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Journal of Molecular Neuroscience Copyright © 2003 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696/03/20:395–406/$25.00

ALZHEIMER’S THERAPEUTICS: Lipid Lowering

Changes in Apolipoprotein E Expression in Response to Dietary and Pharmacological Modulation of Cholesterol Suzana S. Petanceska,*,1,2 Steven DeRosa,1 Ali Sharma,1 Nichole Diaz,1 Karen Duff,1,2 Steven G. Tint,3 Lorenzo M. Refolo ,4 and Miguel Pappolla5 1

Center for Dementia Research, Nathan Kline Institute, Orangeburg NY 10962; Department of Psychiatry, NYU Medical Center, New York, NY, 10016; 3University of Medicine and Dentistry of New Jersey, NJ 08901; 4Institute for the Study of Aging, New York, NY, 10153; 5 University of South Alabama, Mobile, AL 36688

2

Received October 15, 2002; Accepted March 24, 2003

Abstract Apolipoprotein E (ApoE) influences the risk of late onset Alzheimer’s disease (AD) in an isoform-dependent manner, such that the presence of the apoE ε4 allele increases the risk of AD while the presence of the apoE ε2 allele appears to be protective. Although a number of ApoE functions are isoform dependent and may underlie the “risk factor” activity of AD, its ability to bind amyloid β peptides and influence their clearance and/or deposition has gained strong experimental support. Evidence suggests that in addition to genotype, increased ApoE transcription can contribute to AD risk. There is growing evidence in support of the hypothesis that disrupted cholesterol metabolism is an early risk factor for AD. Studies in animal models have shown that chronic changes in cholesterol metabolism associate with changes in brain Aβ accumulation, a process instrumental for establishing AD pathology. ApoE mediates cholesterol homeostasis in the body and is a major lipid carrier in brain. As such, its expression in the periphery and in brain changes in response to changes in cholesterol metabolism. Here, we used a transgenic mouse model of Alzheimer’s amyloidosis to examine whether the diet-induced or pharmacologically induced changes in plasma cholesterol that result in altered brain amyloidosis also affect ApoE content in liver and in brain. We found that chronic changes in total cholesterol in plasma lead to changes in ApoE mRNA levels in brain. We also found that cholesterol loading of primary glial cells increases cellular and secreted ApoE levels and that long-term treatment of astrocytes and microglia with statins leads to a decrease in the cellular and/or secreted ApoE. These observations suggest that disrupted cholesterol metabolism may increase the risk of developing AD in part due to the effect of cholesterol on brain ApoE expression. Index Entries: Alzheimer’s disease, ApoE, cholesterol, statin, transgenic mice.

Introduction Cerebral accumulation of amyloid-β (Aβ) peptides is an early and perhaps necessary event for the establishment of Alzheimer’s disease (AD) pathology (Naslund et al., 2000; Parvathy et al. 2001). Consequently, therapeutic strategies aimed at reducing brain Aβ production and accumulation or accelerating its clearance might delay the onset or retard the clinical

progression of the disease. Aβ peptides are generated by proteolytic processing of the amyloid precursor protein (APP), a type I, single-transmembrane glycoprotein, as a result of the action of β- and γ-secretase activities. Alternatively, APPcan be cleaved by α-secretase, which precludes the formation of Aβ. The relative utilization of these two alternative processing routes can be modulated by numerous signal transduction pathways (Gandy and Petanceska, 2000).

*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]

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396 The overwhelming majority of AD cases are sporadic; therefore, identification of risk factors is essential for understanding the mechanisms that underlie AD pathology and for developing efficient, rational therapies. Anumber of epidemiological studies have identified a significant correlation between total plasma cholesterol and AD risk (Jarvik et al., 1994, 1995; Notkola et al. 1998; Kivipelto et al., 2001, 2002); other studies, however, have reported no significant correlation between hypercholesterolemia and AD (Yoshitake et al., 1995; Romas et al., 1999; Prince et al., 2000). Although it is still a matter of controversy whether hypercholesterolemia is a risk factor for AD, there is a growing consensus that AD is a disease in which cholesterol homeostasis—brain cholesterol homeostasis in particular—is disrupted (Koudinov et al., 2002). This hypothesis has gained strong support in the findings that the levels of 24S-hydroxycholesterol, an elimination metabolite of cholesterol in brain, are elevated in patients with AD (Jessen et al., 2000) and in the recent identification of a polymorphism in the gene for 24S-hydroxylase as a risk factor for late-onset AD (Kolsch et al., 2002; Papassotiropoulos et al., 2003). Findings from in vitro and in vivo studies suggest that a likely mechanism by which disrupted cholesterol mechanism influences the risk of AD is by modulating Aβ generation and accumulation. Increased cellular cholesterol content increases the production of Aβ; decrease in cellular cholesterol or in the ratio of cholesterol to cholesterol esters stimulates the utilization of the nonamyloidogenic processing pathway and results in decreased production of Aβ peptides (reviewed in Hartmann 2001). Evidence suggests that the effects of changes in cellular cholesterol content affect Aβ production by influencing the activity, subcellular distribution, or expression of the enzymes involved in the processing of the Aβ precursor protein, APP (Kojro et al., 2001; Runz et al., 2002; Wahrle et al., 2002; Ehehalt et al., 2003). Studies in various animal models, such as rabbits, guinea pigs, and Aβ-depositing transgenic mice, have provided further evidence that changes in cholesterol metabolism modulate brain Aβ accumulation (Sparks 1996; Howland et al., 1998; Refolo et al., 2000, 2001; Fassbender et al., 2001; Petanceska et al., 2002; Shie et al., 2002). The results of several retrospective epidemiological studies showing significantly lower prevalence of AD in populations using the cholesterollowering drugs, statins, for the treatment of


