Enzymatically Formed Oxysterols and Cell Death.

The side-chain hydroxylation of cholesterol by specific enzymes produces 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and other products. These enzymatically formed side-chain oxysterols act as intermediates in the biosynthesis of bile acids and serve as signaling molecules that regulate cholesterol homeostasis. Besides these intracellular functions, an imbalance in oxysterol homeostasis is implicated in pathophysiology. Furthermore, growing evidence reveals that oxysterols affect cell proliferation and cause cell death. This chapter provides an overview of the pathophysiological role of side-chain oxysterols in developing human diseases. We also summarize our understanding of the molecular mechanisms underlying the induction of various forms of cell death by side-chain oxysterols.

Identification of a novel endogenous long non-coding RNA that inhibits selenoprotein P translation.

Selenoprotein P (SELENOP) is a major plasma selenoprotein that contains 10 Sec residues, which is encoded by the UGA stop codon. The mRNA for SELENOP has the unique property of containing two Sec insertion sequence (SECIS) elements, which is located in the 3' untranslated region (3'UTR). Here, we coincidentally identified a novel gene, CCDC152, by sequence analysis. This gene was located in the antisense region of the SELENOP gene, including the 3'UTR region in the genome. We demonstrated that this novel gene functioned as a long non-coding RNA (lncRNA) that decreased SELENOP protein levels via translational rather than transcriptional, regulation. We found that the CCDC152 RNA interacted specifically and directly with the SELENOP mRNA and inhibited its binding to the SECIS-binding protein 2, resulting in the decrease of ribosome binding. We termed this novel gene product lncRNA inhibitor of SELENOP translation (L-IST). Finally, we found that epigallocatechin gallate upregulated L-IST in vitro and in vivo, to suppress SELENOP protein levels. Here, we provide a new regulatory mechanism of SELENOP translation by an endogenous long antisense ncRNA.

Astrocytes release glutamate via cystine/glutamate antiporter upregulated in response to increased oxidative stress related to sporadic amyotrophic lateral sclerosis.

A vast body of evidence implicates increased oxidative stress and extracellular glutamate accumulation in the pathomechanism of sporadic amyotrophic lateral sclerosis (ALS). Cystine/glutamate antiporter (xCT) carries extracellular cystine uptake and intracellular glutamate release (cystine/glutamate exchange) in the presence of oxidative stress. The aim of the present study was to determine the involvement of xCT in ALS. Immunohistochemical observations in the spinal cord sections demonstrated that xCT was mainly expressed in astrocytes, with staining more intense in 12 sporadic ALS patients as compared to 12 age-matched control individuals. Western blot and densitometric analyses of the spinal cord samples revealed that the relative value of xCT/ß-actin optical density ratio was significantly higher in the ALS group as compared to the control group. Next, we conducted cell culture experiments using a human astrocytoma-derived cell line (1321N1) and a mouse motor neuron/neuroblastoma hybrid cell line (NSC34). In 1321N1 cells, the normalized xCT expression levels in cell lysates were significantly increased by H2 O2 treatment. Glutamate concentrations in 1321 N1 cell culture-conditioned media were significantly elevated by H2 O2 treatment, and the H2 O2 -driven elevations were completely canceled by the xCT inhibitor erastin pretreatment. In motor neuron-differentiated NSC34 cells (NSC34d cells), both the normalized xCT expression levels in the cell lysates and glutamate concentrations in the cell-conditioned media were constant with or without H2 O2 treatment. The present results provide in vivo and in vitro evidence that astrocytes upregulate xCT expression to release glutamate in response to increased oxidative stress associated with ALS, contributing to extracellular glutamate accumulation.

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

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-OHC-induced cell death.

Synthesis of 24(S)-hydroxycholesterol esters responsible for the induction of neuronal cell death.

We synthesized several candidates of 24(S)-hydroxycholesterol (24S-OHC) esters, which are involved in neuronal cell death, through catalysis with acyl-CoA:cholesterol acyltransferase-1 (ACAT-1). We studied the regioselectivity of the acylation of the secondary alcohol at the 3- or 24-position of 24S-OHC. The appropriate saturated and unsaturated long-chain fatty acids were esterified with the protected 24S-OHC and then de-protected to afford the desired esters at a satisfactory yield. We then confirmed by HPLC monitoring that the retention times of four esters of 24S-OHC, namely 3-oleate, 3-linoleate, 3-arachidonoate and 3-docosahexaenoate, were consistent with those of 24S-OHC esters observed in 24S-OHC-treated SH-SY5Y cells.

