Endosomal-Lysosomal Cholesterol Sequestration by U18666A Differentially Regulates Amyloid Precursor Protein (APP) Metabolism in Normal and APP-Overexpressing Cells

Abstract

Amyloid β (Aβ) peptide, derived from amyloid precursor protein (APP), plays a critical role in the development of Alzheimer's disease. Current evidence indicates that altered levels or subcellular distribution of cholesterol can regulate Aβ production and clearance, but it remains unclear how cholesterol sequestration within the endosomal-lysosomal (EL) system can influence APP metabolism. Thus, we evaluated the effects of U18666A, which triggers cholesterol redistribution within the EL system, on mouse N2a cells expressing different levels of APP in the presence or absence of extracellular cholesterol and lipids provided by fetal bovine serum (FBS). Our results reveal that U18666A and FBS differentially increase the levels of APP and its cleaved products, the α-, β-, and η-C-terminal fragments, in N2a cells expressing normal levels of mouse APP (N2awt), higher levels of human wild-type APP (APPwt), or "Swedish" mutant APP (APPsw). The cellular levels of Aβ/Aβ were markedly increased in U18666A-treated APPwt and APPsw cells. Our studies further demonstrate that APP and its cleaved products are partly accumulated in the lysosomes, possibly due to decreased clearance. Finally, we show that autophagy inhibition plays a role in mediating U18666A effects. Collectively, these results suggest that altered levels and distribution of cholesterol and lipids can differentially regulate APP metabolism depending on the nature of APP expression.

Acidifying Endolysosomes Prevented Low-Density Lipoprotein-Induced Amyloidogenesis

“…Currently, it is not understood what leads to such a discrepancy. In addition, U18666A, an agent that leads to cholesterol accumulation in endolysosomes, de-acidifies endolysosomes [108], and increases Aβ levels [109]. On the other hand, restoring endolysosomal acidification by GSK3 inhibition [110] or acidic nanoparticle [111–113] has been shown to improve amyloid pathology.…”

Implications of exosomes derived from cholesterol-accumulated astrocytes in Alzheimer's disease pathology

“…1C and D). Since cholesterol sequestration enhances APP processing leading to increased secretion of Aβ peptides (Chung et al, 2018;Yang et al, 2017), we wanted to establish if exosomes Disease Models & Mechanisms • DMM • Accepted manuscript derived from UA-treated astrocytes may have a role in the development of AD pathology.…”

“…1 C,D). Because cholesterol sequestration enhances APP processing, leading to increased secretion of Aβ peptides ( Chung et al, 2018 ; Yang et al, 2017 ), we wanted to establish whether exosomes derived from U18666A-treated astrocytes may have a role in the development of AD pathology. As a first step, we revealed that exosomes isolated from control astrocytes using polyethylene glycol (PEG)-based precipitation method ( Rider et al, 2016 ) display established exosomal markers flotillin-1, ALIX, TSG101, CD63 and CD81 ( Perez-Gonzalez et al, 2012 ; Raposo and Stoorvogel, 2013 ), but not the negative marker calnexin ( Zhang et al, 2019 ) ( Fig.…”

“…To highlight the significance of cholesterol to the secretion of exosomes, cultured astrocytes were exposed or unexposed to Dil and then treated for 24 h with various modulators of cellular cholesterol levels, such as cholesterol, methyl-β-cyclodextrin (MBCD) and wortmannin ( Chung et al, 2018 ; Costa Verdera et al, 2017 ; Maulik et al, 2012 ; Tobert, 2003 ). As expected, total cholesterol levels in astrocytes detected using gas-liquid chromatography ( Fig.…”

“…The observed changes are due to cholesterol sequestration rather than cellular degeneration as viability of astrocytes was not found to be affected following treatment with 5µg/ml U18666A over 24hrs. Although previous studies have shown that U18666A-induced cholesterol sequestration can lead an increased levels of APP and/or its cleaved products in cultured astrocytes/neurons/ cell lines (Boland et al, 2010;Chung et al, 2018;Davis, 2008;Jin et al, 2004;Koh and Cheung, 2006;Runz et al, 2002;Yamazaki et al, 2001;Yang et al, 2017), there is some discrepancy in the results which are likely due to cell types used in the studies along with the concentration and duration of the U18666A treatment.…”