Petanceska et al. hypercholesterolemia and hyperlipidemia-related coronary arterial disease (Wolozin et al., 2000; Jick et al., 2000; Rockwood 2002; Yaffe et al., 2002) also support the hypothesis that disrupted cholesterol homeostasis is an early risk factor for AD. We have reported that chronic treatment of PSAPP mice with atorvastatin can delay the onset of brain Aβ deposition (Petanceska et al., 2002). This finding, together with results of studies in guinea pigs (Fassbender et al., 2001) and results from studies in humans (Buxbaum et al., 2002; Simons et al., 2002), supports the hypothesis that statins reduce the risk of AD by modulating Aβ metabolism. There is evidence that changes in peripheral cholesterol metabolism associate with altered APP processing in brain, possibly as a result of alterations in brain cholesterol content (Refolo et al., 2000, 2001). Another way that changes in cholesterol metabolism can modulate brain Aβ accumulation is by affecting the process of Aβ fibrillization and subsequent deposition, or the process of Aβ clearance. Apolipoprotein E (ApoE) is a constituent of several cholesterol-carrying particles and is the major lipid carrier in the brain. The three alleles of ApoE differentially influence the risk of developing lateonset AD such that the presence of the ApoE ε4 allele increases AD risk while the presence of the ApoE ε2 allele is protective (Corder et al., 1993, 1994). ApoE isoforms also associate with different serum lipoprotein profiles and differentially modulate lipoprotein profiles in response to dietary fat intake and cholesterol-lowering therapies (Hagberg et al., 2000; Ordovas and Mooser 2002). ApoE ε4 carriers are not only at increased risk of developing late-onset AD but are also hypercholesterolemic and poorly respond to statin treatment for the reduction of serum low-density lipid (LDL) cholesterol (Jarvik et al., 1995; Notkola et al., 1998; Ordovas and Mooser 2002). Anumber of activities of ApoE are isoform dependent and may contribute to its differential effect on AD risk (Fagan and Holtzman 2000). The isoformdependent role of ApoE as a pathological chaperone for Aβ involved in its fibrillization and clearance has gained strong experimental support (Poirier, 2000; Holtzman 2001). Genetic studies in humans and studies in animal models suggest that in addition to genotype, increased ApoE levels can confer greater risk of AD by impact on brain Aβ deposition. Higher levels of ApoE have been observed in the brains and plasma of AD patients compared with that of agematched controls (Jarvik et al., 1994, 1995; Notkola

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Cholesterol Regulates ApoE Expression et al. 1998); in addition, certain ApoE promoter polymorphisms conferring higher ApoE expression are associated with increased risk for AD, independent of the ApoE ε4 allele (Artiga et al., 1998; Bullido et al., 2000; Wang et al. 2000; Laws et al., 2000; Lambert et al., 2001). Studies in Aβ-depositing transgenic mice have shown that given the same amount of brain Aβ, the density of neuritic plaques depends on the amount of ApoE in brain (Bales et al., 1999; Holtzman et al., 2000). In addition, in plaque-forming mice that express human ApoE, the presence of ApoE ε4 enhances amyloid deposition compared to ApoE ε3 (Holtzmann et al., 2000b). There is also evidence that both human and mouse ApoE significantly affect the age of onset, as well as the levels, structure, and anatomical distribution, of brain Aβ deposits in a transgenic mouse model of Alzheimer’s amyloidosis (Fagan et al., 2002). However, in the environment of the mouse brain, human ApoE isoforms delay the onset of Aβ deposition compared to mouse ApoE (Holtzman et al., 1999), possibly as a result of differences in the composition of mouse vs human brain ApoE lipoproteins (Fagan et al. 1999). There is evidence that the expression of ApoE in the periphery (Driscoll et al., 1990; Srivastava et al., 1996a; Wong and Rubenstein, 1997; Santillo et al., 1999) and in brain (Sparks et al., 1995; Howland et al., 1998; Levin-Allerand et al., 2002; Shie at al. 2002) is sensitive to changes in plasma cholesterol. It is therefore plausible that ApoE mediates at least some of the effects of long-term changes in peripheral cholesterol metabolism on brain Aβ accumulation. In the study reported here, we investigated how dietary and pharmacological modulation of plasma cholesterol in the PSAPP transgenic model of Alzheimer ’s amyloidosis that associated with changes in brain Aβ accumulation reflect on ApoE content in liver and in brain. In an effort to begin to understand the mechanisms by which statins attenuate brain Aβ accumulation, we examined how chronic treatment of PSAPP mice with atorvastatin (Lipitor®) that resulted in reduced brain Aβ levels and reduced Aβ burden affects APP processing and ApoE levels. Finally, given the importance of glialderived cholesterol and ApoE for synaptic transmission, as well as neuronal regeneration, and the involvement of ApoE in the modulation of neuroinflammation, we examined the effect of cholesterol loading and statin treatment on cellular and secreted ApoE content in primary mixed glial cultures and in BV2 microglial cells.


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Materials and Methods Animals, Treatment Paradigms, and Tissue Processing We used brain and liver tissues from animals used in our previous hypercholesterolemia, BM15.766, and atorvastatin studies (Refolo et al., 2000, 2001; Petanceska et al., 2002). In each of these studies, double transgenic PSAPP mice obtained by crossing hemizygous APP transgenic from the Tg2576 line (human APP Swe transgene) with homozygous PS1 mice, from the H8.9 line (human PS1M146L transgene) were used (Holcomb et al., 1998). The double transgenic animals from this cross develop amyloid deposits at 10–12 wk of age. For the hypercholesterolemia study, 5 wk-old PSAPP mice received a basal diet or a high cholesterol diet (Purina Test Diets) for 7 wk. For the BM15.766 study, PSAPP animals at 8 wk of age received BM15.766 (250 mg/kg body weight [BW]/day administered by oral gavage) or vehicle (sesame oil) for 7 wk. For the atorvastatin study, PSAPP animals at 8 wk of age received Lipitor® (30 mg atorvastatin/kg BW/day, administered orally) or vehicle (0.5% methylcellulose in strawberry Kool-Aid) for 8 wk. In all studies, each experimental group contained between 6 and 10 mice and an equal number of male vs female mice. Throughout each study the animals had access to food and water ad libitum. At the end of the treatments the animals were anesthetized and sacrificed by transcardial perfusion with phosphate buffer. Blood, livers, and brains were collected. Livers were minced and flash frozen. The brains were separated in hemibrains; one hemibrain was flash frozen and used for various biochemical analyses (Aβ ELISA, Western blotting, and immunoprecipitation/Western blotting), whereas the other hemibrain was postfixed in formalin and used for Aβ immunohistochemistry and amyloid load measurements. Detection of flAPP, sAPPα, and β-CTF-APP Full-length APP (flAPP) and its processing derivatives were determined in the formic acid extracts used for Aβ ELISA, as described previously (Refolo et al., 2000, 2001). ApoE Detection Apolipoprotein E levels in brain and liver tissue were determined by direct Western blotting with a goat anti-ApoE antibody (Calbiochem, La Jolla, CA).