Aß induces oxidative stress in senescence-accelerated (SAMP8) mice.

According to the amyloid hypothesis, amyloid ß accumulates in brains with Alzheimer's disease (AD) and triggers cell death and memory deficit. Previously, we developed a rice Aß vaccine expressing Aß, which reduced brain Aß levels in the Tg2576 mouse model of familial AD. We used senescence-accelerated SAMP8 mice as a model of sporadic AD and investigated the relationship between Aß and oxidative stress. Insoluble Aß and 4-hydroxynonenal (4-HNE) levels tended to be reduced in SAMP8 mice-fed the rice Aß vaccine. We attempted to clarify the relationship between oxidative stress and Aß in vitro. Addition of Aß peptide to the culture medium resulted in an increase in 4-HNE levels in SH-SY5Y cells. Tg2576 mice, which express large amounts of Aß in their brain, also exhibited increased 4-HNE levels; this increase was inhibited by the Aß vaccine. These results indicate that Aß induces oxidative stress in cultured cells and in the mouse brain.

Diverse functions of 24(S)-hydroxycholesterol in the brain.

24(S)-hydroxycholesterol (24S-OHC) which is enzymatically produced in the brain plays important physiological roles in maintaining brain cholesterol homeostasis. We found that 24S-OHC at sub-lethal concentrations down-regulated amyloid precursor protein (APP) trafficking via enhancement of the complex formation of APP with up-regulated glucose-regulated protein 78, an endoplasmic reticulum chaperone. In accordance with this mechanism, 24S-OHC suppressed amyloid-ß production in human neuroblastoma SH-SY5Y cells. Furthermore, 24S-OHC at sub-lethal concentrations induced adaptive responses via transcriptional activation of the liver X receptor signaling pathway, thereby protecting neuronal cells against the forthcoming oxidative stress induced by 7-ketocholesterol. On the other hand, we found that high concentrations of 24S-OHC induced apoptosis in T-lymphoma Jurkat cells which endogenously expressed caspase-8, and induced necroptosis - a form of programmed necrosis - in neuronal SH-SY5Y cells which expressed no caspase-8. In this Article, we show the diverse functions of 24S-OHC and consider the possible importance of controlling 24S-OHC levels in the brain for preventing neurodegenerative diseases.

Suppression of amyloid-ß production by 24S-hydroxycholesterol via inhibition of intracellular amyloid precursor protein trafficking.

Cholesterol can be converted to 24S-hydroxycholesterol (24SOHC) by neuronal cholesterol 24-hydroxylase. In mouse models of Alzheimer's disease (AD), increasing 24SOHC levels reduced AD pathology. However, mechanisms underlying the effects of 24SOHC on amyloid-ß (Aß) production have remained unclear. Here we report that 24SOHC treatment reduces Aß production and increases endoplasmic reticulum (ER)-resident immature amyloid precursor protein (APP) levels in human neuroblastoma SH-SY5Y cells and CHO cells stably expressing human APP. Treatment with 1-10 µM 24SOHC (equivalent to the concentrations detected in human brain homogenates) diminished Aß production (IC50=4.6 µM for Aß40) without affecting secretase activities. To evaluate the intracellular APP transport, we established an in vitro vesicle formation assay. We found that APP budding via COPII vesicles was diminished by 70% in 24SOHC-treated cells. The proteomics and immunoblotting analysis revealed that 24SOHC induced the expression of glucose-regulated protein 78 (GRP78), an ER chaperone, through unfolded protein response pathways, and enhanced the formation of the APP/GRP78 complex. Knockdown of GRP78 diminished the inhibitory effects of 24SOHC on Aß production. These results suggest that 24SOHC down-regulates APP trafficking via enhancement of the complex formation of APP with up-regulated GRP78 in the ER, resulting in suppression of Aß production.

α-Tocopherol suppresses 24(S)-hydroxycholesterol-induced cell death via inhibition of endoplasmic reticulum membrane disruption.

