Use of in Vivo Models to Study the Role of Cholesterol in the Etiology of Alzheimer's Disease

Cholesterol has been implicated in the pathogenesis of Alzheimer's disease, both through intracellular effects, and through an extracellular effect due to its physical interaction with plaque associated amyloid. Epidemiology studies have implicated high cholesterol as a risk factor for AD, and have shown that the use of cholesterol reducing agents (statins) can be protective against the disease. We, and others have shown that cholesterol levels modulate the processing of the amyloid precursor protein (APP) both in vivo and in vitro, affecting the accumulation of Abeta (A) peptides which may directly impact the risk of AD. This review describes the biology of sterols, and identifies how cholesterol may exacerbate the pathogenesis of AD. Data from in vivo and in vitro studies will then be presented to describe how treatments aimed at modulating lipid levels may be efficacious in treating AD.     

Regulatory effects of simvastatin and apoJ on APP processing and amyloid-β clearance in blood-brain barrier endothelial cells

Amyloid-β peptides (Aβ) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aβ metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aβ. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aβ metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aβ oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aβ clearance across the pBCEC monolayer. Treatment of pBCEC with Aβ_(1–40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3 × Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aβ oligomers and reduced Aβ uptake and cell-associated Aβ oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aβ processing and clearance at the BBB.     

ACAT inhibition and amyloid beta reduction

Alzheimer's disease (AD) is a devastating neurodegenerative disorder. Accumulation and deposition of the beta-amyloid (Aβ) peptide generated from its larger amyloid precursor protein (APP) is one of the pathophysiological hallmarks of AD. Intracellular cholesterol was shown to regulate Aβ production. Recent genetic and biochemical studies indicate that not only the amount, but also the distribution of intracellular cholesterol is critical to regulate Aβ generation. Acyl-coenzyme A: cholesterol acyl-transferase (ACAT) is a family of enzymes that regulates the cellular distribution of cholesterol by converting membrane cholesterol into hydrophobic cholesteryl esters for cholesterol storage and transport. Using pharmacological inhibitors and transgenic animal models, we and others have identified ACAT1 as a potential therapeutic target to lower Aβ generation and accumulation. Here we discuss data focusing on ACAT inhibition as an effective strategy for the prevention and treatment of AD.     

Direct binding of cholesterol to the amyloid precursor protein: An important interaction in lipid–Alzheimer's disease relationships?

It is generally believed that cholesterol homoeostasis in the brain is both linked to and impacted by Alzheimer's disease (AD). For example, elevated levels of cholesterol in neuronal plasma and endosome membranes appear to be a pro-amyloidogenic factor. The recent observation that the C-terminal transmembrane domain (C99, also known as the β-C-terminal fragment, or β-CTF) of the amyloid precursor protein (APP) specifically binds cholesterol helps to tie together previously loose ends in the web of our understanding of Alzheimer's–cholesterol relationships. In particular, binding of cholesterol to C99 appears to favor the amyloidogenic pathway in cells by promoting localization of C99 in lipid rafts. In turn, the products of this pathway—amyloid-β and the intracellular domain of the APP (AICD)—may down-regulate ApoE-mediated cholesterol uptake and cholesterol biosynthesis. If confirmed, this negative-feedback loop for membrane cholesterol levels has implications for understanding the function of the APP and for devising anti-amyloidogenic preventive strategies for AD.     

Alterations of cholesterol precursor levels in Alzheimer's disease

Cerebral and extracerebral cholesterol metabolism are altered in Alzheimer's disease (AD) as indicated by reduced plasma levels of the cholesterol elimination products 24S-hydroxycholesterol, which is of cerebral origin, and of 27-hydroxycholesterol, which is formed extracerebrally. However, it has to be evaluated, if changes of cholesterol metabolism in the whole body or in the CNS are exclusively due to the altered elimination of cholesterol or are also due to altered de novo synthesis in AD. We investigated CSF and plasma levels of cholesterol and of its precursors lanosterol, lathosterol and desmosterol in AD patients and non-demented controls. We found CSF levels of cholesterol (p = 0.011), absolute levels of all investigated cholesterol precursors (each p < 0.001) and ratios of cholesterol precursors/cholesterol (each < 0.01) to be lower in AD patients as compared to controls. In plasma, the absolute levels of lanosterol (p = 0.026) and lathosterol (p < 0.001) and the ratio of lathosterol/cholesterol (p = 0.002) but none of the other investigated parameters were reduced in AD patients (p > 0.1). Furthermore, ratios of desmosterol/lathosterol in CSF (p = 0.023) and plasma (p = 0.009) were higher in AD patients as compared to controls. Our data support the hypothesis that cholesterol metabolism is altered in AD and further suggest that especially cholesterol de novo synthesis within the CNS of AD patients might be reduced. These findings raise doubt on a beneficial effect of cholesterol lowering treatment in manifest AD.     