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398 To measure ApoE levels in brain, aliquots of the 70% formic acid extracts used for Aβ ELISA were reconstituted in 2% SDS/PBS as described (Refolo et al., 2001); from each animal, 30 µg of protein was used for immunoblotting. The signal was developed using a chemiluminescent kit (Pierce) and quantified using Scan Analysis Software. Signal intensity was expressed in relative densitometric units (r.d.u). To measure ApoE levels in liver, the flash-frozen tissue was extracted in 2% SDS/PBS buffer containing a cocktail of protease inhibitors (Complete; Boehringer Manheim), and 30 µg of protein from each animal was used for immunoblotting. Apolipoprotein E mRNAlevels in brain were measured by slot blot Northern analysis. For this, total RNA was prepared from flash-frozen brain tissue using the RNAEasy RNA extraction kit (Quiagen). Ten micrograms of total RNA was applied onto a nitrocellulose membrane and probed with a 32 P-radiolabeled mouse ApoE cDNA probe (clone p2C1, ATCC), as described previously (Petanceska et al., 1996).

Glial Culture Experiments Primary mixed glial cultures were prepared from 1-d-old C57/BL6 mouse pups, as described previously (Canoll et al., 1996). The cultures contained >85% astrocytes; the remaining cells were microglia and O2Aprogenitor cells, as determined by immunofluorescent staining with marker antibodies for each cell type (glial fibrillary acidic protein [GFAP], cd11b, and A2B5, respectively). After 10 d in culture, the cells were plated in 35-mm dishes at a density of 2 × 105 cells/well. Twenty-four hours later, the cells were treated with cholesterol (Sigma, St. Louis, MO) at 20 µg/mL, lovastatin (Sigma) at 5 µM, a combination of cholesterol and lovastatin or vehicle (0.01% ethanol) in serum-free medium containing N2 supplement (GIBCO) for 20 h. Each treatment was done in triplicate. BV2 cells (gift from Dr. Cindy Lemere, Harvard Medical School) were maintained in 10% fetal bovine serum in Dulbecco modified Eagle’s medium (DMEM). For experiments, the cells were plated in 35-mm dishes at a density of 2 × 105 cells/ well and treated with 5 µM atorvastatin of vehicle (0.01% dimethylsulfoxide) for 20 h. Media were collected and flash-frozen. Cells were lysed in 1% Triton X-100/PBS buffer containing a cocktail of protease inhibitor (Complete; Boehringer Manheim), and cell extracts were prepared by centrifugation at 14,000 rpm. Protein concentration was determined with a BCA protein assay kit (Pierce). From each Journal of Molecular Neuroscience

Petanceska et al. sample, 25 µL of media and 30 µg of cell lysate material were run on 10–20% Tris-tricine gels. After electrotransfer to PVDF membranes, ApoE was detected by immunoblotting with a goat anti-ApoE antibody (Calbiochem, La Jolla, CA). Relative intensity of the ApoE signal developed by chemiluminescence (ECL kit from Pierce), was determined using Scan Analysis software.

Results Previously, we reported that PSAPP mice placed on a high-cholesterol diet during the onset of Aβ deposition became hypercholesterolemic and accumulated significantly more Aβ40 and Aβ42 peptides in brain reflected in significantly greater amyloid load as compared to PSAPP mice receiving regular mouse chow (Refolo et al., 2000). Treatment of PSAPP mice with the cholesterol-lowering drug BM15.766, an inhibitor of the last step of the de novo cholesterol biosynthesis pathway, caused hypocholesterolemia and was associated with reduced brain amyloidosis (Refolo et al., 2001) In both studies we reported modest but significant changes in total cholesterol content in brain (~9% increase and ~12% decrease, respectively), as well as changes in steady-state levels of the APPα processing derivatives sAPPα and β-CTF-APP. More specifically, diet-induced hypercholesterolemia was associated with increased levels of the amyloidogenic β-CTF-APP product and decreased levels of sAPPα. In turn, the BM15.766induced hypocholesterolemia was associated with a decrease in the amyloidogenic processing of APP and a concomitant increase in nonamyloidogenic APP processing. These findings suggested that the observed changes in brain amyloidosis in both of these studies are attributable, at least in part, to changes in Aβ generation. To gain more information regarding the mechanisms that underlie the observed changes in brain amyloidosis in response to the above treatments, we measured the levels of ApoE in brain and in liver tissue from the PSAPP mice used in the hypercholesterolemia and BM15.766 studies, using semiquantitative Western blotting. Both liver and brain ApoE content were significantly elevated (~160% of control) in the PSAPP mice with diet-induced hypercholesterolemia compared with the normocholesterolemic group (basal diet) (Fig. 1). The PSAPP mice with BM15-766-induced hypocholesterolemia had significantly lower levels (~70% of control levels) of ApoE in both liver and brain compared with mice

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Fig. 1. Diet-induced hypercholesterolemia and pharmacologically induced hypocholesterolemia are associated with changes in ApoE levels in liver and in brain. Formic acid brain extracts (70%) were reconstituted in 2% SDS/PBS, as described previosly (Refolo et al, 2000); 30 µg of protein from brain and liver extracts was used for direct Western blotting with a goat anti-ApoE antibody (Calbiochem, La Jolla, CA).