The brain-specific cholesterol metabolite 24(S)-hydroxycholesterol (24S-OHC) has been shown to cause neuronal cell death when subjected to esterification by acyl-CoA:cholesterol acyltransferase 1 (ACAT1). Accumulating 24S-OHC esters in the endoplasmic reticulum (ER) provoked ER membrane disruption and an integrated stress response (ISR), a signaling pathway that regulates adaptation to various stresses. We have previously reported that α-tocopherol (α-Toc) but not α-tocotrienol (α-Toc3), among vitamin E homologs, suppressed 24S-OHC-induced cell death without affecting ACAT1 activity in human neuroblastoma SH-SY5Y cells. However, the precise mechanisms underlying the inhibitory activity of α-Toc have yet to be elucidated. In the present study, we aimed to investigate the effects of α-Toc on the 24S-OHC-induced cell death machinery. We showed that α-Toc, but not α Toc3, suppressed 24S-OHC-induced ISR and downstream eukaryotic translation initiator factor 2α (eIF2α) phosphorylation. We also found that α-Toc inhibited stress granule formation and robust downregulation of nascent protein synthesis, which were induced by 24S-OHC treatment. Furthermore, disruption of ER membrane integrity was suppressed by α-Toc, but not by α-Toc3. Our findings suggest that the inhibitory effects of α-Toc on 24S-OHC-induced cell death may be attributed to its protective function against ER membrane disruption.

24(S)-hydroxycholesterol induces neuronal cell death through necroptosis, a form of programmed necrosis.

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.

Integrated stress response is involved in the 24(S)-hydroxycholesterol-induced unconventional cell death mechanism.

Perturbation of proteostasis triggers the adaptive responses that contribute to the homeostatic pro-survival response, whereas disruption of proteostasis can ultimately lead to cell death. Brain-specific oxysterol-i.e., 24(S)-hydroxycholesterol (24S-OHC)-has been shown to cause cytotoxicity when esterified by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) in the endoplasmic reticulum (ER). Here, we show that the accumulation of 24S-OHC esters caused phosphorylation of eukaryotic translation initiator factor 2α (eIF2α), dissociation of polysomes, and formation of stress granules (SG), resulting in robust downregulation of global protein de novo synthesis in human neuroblastoma SH-SY5Y cells. We also found that integrated stress response (ISR) activation through PERK and GCN2 activation induced by 24S-OHC treatment caused eIF2α phosphorylation. 24S-OHC-inducible SG formation and cell death were suppressed by inhibition of ISR. These results show that ACAT1-mediated 24S-OHC esterification induced ISR and formation of SG, which play crucial roles in 24S-OHC-inducible protein synthesis inhibition and unconventional cell death.

Suppression of Amyloid-ß Adsorption on Endoplasmic Reticulum Stress-Mimicking Membranes by α-Tocopherol and α-Tocotrienol.

Two forms of hydrophobic vitamin E (VE), α-tocopherol (Toc) and α-tocotrienol (Toc3), have been proposed to be effective against Alzheimer's disease (AD), the etiology of which is thought to involve endoplasmic reticulum (ER) stress. However, previous studies reported conflicting effects of Toc and Toc3 on the risk of AD. We prepared liposomes mimicking the phase separation of the ER membrane (solid-ordered/liquid-disordered phase separation) and studied how VE can influence the interaction between amyloid-ß (Aß) and the ER membrane. We found that Toc could inhibit the formation of the solid-ordered phase more significantly than Toc3. Furthermore, Aß protofibril adsorption on ER stress-mimicking membranes was more strongly suppressed by Toc compared with Toc3. Therefore, we concluded that VE can relieve ER stress by destabilizing the solid-ordered phase of the ER membrane and subsequently reducing the amount of Aß adsorbed on the membrane. Moreover, Toc exerted a stronger effect than Toc3.

Therapeutic effect of a novel curcumin derivative GT863 on a mouse model of amyotrophic lateral sclerosis.

The present study investigated the therapeutic effects of the curcumin derivative 3-[(1E)-2-(1H-indol-6-yl)ethenyl]-5-[(1E)-2-[2-methoxy-4-(2-pyridylmethoxy)phenyl]ethenyl]-1H-pyrazole (GT863) in amyotrophic lateral sclerosis (ALS). The inhibitory effect of GT863 on superoxide dismutase 1 (SOD1) aggregation was evaluated in cell-free assays. GT863 interfered with the conformational changes of the SOD1 protein and later, oligomeric aggregation. Furthermore, its antioxidant, anti-inflammatory, and neuroprotective effects were evaluated in cell-free and cultured cell assays. GT863 inhibited H2O2- and glutamate-induced cytotoxicity and activated an antioxidant responsive element pathway. Additionally, in vivo effects of GT863 in the ALS mice model were evaluated by its oral administration to H46R mutant SOD1 transgenic mice. Rotarod test showed that GT863 administration significantly slowed the progression of motor dysfunction in the mice. In addition, GT863 substantially reduced highly-aggregated SOD1, further preserving large neurons in the spinal cord of GT863-treated mice. Collectively, these results indicated that GT863 could be a viable therapeutic agent with multiple vital actions for the treatment of ALS.

