There is growing interest in the potential contribution of cholesterol to the pathogenesis of Alzheimer's disease (AD). Treatment with cholesterol-lowering statins appears to lower the risk of developing AD. Furthermore, Abeta production has been directly associated with cholesterol-rich domains (lipid rafts) and cholesterol levels have been shown to modulate APR processing and Abeta generation. The link between cholesterol and Abeta has recently been revealed in Niemann Pick Type C (NPC) diseases as well as AD. Deficiency in NPC1 protein causes intracellular accumulation of unesterified cholesterol in late endosomal/lysosomal compartments that is accompanied by a significant increase in Abeta production and a shift in presenilin 1 (PS1) localization to early/late endosomes. Contradictory results were reported on whether changes in cholesterol content may directly affect gamma-secretase cleavage of APP. The goal of this project is to elucidate the mechanisms by which cholesterol affects APP metabolism. We hypothesize that cholesterol modulates APP processing in a similar manner in wild-type and NPC1 knock-out cells. We also hypothesize that cholesterol levels regulate intracellular trafficking of APP, BACE1 and PS1, three key players in the pathogenesis of Alzheimer's disease. We speculate that cholesterol-dependent association with lipid rafts of the three proteins regulates their endocytosis and thus generation of Abeta. In addition, we hypothesize that levels of cholesterol and/or apolipoprotein E (apoE) modulate APP processing directly regulating gamma-secretase activity. To analyze the parallels between cholesterol and APP processing in wild-type and NPC1-/- cells we will test whether an increase or a decrease in cholesterol levels modulates APP metabolism in a similar manner. Comparing cellular localization and association with lipid rafts of APP, BACE1 and PS1 in wt and in NPC1 Knock out cells under sterol-starved and sterol-fed conditions we will elucidate whether the cholesterol-effect on aberrant APP processing is mediated via specific subcellular or lipid raft compartment(s). To test whether gamma-secretase activity per se can be regulated by cholesterol and/or apoE levels we will perform in vitro gamma-assay. If gamma-secretase activity is regulated by cholesterol levels and/or apoE we will test whether this feature is specific for gamma-cleavage of APP or is common among other gamma-secretase substrates (e.g. Notch 1). This research will be done primarily in Croatia at the Rudjer Boskovic Institute in collaboration with Dr. Silva Hecimovic as an extension of NIH grant #R01AG016208. Through these studies we will elucidate the molecular mechanisms of cholesterol action in Alzheimer's disease. Finding the molecular link(s) between cholesterol and AD is important both for treating Alzheimer's disease as well as for understanding normal APP function.