Fig. 2. Positive correlation between total plasma cholesterol levels, brain ApoE levels, and brain Aβ. Data points for total plasma cholesterol, brain ApoE, and total brain Aβ from the high-cholesterol experimental group, the BM15.766treated group, and the basal-diet group of PSAPP mice were used for the correlation analysis.

receiving vehicle only (Fig. 1). Correlation analysis of the data points for plasma cholesterol, brain Aβ, and brain ApoE for individual animals from these studies revealed a strong positive correlation between total plasma cholesterol, brain ApoE levels, and the levels of formic-acid extractable brain Aβ (Fig. 2). Slot-blot Northen analysis of total RNA isolated from littermate animals containing only the PS1 transgene revealed that the observed changes in ApoE protein levels in brain in response to the high-cholesterol diet or BM15.766 treatment were accompanied with changes in ApoE mRNA levels (Fig. 3). We have also reported that chronic treatment of predepositing PSAPP mice with the 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA reductase inhibitor atorvastatin (Lipitor®) resulted in attenuated brain amyloidosis (Petanceska et al., 2002). The


atorvastatin treatment induced hypocholesterolemia, without altering the total cholesterol content in brain. The latter observation is likely due to the relative hydrophilicity and poor BBB permeability of atorvastatin (Hamelin and Turgeon, 1998). Chronic treatment of PSAPP mice with BM15.766, a relatively lipophylic drug with high BBB permeability (Honda et al., 1998), reduced total brain cholesterol content (Refolo et al., 2001). Similarly, treatment of C57/BL6 mice with the lipophylic statin, lovastatin, also resulted in reduced brain cholesterol content (Eckert et al., 2001). The atorvastatin-induced hypocholesterolemia was associated with a significant reduction in the levels of formic-acid extractable Aβ40 and Aβ42 in brain and a comparable reduction in amyloid burden (Petanceska et al., 2002). Western blotting analysis of brain extracts from animals used in this study

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Fig. 3. Changes in plasma cholesterol are associated with changes in brain ApoE mRNA expression. Littermate animals carrying only the PS1 transgene were subjected to vehicle high cholesterol diet and BM15.766 treatment for 8 wk. Total brain RNA was isolated, and slot-blot Northern analysis was performed using a 32P radiolabeled cDNA probe for mouse ApoE (see Materials and Methods).

Petanceska et al. The highest expression of ApoE is in the liver, followed by brain (Srivastava et al. 1996b). ApoE in brain is synthesized and secreted by astrocytes and microglia in the form of unique lipoprotein particles (Fagan et al., 1999; Xu et al., 2000). We investigated the effect of cholesterol loading and statin treatment on cellular and secreted ApoE levels in primary glial cultures and in BV2 microglia. We found that increasing cellular cholesterol content by cholesterol loading led to increases in the levels of cellular and secreted ApoE in primary glia (Fig. 6). Long-term (20 h) treatment with lovastatin resulted in decreased secretion of ApoE. Lovastatin treatment also prevented the cholesterol-induced increase in ApoE levels in these cultures (Fig. 6). Long-term (20 h) treatment of BV2 microglia with atorvastatin decreased the levels of both cellular and secreted ApoE (Fig. 7).

Discussion

Fig. 4. Atorvastatin treatment decreases brain ApoE levels. Aliquots of the formic acid brain extracts used for Aβ ELISAs (see Fig. 2) were reconstituted in 2% SDS/PBS buffer and used for Western blotting (30 µg protein/lane) with an antiApoE antibody (Calbiochem).

revealed that similar to the effect of BM15.766, treatment with atorvastatin resulted in reduced ApoE levels in brain (Fig. 4). However, in contrast to BM15.766, atorvastatin treatment did not significantly alter the steady-state levels of the APPprocessing derivatives, sAPPα and β-CTF-APP (Fig. 5), suggesting that the effect of atorvastatin on brain amyloidosis was primarily attributable to changes in ApoE-mediated Aβ deposition/clearance rather than Aβ generation.


The hypothesis that disrupted cholesterol homeostasis underlies the neurodegeneration observed in AD gained the strongest support to date with the recent discovery that a polymorphism in the gene for 24S-hydroxylase, an enzyme that controls brain cholesterol homeostasis, strongly segregates with late-onset AD (Kolsch et al., 2002; Papassotiropoulos et al., 2003). This polymorphism also associates with increased brain Aβ accumulation. Moreover, the additional presence of the ApoE ε4 allele synergistically increased the risk of late-onset AD (Papassotiropoulos et al., 2003). ApoE is quickly up-regulated in response to various types of neuronal injury, suggesting a neuroprotective role; however, overexpression of ApoE appears to be neurotoxic (Laskowitz et al., 1998). These observations suggest that in addition to genotype, ApoE levels determine how it influences both normal and pathological processes. This is supported by genetic epidemiological studies that have observed association between polymorphisms in the ApoE gene promoter that confer greater transcriptional activity and increased risk of late-onset AD (reviewed by Bullido and Valdivieso, 2000). Experimental evidence suggests that hypercholesterolemia leads to increased brain ApoE content. This was first observed in rabbits (Sparks et al., 1995). Increased brain ApoE levels in response to dietary hypercholesterolemia have also been reported in different human APP-carrying transgenic mouse