Sterol-regulatory-element-binding protein 2 and nuclear factor Y control human farnesyl diphosphate synthase expression and affect cell proliferation in hepatoblastoma cells.

FDPS (farnesyl diphosphate synthase) catalyses the formation of farnesyl diphosphate, a key intermediate in the synthesis of cholesterol and isoprenylated cellular metabolites. FDPS is also the molecular target of nitrogen-containing bisphosphonates, which are used as bone-antiresorptive drugs in various disorders. In the present study, we characterized the sterol-response element and NF-Y (nuclear factor Y)-binding site in the human FDPS promoter. Using a luciferase assay, electrophoretic mobility-shift assay and chromatin immunoprecipitation assay, we demonstrated that these elements are responsible for the transcription of the FDPS gene, and that its transcriptional activation is mediated by SREBP-2 (sterol-regulatory-element-binding protein 2) and NF-Y. We also investigated whether sterol-mediated FDPS expression is involved in the cell proliferation induced by zoledronic acid, an FDPS inhibitor. We show that the SREBP-2- and NF-Y-mediated regulation of FDPS gene transcription modulates cell proliferation. These results suggest that SREBP-2 and NF-Y are required to trigger cell proliferation through the induction of FDPS expression and that the pharmacological action of zoledronic acid is involved in this pathway.

Transport of LDL-derived cholesterol from the NPC1 compartment to the ER involves the trans-Golgi network and the SNARE protein complex.

Mammalian cells acquire cholesterol mainly from LDL. LDL enter the endosomes, allowing cholesteryl esters to be hydrolyzed by acid lipase. The hydrolyzed cholesterol (LDL-CHOL) enters the Niemann-Pick type C1 (NPC1)-containing endosomal compartment en route to various destinations. Whether the Golgi is involved in LDL-CHOL transport downstream of the NPC1 compartment has not been demonstrated. Using subcellular fractionation and immunoadsorption to enrich for specific membrane fractions, here we show that, when parental Chinese hamster ovary (CHO) cells are briefly exposed to (3)H-cholesteryl linoleate (CL) labeled-LDL, newly liberated (3)H-LDL-CHOL appears in membranes rich in trans-Golgi network (TGN) long before it becomes available for re-esterification at the endoplasmic reticulum (ER) or for efflux at the plasma membrane. In mutant cells lacking NPC1, the appearance of newly liberated (3)H-LDL-CHOL in the TGN-rich fractions is much reduced. We next report a reconstituted transport system that recapitulates the transport of LDL-CHOL to the TGN and to the ER. The transport system requires ATP and cytosolic factors and depends on functionality of NPC1. We demonstrate that knockdown by RNAi of 3 TGN-specific SNAREs (VAMP4, syntaxin 6, and syntaxin 16) reduces >/=50% of the LDL-CHOL transport in intact cells and in vitro. These results show that vesicular trafficking is involved in transporting a significant portion of LDL-CHOL from the NPC1-containing endosomal compartment to the TGN before its arrival at the ER.

Different functions of vitamin E homologues in the various types of cell death induced by oxysterols.

24(S)-Hydroxycholesterol (24S-OHC) and 25-hydroxycholesterol (25-OHC) are produced by cholesterol 24-hydroxylase and cholesterol 25-hydroxylase, respectively. The purpose of the present study was to determine the type of cell death induced by these oxysterols in neuronal cells, hepatic cells, and keratinocytes, and to elucidate the inhibitory effect of vitamin E homologues on various types of cell death. In human neuronal cells (SH-SY5Y cells), 24S-OHC and 25-OHC caused a cell death that was independent of caspase activation. We reported previously that the esterification of 24S-OHC by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) and the resulting formation of a lipid droplet (LD)-like structure are responsible for the 24S-OHC-induced neuronal cell death. Here, we found that 25-OHC also induced ACAT1-mediated 25-OHC esterification and LD formation in neuronal cells. 25-OHC-induced cell death was inhibited by α-tocopherol (α-Toc) but not by α-tocotrienol (α-Toc3), as observed for 24S-OHC-induced cell death in SH-SY5Y cells. In human hepatic cells (HepG2 cells), these oxysterols caused a cell death that was caspase- and oxysterol-esterification-independent. This cell death was suppressed by both α-Toc and α-Toc3, suggesting the involvement of free-radical-mediated lipid peroxidation in the cell death induced by these oxysterols in hepatic cells. In human keratinocytes (HaCaT cells), these oxysterols caused a caspase-dependent but oxysterol-esterification-independent cell death that was inhibited by α-Toc but not by α-Toc3. These results suggest that α-Toc and α-Toc3 act as radical-scavenging antioxidants against oxysterol-induced cell death in the same way in hepatic cells, whereas their behavior is different in inhibition of cell death in neuronal cells and keratinocytes. Collectively, these results demonstrated that 24S-OHC and 25-OHC induced the same type of cell death in each of the cell types examined, and that α-Toc and α-Toc3 exerted different effects, depending on the type of cell death.

