Esterification of 24S-OHC induces formation of atypical lipid droplet-like structures, leading to neuronal cell death

Abstract

The brain contains about 25% of the total amount of cholesterol in the human body (1). As the blood-brain barrier prevents cholesterol translocation between the brain and the circulation, brain cholesterol is locally produced. To maintain cholesterol homeostasis in the brain, cholesterol 24-hydroxylase (CYP46A1) converts excess amounts of cholesterol into 24(S)-hydroxycholesterol (24S-OHC), which readily crosses the blood-brain barrier (2). CYP46A1 mRNA and protein are present in neurons, but not in support cells (3). It has been reported that free 24S-OHC is present at concentrations of about 4-15 ng/mg wet weight in the human brain (4). Another report showed that 24S-OHC in four different brain areas of healthy controls was about 20 ng/mg tissue (5); assuming a volume of 1 l per 1 mg tissue, this would imply a concentration of 24S-OHC in brain tissue that is approximately 50 M. Several lines of evidence suggest that dysregulation of cholesterol and 24S-OHC metabolism in the brain has been linked to the development of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease Selective expression of CYP46A1 around neuritic plaques has also been reported Abstract The 24(S)-hydroxycholesterol (24S-OHC), which plays an important role in maintaining brain cholesterol homeostasis, has been shown to possess neurotoxicity. We have previously reported that 24S-OHC esterification by ACAT1 and the resulting lipid droplet (LD) formation are responsible for 24S-OHC-induced cell death. In the present study, we investigate the functional roles of 24S-OHC esters and LD formation in 24S-OHC-induced cell death, and we identify four long-chain unsaturated fatty acids (oleic acid, linoleic acid, arachidonic acid, and DHA) with which 24S-OHC is esterified in human neuroblastoma SH-SY5Y cells treated with 24S-OHC. Here, we find that cotreatment of cells with 24S-OHC and each of these four unsaturated fatty acids increases prevalence of the corresponding 24S-OHC ester and exacerbates induction of cell death as compared with cell death induced by treatment with 24S-OHC alone. Using electron microscopy, we find in the present study that 24S-OHC induces formation of LD-like structures coupled with enlarged endoplasmic reticulum (ER) lumina, and that these effects are suppressed by treatment with ACAT inhibitor. Collectively, these results illustrate that ACAT1-catalyzed esterification of 24S-OHC with long-chain unsaturated fatty acid followed by formation of atypical LD-like structures at the ER membrane is a critical requirement for 24S-OHCinduced cell death.-