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Fig. 5. Atorvastatin does not alter whole-brain APP processing. Aliquots of formic acid brain extracts used for Aβ ELISAs were reconstituted in 2% SDS/PBS buffer (Refolo et al., 2000) and used for Western blotting (50 µg protein/lane) or immunoprecipitation (IP)/Western blotting (750 µg protein for each IP). Full-length APP was detected with antibody 369 (gift from Dr. Sam Gandy, Thomas Jefferson University), sAPPα was detected by IP/Western blotting using antibodies 207 (gift from Dr. Barry Greenberg, Astra Zeneca) and 6E10 (Signet); β-CTF-APP was detected by IP/Western blotting using antibodies 369 and 6E10, respectively.

models (Howland et al., 1998; Shie et al., 2002; LevinAllerhand et al. 2002). Here, we report that in a transgenic mouse model of Alzheimer’s amyloidosis, the diet-induced and pharmacologically induced changes in plasma cholesterol that associated with alterations in brain Aβ deposition and APP processing (Refolo et al., 2000, 2001) are also accompanied by changes in the levels of liver and brain ApoE. Similar to findings by others (Howland et al., 1998; Shie at al., 2002), we observed a strong positive correlation between total cholesterol in plasma and brain ApoE levels and between ApoE and Aβ levels in brain. In addition, we show that brain ApoE mRNA levels change in response to these changes in the levels of total cholesterol in plasma. We also find that chronic treatment of PSAPP mice with atorvastatin (Lipitor®), which associated with attenuated brain amyloidosis, was accompanied


with a decrease in brain ApoE levels. This suggests that statins may delay the onset of brain amyloidosis by affecting ApoE-mediated Aβ deposition and/or clearance and, in doing so, reduce the risk of AD. Our finding that atorvastatin reduced brain ApoE content but did not significantly alter APP processing implies that its effect on brain amyloidosis is largely a result of increased Aβ clearance or decreased Aβ deposition. Further studies are needed to determine whether the effects of statins on Aβ deposition are localized to the brain parenchyma or whether they are a result of changes in the dynamic equilibrium between the Aβ pools in the central nervous system (CNS) and plasma. Astrocytes and microglia are the major source of ApoE in the CNS (Stone et al.,1997; Fagan et al., 1999; Xu et al., 2000). Neurons depend on glial-derived cholesterol, delivered in the form of ApoE-containing

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Fig. 6. Cholesterol regulates ApoE levels in primary mixed glial cultures. Primary mixed glial cultures were prepared from 1-d-old C57Bl/6 mouse pups, as described (Canoll et al., 1996). In a typical prep, the majority of cells (>85%) are astrocytes (GFAP positive); the remaining cells are microglia and O2A progenitors. After 10 d in vitro, the cells were passaged into triplicate 35-mm wells (105 cells/well), transferred into lipid-free medium, and treated with vehicle (control), cholesterol 20 µg/mL, lovastatin (5 µM), or a combination of cholesterol and lovastatin for 24 h. Media and lysates were harvested and assayed for ApoE levels by Western blotting with a goat anti-ApoE antibody (Calbiochem, La Jolla, CA). The ApoE signal was quantified using Scan Analysis software.

lipoproteins for the formation and maintenance of efficient synapses (Mauch et al., 2001). Numerous paradigms of neuronal injury result in glial activation accompanied by increased ApoE expression, a process necessary for neuronal regeneration (Laskowitz et al., 1998; Lynch et al., 2001). There is also evidence that the induction of ApoE in glial cells in response to different activation stimuli limits neuroinflammation (LaDu et al. 2001). Whereas Aβ peptides can induce glial ApoE production (Meda et al., 1995), exogenously added ApoE can attenuate Aβinduced glial activation (LaDu et al. 2001). However, overproduction of ApoE by activated glia can exacerbate certain proinflammatory responses (Guo et al., 2002). This is of relevance, as chronic neuroinflammation characterized by glial activation is one of the salient features of AD pathology (McGeer and McGeer, 2001). Glial cells, microglia in particular, are


also involved in the processes of Aβ deposition and Aβ clearance (Neuroinflammation Working Group, 2000). ApoE-containing lipoproteins might influence the uptake and degradation of Aβ by microglia in an isoform-dependent manner (Cole and Ard, 2000). Because of the critical involvement of ApoE in the above processes, it is important to understand how ApoE expression is modulated by long-term changes in brain cholesterol homeostasis. Here, we show that cholesterol loading of primary mixed glial cultures increased both cellular and secreted ApoE, whereas statin (lovastatin) treatment reduced only ApoE secretion. Lovastatin also prevented the high-cholesterol-induced increase in ApoE levels in these cultures. Treatment of BV2 microglial cells with atorvastatin reduced the levels of both cellular and secreted ApoE. These changes in ApoE might be a result of changes in the cellular-

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Fig. 7. Atorvastatin reduces cellular and secreted ApoE levels in BV2 microglia. BV2 microglial cells were treated with atorvastatin (5 µM), lipopolysacharide (LPS), (1 µg/mL), or vehicle for 20 h. The levels of ApoE (cellular and secreted) were detected by Western blotting and quantified by densitometry using Scan Analysis software. LPS treatment was used as a positive control (Werb and Chin, 1983).

free cholesterol content, as there is evidence that ApoE expression in astrocytoma cells and in macrophages is positively regulated by the cellular content of free cholesterol (Mazzone et el. 1989). The molecular mechanisms that regulate ApoE gene expression in response to changes in cellular cholesterol content, however, have not been elucidated. In macrophages and adipocytes, ApoE gene expression is regulated by oxysterols—hydroxylation metabolites of cholesterol (Laffite et al., 2001). Oxysterols bind to the nuclear receptors LXRα and LXRβ, which in turn hetrodimerize with the retinoic nuclear receptors RXR and transactivate expression of ApoE and other genes involved in cholesterol efflux (Laffite et al., 2001). It is still debated whether oxysterols are the physiological ligands of the LXR receptors. It is of interest, however, that oxysterols are found in the circulation and are able to pass cell membranes and the BBB (Bjorkhem et al. 2002). A number of oxysterols are also produced in the brain. Of them, 25S-hydroxycholesterol is the most potent regulator of gene transcription; 25S-hydroxycholesterol induces the expression of ApoE in macrophages and in astrocytoma cells (Mazzone et al. 1987; Gueguen et al., 2001). Lipid reduction may mediate the observed effect of statins on the cellular and secreted ApoE content