NRF3 upregulates gene expression in SREBP2-dependent mevalonate pathway with cholesterol uptake and lipogenesis inhibition.

Lipids, such as cholesterol and fatty acids, influence cell signaling, energy storage, and membrane formation. Cholesterol is biosynthesized through the mevalonate pathway, and aberrant metabolism causes metabolic diseases. The genetic association of a transcription factor NRF3 with obesity has been suggested, although the molecular mechanisms remain unknown. Here, we show that NRF3 upregulates gene expression in SREBP2-dependent mevalonate pathway. We further reveal that NRF3 overexpression not only reduces lanosterol, a cholesterol precursor, but also induces the expression of the GGPS1 gene encoding an enzyme in the production of GGPP from farnesyl pyrophosphate (FPP), a lanosterol precursor. NRF3 overexpression also enhances cholesterol uptake through RAB5-mediated macropinocytosis process, a bulk and fluid-phase endocytosis pathway. Moreover, we find that GGPP treatment abolishes NRF3 knockdown-mediated increase of neutral lipids. These results reveal the potential roles of NRF3 in the SREBP2-dependent mevalonate pathway for cholesterol uptake through macropinocytosis induction and for lipogenesis inhibition through GGPP production.

Single chain variable fragment against nicastrin inhibits the gamma-secretase activity.

Gamma-secretase is a membrane protein complex that catalyzes intramembrane proteolysis of a variety of substrates including the amyloid beta precursor protein of Alzheimer disease. Nicastrin (NCT), a single-pass membrane glycoprotein that harbors a large extracellular domain, is an essential component of the gamma-secretase complex. Here we report that overexpression of a single chain variable fragment (scFv) against NCT as an intrabody suppressed the gamma-secretase activity. Biochemical analyses revealed that the scFv disrupted the proper folding and the appropriate glycosyl maturation of the endogenous NCT, which are required for the stability of the gamma-secretase complex and the intrinsic proteolytic activity, respectively, implicating the dual role of NCT in the gamma-secretase complex. Our results also highlight the importance of the calnexin cycle in the functional maturation of the gamma-secretase complex. The engineered intrabodies may serve as rationally designed, molecular targeting tools for the discovery of novel actions of the membrane proteins.

Curcumin Derivative GT863 Inhibits Amyloid-Beta Production via Inhibition of Protein N-Glycosylation.

Amyloid-ß (Aß) peptides play a crucial role in the pathogenesis of Alzheimer's disease (AD). Aß production, aggregation, and clearance are thought to be important therapeutic targets for AD. Curcumin has been known to have an anti-amyloidogenic effect on AD. In the present study, we performed screening analysis using a curcumin derivative library with the aim of finding derivatives effective in suppressing Aß production with improved bioavailability of curcumin using CHO cells that stably express human amyloid-ß precursor protein and using human neuroblastoma SH-SY5Y cells. We found that the curcumin derivative GT863/PE859, which has been shown to have an inhibitory effect on Aß and tau aggregation in vivo, was more effective than curcumin itself in reducing Aß secretion. We further found that GT863 inhibited neither ß- nor γ-secretase activity, but did suppress γ-secretase-mediated cleavage in a substrate-dependent manner. We further found that GT863 suppressed N-linked glycosylation, including that of the γ-secretase subunit nicastrin. We also found that mannosidase inhibitors that block the mannose trimming step of N-glycosylation suppressed Aß production in a similar fashion, as was observed as a result of treatment with GT863. Collectively, these results suggest that GT863 downregulates N-glycosylation, resulting in suppression of Aß production without affecting secretase activity.

Acyl-coenzyme A:cholesterol acyltransferases.

The enzymes acyl-coenzyme A (CoA):cholesterol acyltransferases (ACATs) are membrane-bound proteins that utilize long-chain fatty acyl-CoA and cholesterol as substrates to form cholesteryl esters. In mammals, two isoenzymes, ACAT1 and ACAT2, encoded by two different genes, exist. ACATs play important roles in cellular cholesterol homeostasis in various tissues. This chapter summarizes the current knowledge on ACAT-related research in two areas: 1) ACAT genes and proteins and 2) ACAT enzymes as drug targets for atherosclerosis and for Alzheimer's disease.