Interaction of APP with BACE protease can lead to the production of the pathogenic form of Ab. Since PLAIs lead to the colocalization of cholesterol and APP, we investigated the colocalization of APP and BACE when cells were grown in different levels of LDL and treated with the PLAI, ONO RS-082 or simvastatin, which inhibits cholesterol synthesis. Since PLA2 enzymes are associated with different compartments involved in membrane trafficking, they may also play a role in the trafficking of newly-synthesized cholesterol and affect the trafficking or processing of APP. SH-SY5Y, HeLa, and NPC1 cells were grown in low, moderate, or high levels of LDL, then treated with ONO and simvastatin alone or in combination. Immunofluorescence confocal microscopy revealed colocalization of APP and BACE in a small fraction of juxtanuclear puncta only in untreated cells grown in low-LDL media. No colocalization was observed in cells grown in higher levels of LDL or treated with ONO or simvastatin. This suggests that how cells obtain cholesterol may influence cellular processes.