403 in glial cells; however, it is equally plausible that statin activities other than lipid lowering might mediate this effect. A recent study that examined the regulation of ApoE secretion by glial cells demonstrated that the inhibition of ApoE secretion by glial cells and by BV2 microglia in response to lovastatin is independent of its cholesterol-lowering activity and is instead a result of its inhibition of mevalonate synthesis and consequent inhibition of geranyl geranylation of a key regulator of ApoE secretion (Naidu et al., 2002). It is of note that we observed a decrease in both cellular and secreted ApoE in BV2 cells in response to atorvastatin treatment, whereas lovastatin treatment reduced only ApoE secretion in primary glial cultures (this study) and in BV2 cells (Naidu et al., 2002), implying that different statins can differentially modulate ApoE production and secretion by glial cells. Taken together, our findings suggest that long-term changes in plasma cholesterol induced by diet or induced pharmacologically can lead to changes in brain ApoE expression and provide a plausible mechanism for the proposed link between hypercholesterolemia and AD risk. Further studies are needed to elucidate the mechanism(s) by which changes in plasma cholesterol affect brain ApoE expression in different brain regions and in different cell types.

Acknowledgments This work was supported by the Institute for the Study of Aging.

References Artiga M. J., Bullido M. J., Frank A., Sastre I., Recuero M., Garcia M. A., et al. (1998) Risk for Alzheimer’s disease correlates with transcriptional activity of the APOE gene. Hum. Mol. Genet. 12, 1887–1892. Bales K. R., Verina T., Cummins D. J., Du Y., Dodel R. C., Saura J., et al. (1999) Apolipoprotein E is essential for amyloid deposition in the APPV717F transgenic mouse model of Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 96, 15,233–15,238. Bjorkhem I., Meaney S., and Diczfalusy U. (2002) Oxysterols in human circulation: which role do they have? Curr. Opin. Lipidol. 13, 247–253. Bullido M. J. and Valdivieso F. (2000) Apolipoprotein E gene polymorphisms in Alzheimer’s disease. Microsc. Res. Tech. 50, 261–267. Buxbaum J. D., Cullen E. I., and Friedhoff L. T. (2002) Pharmacological concentrations of the HMGCoAreductase inhibitor lovastatin decrease the formation of the Alzheimer β-amyloid peptide in vitro and in patients. Front Biosci. 7, 50–59.

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404 Canoll P. D., Petanceska S., Schlessinger J., and Musacchio J. M. (1996) Three forms of RPTP-beta are differentially expressed during gliogenesis in the developing rat brain and during glial cell differentiation in culture. J. Neurosci. Res. 44, 199–215. Cole G. and Ard M. D. (2000) Influence of lipoproteins on microglial degradation of Alzheimer’s amyloid betaprotein. Microsc. Res. Tech. 15, 316–324. Corder E. H., Saunders A. M., Strittmatter W. J., et al. (1993) Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 261, 921–923. Corder E. H., Saunders A. M., Risch N. J., et al. (1994) Protective effect of apolipoprotein E type 2 allele for late onset Alzheimer disease. Nat. Genet. 2, 180–184. Driscoll D. M., Mazzone T., Matsushima T., and Getz G. S. (1990) Apoprotein E biosynthesis in the cholesterolfed guinea pig. Arteriosclerosis 10, 31–39. Eckert G. P., Kirsch C., and Mueller, W. E. (2001) Differential effects of lovastatin treatment on brain cholesterol levels in normal and Apo-E deficient mice. Neuropharmacol. Neurotoxicol. 12, 883–887. Ehehalt R., Keller P., Haass C., Thiele C., and Simons K. (2003) Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts. J. Cell Biol. 160, 113–123. Fagan A. and Holtzman D. M. (2000) Astrocyte Lipoproteins, Effects of ApoE on neuronal function, and role of ApoE in amyloid β deposition in vivo. Microsc. Res. Tech. 50, 297–304. Fagan A. M., Holtzman D. M., Munson G., Mathur T., Schneider D., Chang L. K., et al. (1999) Unique lipoproteins secreted by primary astrocytes from wild type, ApoE (–/–), and human ApoE transgenic mice. J. Biol. Chem. 274, 30,001–30,007. Fagan A. M., Watson M., Parsadanian M., Bales K. R., Paul S. M., and Holtzman D. M. (2002) Human and murine ApoE markedly alters Abeta metabolism before and after plaque formation in a mouse model of Alzheimer’s disease. Neurobiol. Dis. 9, 305–318. Fassbender K., Simons M., Bergmann C., Stroick M., Lutjohann D., Keller P., et al. (2001) Simvastatin strongly reduces levels of Alzheimer’s disease beta-amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo. Proc. Natl. Acad. Sci. U.S.A. 98, 5856–5861. Gandy S. and Petanceska S. (2000) Regulation of Alzheimer beta-amyloid precursor trafficking and metabolism. Biochim. Biophys. Acta 502, 44–52. Gueguen Y., Bertrand P., Ferrari L., Batt A. M., and Siest G. (2001) Control of apolipoprotein E secretion by 25-hydroxycholesterol and proinflammatory cytokines in the human astrocytoma cell line CCF-STTG1. Cell Biol. Toxicol. 17, 191–199. Guo L., LaDu M. J., and Van Eldik L. J. (2002) ApoE down regulates pro-inflammatory responses induced by oligomeric Aβ in activated glia. Soc. Neurosci. Abstr. 883, 12.