Amyloid beta-protein interactions with membranes and cholesterol: causes or casualties of Alzheimer's disease

Amyloid beta-protein (Aβ) is thought to be one of the primary factors causing neurodegeneration in Alzheimer's disease (AD). This protein is an amphipathic molecule that perturbs membranes, binds lipids and alters cell function. Several studies have reported that Aβ alters membrane fluidity but the direction of this effect has not been consistently observed and explanations for this lack of consistency are proposed. Cholesterol is a key component of membranes and cholesterol interacts with Aβ in a reciprocal manner. Aβ impacts on cholesterol homeostasis and modification of cholesterol levels alters Aβ expression. In addition, certain cholesterol lowering drugs (statins) appear to reduce the risk of AD in human subjects. However, the role of changes in the total amount of brain cholesterol in AD and the mechanisms of action of statins in lowering the risk of AD are unclear. Here we discuss data on membranes, cholesterol, Aβ and AD, and propose that modification of the transbilayer distribution of cholesterol in contrast to a change in the total amount of cholesterol provides a cooperative environment for Aβ synthesis and accumulation in membranes leading to cell dysfunction including disruption in cholesterol homeostasis.     

Alzheimer Disease—No Target for Statin Treatment. A Mini Review

Nosologically, Alzheimer disease (AD) is not a single disorder. A minority of around 400 families worldwide can be grouped as hereditary in origin, whereas the majority of all Alzheimer cases (approx. 25 million worldwide) are sporadic in origin. In the pathophysiology of the latter type, a number of susceptibility genes contribute to the disease among which are allelic abnormalities of the apolipoprotein E4 gene pointing to a link between disturbed cholesterol metabolism and sporadic AD. Cholesterol is a main component of membrane composition enriched in microdomains and is functionally linked to the proteolytic processing of amyloid precursor protein (APP). In sporadic AD, a marked diminution of both membrane phospholipids and cholesterol has been found. Evidence has been provided that high plasma cholesterol may protect from AD. In contrast to these well documented abnormalities observed in AD patients, it was assumed that an elevated cholesterol concentration might favour the generation of β-amyloid and, thus, AD. However, a series of in vitro-and in vivo-studies did not provide evidence for the assumption that an enhanced cholesterol concentration increased βA4-production. A harsh reduction of membrane cholesterol only caused a “beneficial” effect of APP metabolism. However, this experimentally induced condition may not be compatible to sporadic AD. The application of statins in sporadic AD did not yield results to assume that this therapeutic strategy may prevent or treat successfully sporadic AD. Dedicated to Professor John P. Blass.     

Membrane traffic in sphingolipid storage diseases

In this review, we summarize our studies of membrane lipid transport in sphingolipid storage disease (SLSD) fibroblasts. We recently showed that several fluorescent SL analogs were internalized from the plasma membrane predominantly to the Golgi complex of normal cells, while in ten different SLSD cell types, these lipids accumulated in endosomes and lysosomes (The Lancet 1999;354: 901-905). Additional studies showed that cholesterol homeostasis is perturbed in multiple SLSDs secondary to SL accumulation and that mistargeting of SL analogs was regulated by cholesterol (Nature Cell Biol 1999;1: 386-388). Based on these findings, we hypothesize that endogenous sphingolipids, which accumulate in SLSD cells due to primary defects in lipid catabolism, result in an altered intracellular distribution of cholesterol, and that this alteration in membrane composition then results in defective sorting and transport of SLs. The importance of SL/cholesterol interactions and potential mechanisms underlying the regulation of lipid transport and targeting are also discussed. These studies suggest a new paradigm for regulation of membrane lipid traffic along the endocytic pathway and could have important implications for future studies of protein trafficking as well as lipid transport. This work may also lead to important future clinical developments (e.g. screening tests for SLSD, new methodology for screening drugs which abrogate lipid storage, and possible therapeutic approaches to SLSD).     