Petanceska et al. Hagberg J. M., Kenneth R. W., and Ferrell R. E. (2000) APOE gene and gene-environment effects on plasma lipoprotein-lipid levels. Physiol. Genomics 4, 101–108. Hamelin B. A. and Turgeon T. (1998) Hydrophilicity/ lipophilicity: relevance for the pharmacology and clinical effects of HMG-CoA reductase inhibitors. Trends Pharmacol. Sci. 19, 26–37. Hartmann T. (2001) Cholesterol, A beta and Alzheimer’s disease. Trends Neurosci. 11(Suppl.), S45–S48. Holcomb L., Gordon M. N., McGowan E., Yu X., Benkovic S., Jantzen P., et al. (1998) Accelerated Alzheimer’s type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin transgenes. Nat. Med. 4, 97–100. Holtzman D. M. (2001) Role of apoe/Abeta interactions in the pathogenesis of Alzheimer’s disease and cerebral amyloid angiopathy. J. Mol. Neurosci. 17, 147–155. Holtzman D. M., Bales K. R., Wu S., Bhat P., Parsadanian M., Fagan A. M., et al. (1999) Expression of human apolipoprotein E reduces amyloid-beta deposition in a mouse model of Alzheimer’s disease. J. Clin. Invest. 103, R15–R21. Holtzman D. M., Fagan A. M., Mackey B., Tenkova T., Sartorius L., Paul S. M., et al. (2000a) Apolipoprotein E facilitates neuritic and cerebrovascular plaque formation in an Alzheimer’s disease model. Ann. Neurol. 47, 739–747. Holtzman D. M., Bales K. R., Tenkova T., Fagan A. M., Parsadanian M., Sartorius L. J., et al. (2000b) Apolipoprotein-isoform dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 97, 2892–2897. Honda A., Salen G., Nguyen L. B., Xu G., Tint G. S., Batta A. K., and Shefer S. (1998) Regulation of early cholesterol biosynthesis in rat liver: effects of sterols, bile acids, lovastatin, and BM 15.766 on 3-hydroxy3-methylglutaryl coenzyme Asynthase and acetoacetyl coenzyme A thiolase activities. Hepatology 27, 154–159. Howland D. S., Trusko S. P., Savage M. J., et al. (1998) Modulation of secreted beta-amyloid precursor protein and amyloid beta-peptide in brain by cholesterol. J. Biol. Chem. 273, 16,576–16,582. Jarvik G. P., Austin M. A., Fabsitz R. R., Auwerx J., Reed T., Christian J. C., and Deeb S. (1994) Genetic influences on age related change in total cholesterol, low density lipoprotein cholesterol, triglyceride levels longitudinal apolipoprotein E genotype effects. Genet. Epidemiol. 11, 375–384. Jarvik G. P., Wijsman E. M., Kukull W. A., Schellenberg G. D., Yu C., and Larson E. B. (1995) Interactions of apolipoprotein E genotype, total cholesterol level, age, and sex in prediction of Alzheimer’s disease: a casecontrol study. Neurology 45, 1092–1096. Jick H., Zornberg G. L., Jick S. S., Seshadri S., and Drachman D. A. (2000) Statins and the risk of dementia. Lancet 356, 1627–1631.

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Cholesterol Regulates ApoE Expression Jessen F., Rao M. L., von Bergmann K., and Heun R. (2000) Plasma 24S-hydroxycholesterol (cerebrosterol) is increased in Alzheimer and vascular demented patients. J. Lipid. Res. 41, 195–198. Kivipelto M., Helkala E. L., Hanninen T., Laakso M. P., Hallikainen M., Alhainen K., et al. (2001) Midlife vascular risk factors and late life cognitive impairment: A population-based study. Neurology 56, 1683–1689. Kivipelto M., Helkala E. L., Laakso M. P., Hanninen T., Hallikainen M., Alhainen K., et al. (2002) Apolipoprotein E e4 allele, ellevated midlife total cholesterol level and high midlife systolic blood pressure are independent risk factors for late-life Alzheimer’s disease. Ann. Intern. Med. 137, 149–155. Kojro E., Gimpi G., Lammich S., Marz W., and Fahrenzolc (2001) Low cholesterol stimulates the non-amyloidogenic pathway by its effect on the a-secretase ADAM 10. Proc. Natl. Acad. Sci. U.S.A. 98, 5815–5820. Kolsch H., Lutjohann D., Ludwig M., Schulte A., Ptok U., Jessen F., et al. (2002) Polymorphism in the cholesterol 24S-hydroxylase gene is associated with Alzheimer’s disease. Mol. Psychiatry 7, 899–902. Koudinov A. R., Berezov T. T., and Koudinova N. V. (2002) Cholesterol and Alzheimer’s disease: is there a link? Neurology 58, 1135. LaDu M. J., Shah J. A., Reardon C. A., Getz G. S., Bu G., Hu J., et al. (2001) Apolipoprotein E and apolipoprotein E receptors modlulate A beta-induced glial neuroinflammatory responses. Neurochem. Int. 39, 427–434. Laffitte B. A., Repa J. J., Joseph S. B., Wilpitz D. C., Kast H. R., Mangelsdorf D. J., and Tontonoz P. (2001) LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes. Proc. Natl. Acad. Sci. U.S.A. 98, 507–512. Lambert J. C., Mann D., Goumidi L., Harris J., Amouyel P., Iwatsubo T., et al. (2001) Effect of the APOE promoter polymorphisms on cerebral amyloid peptide deposition in Alzheimer’s disease. Lancet 357, 608–609. Laskowitz D. T., Horsburgh K., and Roses A. D. (1998) Apolipoprotein E and the CNS response to injury. J. Cereb. Blood Flow Metab. 18, 465–471. Laws S. M., Clarnette R. M., Taddei K., Martins G., Paton A., Hallmayer J., et al. (2002) APOE-epsilon4 and APOE491A polymorphisms in individuals with subjective memory loss. Mol. Psychiatry 7, 768–775. Levin-Allerhand J. A., Lominska C. E., and Smith J. D. (2002) Increased amyloid-levels in APPSWE transgenic mice treated chronically with a physiological high-fat high-cholesterol diet. J. Nutr. Health Aging 6, 315–319. Lynch J. R., Morgan D., Mance J., Mathew W. D., and Laskowitz D. T. (2001) Apolipoprotein E modulates glial activation and the endogenous central nervous system inflammatory response. J. Neuroimmunol. 114, 107–113. Mazzone T., Basheeruddin K., and Poulos C. (1989) Regulation of macrophage apolipoprotein E gene expression by cholesterol. J. Lipid Res. 30, 1055–1064.