EHD1 regulates cholesterol homeostasis and lipid droplet storage

Endocytic transport is critical for the subcellular distribution of free cholesterol and the endocytic recycling compartment (ERC) is an important organelle that stores cholesterol and regulates its trafficking. The C-terminal EHD protein, EHD1, controls receptor recycling through the ERC and affects free cholesterol distribution in the cell. We utilized embryonic fibroblasts from EHD1 knockout mice (Ehd1(-/-)MEF) and SiRNA in normal MEF cells to assess the role of EHD1 in intracellular transport of cholesterol. Surprisingly, Ehd1(-/-)MEFs displayed reduced levels of esterified and free cholesterol, which returned to normal level upon re-introduction of wild-type, but not dysfunctional EHD1. Moreover, triglyceride and cholesterol storage organelles known as 'lipid droplets' were smaller in size in cells lacking EHD1, indicating that less esterified cholesterol and triglycerides were being stored. Decreased cellular cholesterol and reduced lipid droplet size in Ehd1(-/-)MEFs correlated with ineffectual cholesterol uptake via LDL receptor, suggesting involvement of EHD1 in LDL receptor internalization.     

Ras/mitogen-activated protein kinase (MAPK) signaling modulates protein stability and cell surface expression of scavenger receptor SR-BI

The mitogen-activated protein kinase (MAPK) Erk1/2 has been implicated to modulate the activity of nuclear receptors, including peroxisome proliferator activator receptors (PPARs) and liver X receptor, to alter the ability of cells to export cholesterol. Here, we investigated if the Ras-Raf-Mek-Erk1/2 signaling cascade could affect reverse cholesterol transport via modulation of scavenger receptor class BI (SR-BI) levels. We demonstrate that in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells, Mek1/2 inhibition reduces PPARα-inducible SR-BI protein expression and activity, as judged by reduced efflux onto high density lipoprotein (HDL). Ectopic expression of constitutively active H-Ras and Mek1 increases SR-BI protein levels, which correlates with elevated PPARα Ser-21 phosphorylation and increased cholesterol efflux. In contrast, SR-BI levels are insensitive to Mek1/2 inhibitors in PPARα-depleted cells. Most strikingly, Mek1/2 inhibition promotes SR-BI degradation in SR-BI-overexpressing CHO cells and human HuH7 hepatocytes, which is associated with reduced uptake of radiolabeled and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyane-labeled HDL. Loss of Mek1/2 kinase activity reduces SR-BI expression in the presence of bafilomycin, an inhibitor of lysosomal degradation, indicating down-regulation of SR-BI via proteasomal pathways. In conclusion, Mek1/2 inhibition enhances the PPARα-dependent degradation of SR-BI in hepatocytes.     

Amyloid excess in Alzheimer's disease: what is cholesterol to be blamed for?

A link between alterations in cholesterol homeostasis and Alzheimer's disease (AD) is nowadays widely accepted. However, the molecular mechanism/s underlying such link remain unclear. Numerous experimental evidences support the view that changes in neuronal membrane cholesterol levels and/or subcellular distribution determine the aberrant accumulation of the amyloid peptide in the disease. Still, this view comes from rather contradictory data supporting the existence of either high or low brain cholesterol content. This is of particular concern considering that therapeutical strategies aimed to reduce cholesterol levels are already being tested in humans. Here, we review the molecular mechanisms proposed and discuss the perspectives they open.     

固醇调节元件结合蛋白2信号通路与非酒精性脂肪性肝病

非酒精性脂肪性肝病（non-alcoholic fatty liver disease,NAFLD）是以肝细胞内甘油三酯和胆固醇等脂毒性脂肪过度沉积为主要特征的一种临床获得性代谢综合征。最新研究表明,NAFLD向非酒精性脂肪肝炎(NASH)进展时,肝内胆固醇积累可能较甘油三酯更具有细胞毒性风险。固醇调节元件结合蛋白2（sterol regulatory element-binding protein 2,SREBP2）是脂质代谢重要的核转录因子之一,主要调控胆固醇的生物合成和体内平衡。SREBP2及其靶基因调控的胆固醇异常是引起非酒精性脂肪肝病发生发展的重要因素之一。因此,认识SREBP2信号通路中,上下游各因素的表达调控作用与NAFLD发病机制之间关系,就显得非常重要。本文总结了受SREBP2调控表达的靶基因的特点,着重介绍SREBP2调控胆固醇体内合成与平衡的信号通路与NAFLD发病机制之间关系,为研究和指导治疗NAFLD及其代谢性疾病提供新的思路。     

Cholesterol changes in Alzheimer's disease: methods of analysis and impact on the formation of enlarged endosomes

An increasing number of results implicating cholesterol metabolism in the pathophysiology of Alzheimer's disease (AD) suggest cholesterol as a target for treatment. Research in genetics, pathology, epidemiology, biochemistry, and cell biology, as well as in animal models, suggests that cholesterol, its transporter in the brain, apolipoprotein E, amyloid precursor protein, and amyloid-β all interact in AD pathogenesis. Surprisingly, key questions remain unanswered due to the lack of sensitive and specific methods for assessing cholesterol levels in the brain at subcellular resolution. The aims of this review are not only to discuss the various methods for measuring cholesterol and its metabolites and to catalog results obtained from AD patients but also to discuss some new data linking high plasma membrane cholesterol with modifications of the endocytic compartments. These studies are particularly relevant to AD pathology, since enlarged endosomes are believed to be the first morphological change observed in AD brains, in both sporadic cases and Down syndrome.     

Lipid carrier proteins and ethanol

Ethanol has a pronounced effect on lipid homeostasis. It is our overall hypothesis that certain lipid carrier proteins are targets of acute and chronic ethanol exposure and that perturbation of these proteins induces lipid dysfunction leading to cellular pathophysiology. These proteins include both intracellular proteins and lipoproteins. This paper examines recent data on the interaction of ethanol with these proteins. In addition, new data are presented on the stimulatory effects of ethanol on low-density-lipoprotein (LDL)-mediated cholesterol uptake into fibroblasts and direct perturbation of the LDL apolipoprotein, apolipoprotein B. A cell model is presented that outlines potential mechanisms thought to be involved in ethanol perturbation of cholesterol transport and distribution.     

U18666A Inhibits Intracellular Cholesterol Transport and Neurotransmitter Release in Human Neuroblastoma Cells

To determine if neurochemical function might be impaired in cell models with altered cholesterol balance, we studied the effects of U18666A (3--[(2-diethyl-amino)ethoxy]androst-5-en-17-one) on intracellular cholesterol metabolism in three human neuroblastoma cell lines (SK-N-SH, SK-N-MC, and SH-SY5Y). U18666A (0.2 g/ml) completely inhibited low density lipoprotein (LDL)-stimulated cholesterol esterification in SK-N-SH cells, while cholesterol esterification stimulated by 25-hydroxycholesterol or bacterial sphingomyelinase was unaffected or partially inhibited, respectively. U18666A also blocked LDL-stimulated downregulation of LDL receptor and caused lysosomal accumulation of cholesterol as measured by filipin staining. U18666A treatment for 18 h resulted in 70% inhibition of K⁺-evoked norepinephrine release in phorbol esterdifferentiated SH-SY5Y cells, while release stimulated by the calcium ionophore A23187 was only slightly affected. These results suggest that U18666A may preferentially block a voltage-regulated Ca²⁺ channel involved in norepinephrine release and that alterations in neurotransmitter secretion might be a feature of disorders such as Niemann-Pick Type C, in which intracellular cholesterol transport and distribution are impaired.     

细胞内胆固醇运输

作为生物膜的重要成分,细胞内不同膜上胆固醇含量的高低直接影响生物膜的生物物理特性和细胞信号的传递,与细胞正常的生理功能密切相关。外源内吞的胆固醇和内源合成的胆固醇通过囊泡介导和非囊泡介导的胆固醇运输途径在不同细胞膜之间转运,从而维持了不同细胞器上胆固醇的浓度梯度。一系列胆固醇结合和转运蛋白在细胞内胆固醇的运输中发挥了重要作用。本文旨在总结细胞内胆固醇运输途径与参与胆固醇运输的重要分子及相关作用机制。     

Oxidatively Modified GST and MRP1 in Alzheimer’s Disease Brain: Implications for Accumulation of Reactive Lipid Peroxidation Products

Alzheimer disease (AD) is a neurodegenerative disorder characterized pathologically by intracellular inclusions including neurofibrillary tangles (NFT) and senile plaques. Several lines of evidence implicate oxidative stress with the progression of AD. 4-hydroxy-2-trans-nonenal (HNE), an aldehydic product of membrane lipid peroxidation, is increased in AD brain. The alpha class of glutathione S-transferase (GST) can detoxify HNE and plays an important role in cellular protection against oxidative stress. The export of the glutathione conjugate of HNE is required to fully potentiate the GST-mediated protection. The multidrug resistance protein-1 (MRP1) and GST proteins may act in synergy to confer cellular protection. In the present study, we studied oxidative modification of GST and MRP1 in AD brain by immunoprecipitation of GST and MRP1 proteins followed by Western blot analysis using anti-HNE antibody. The results suggested that HNE is covalently bound to GST and MRP1 proteins in excess in AD brain. Collectively, the data suggest that HNE may be an important mediator of oxidative stress-induced impairment of this detoxifying system and may thereby play a role in promoting neuronal cell death. The results from this study also imply that augmenting endogenous oxidative defense capacity through dietary or pharmacological intake of antioxidants may slow down the progression of neurodegenerative processes in AD.