405 Mazzone T., Gump H., Diller P., and Getz G. S. (1987) Macrophage free cholesterol content regulates apolipoprotein E synthesis. J. Biol. Chem. 262, 11,657–11,662. Mauch D. H., Nagler K., Schumacher S., Goritz C., Muller E. C., Otto A., and Pfrieger F. W. (2001) CNS synaptogenesis promoted by glia-derived cholesterol. Science 294, 1354–1357. McGeer P. L. and McGeer E. G. (2001) Inflammation, autotoxicicty and Alzheimer disease. Neurobiol. Aging 22, 799–809. Meda L., Casatella M. A., Szendrei G. I., Otvos L. Jr., Baron P., Villalba M., et al. (1995) Activation of microglial cells by β-Amyloid protein and interferon-γ Nature 374, 647–650. Naidu A., Xu Q., Catalano R., and Cordell B. (2002) Secretion of apolipoprotein E by brain glia requires protein prenylation and is suppressed by statins. Brain Res. 958, 100–111. Naslund J., Haroutunian V., Mohs R., Davis K. L., Davies P., Greengard P., and Buxbaum J. D. (2000) Correlation between elevated levels of amyloid beta peptide in the brain and cognitive decline. JAMA 283, 1571–1577. Neuroinflammation Working Group (2000) Inflammation and Alzheimer’s disease. Neurobiol. Aging 21, 383–421. Notkola I. L., Sulkava R., Pekkanen J., Erkinjuntti T., Ehnholm C., Kivinen P., et al. (1998) Serum cholesterol, apolipoprotein E epsilon 4 allele, and Alzheimer’s disease. Neuroepidemiology 17, 14–20. Ordovas J. M. and Mooser V. (2002) The ApoE locus and the pharmacogenetics of lipid response. Curr. Opin. Lipidol. 13, 113–117. Papassotiropoulos A., Streffer J. R., Tsolaki M., et al. (2003) Increased brain beta-amyloid load, phosphorylated tau, and risk of Alzheimer disease associated with an intronic CYP46 polymorphism. Arch. Neurol. 60, 29–35. Parvathy S., Davies P., Haroutunian V., Purohit D. P., Davis K. L., Mohs R. C., et al. (2001) Correlation between Abeta x-40, Abeta x-42, and Abeta x-43 containing amyloid plaques and cognitive decline. Arch. Neurol. 58, 2025–2032. Petanceska S. S., DeRosa S., Olm V., et al. (2002) Statin therapy for Alzheimer’s disease: will it work? J. Mol. Neurosci. 19, 155–161. Petanceska S., Canoll P., and Devi L. A. (1996) Expression of rat cathepsin S in phagocytic cells. J. Biol. Chem. 271, 4403–4409. Prince M., Lovestone S., Cervilla J., Joels S., Powell J., Russ C., and Mann A. (2000) The association between ApoE and dementia does not seem to be mediated by vascular factors. Neurology 54, 397–402. Poirier J. (2000) Apolipoprotein E and Alzheimer ’s disease. A role in amyloid catabolism. Ann. N. Y. Acad. Sci. 924, 81–90. Puglielli L., Konopka G., Pack-Chung E., et al. (2001) Acylcoenzyme A: cholesterol acyl transferase modulates the

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406 generation of the amyloid beta-peptide. Nat. Cell. Biol. 3, 905–912. Refolo L. M., Malester B., LaFrancois J., Bryant-Thomas T., Wang R., Tint G. S., et al. (2000) Hypercholesterolemia accelerates the Alzheimer’s amyloid pathology in a transgenic mouse model. Neurobiol. Dis. 7, 321–331. Refolo L. M., Pappolla M. A., LaFrancois J., Malester B., Schmidt S. D., Thomas-Bryant T., et al. (2001) A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model of Alzheimer ’s disease. Neurobiol. Dis. 8, 890–899. Rockwood K., Kirkland S., Hogan D. B., MacKnight, Merry H., Verreault R., et al. (2002) Use of lipid lowering agents, indication bias, and the risk of dementia in community dwelling elderly people. Arch. Neurol. 59, 223–227. Romas S. N., Tang M. X., Berglund L., and Mayeux R. (1999) APOE genotype, plasma lipids, lipoproteins, and AD in community elderly. Neurology 53, 517–521. Runz H., Rietdorf J., Tomic I., et al. (2002) Inhibition of intracellular cholesterol transport alters presenilin localization and amyloid precursor protein processing in neuronal cells. J. Neurosci. 22, 1679–1689. Santillo M., Migliaro A., Mondola P., Laezza C., Damiano S., Stingo S., et al. (1999) Dietary and hypothyroid hypercholesterolemia induces hepatic apolipoprotein E expression in the rat: direct role of cholesterol. FEBS Lett. 463, 83–86. Shie F. S., Jin L. W., Cook D. G., Leverenz J. B., and LeBoeuf R. C. (2002) Diet-induced hypercholesterolemia enhances brain Abeta accumulation in transgenic mice. Neuroreport 13, 455–459. Simons M., Schwarzler F., Lutjohann D., von Bergmann C., Beyreuther K., Dichgans J., et al. (2002) Treatment with simvastatin in normocholesterolemic patients with Alzheimer ’s disease: a 26 week randomized, placebo-controlled, double-blind trial. Ann. Neurol. 52, 346–350. Sparks D. L. (1996) Intraneuronal beta-amyloid immunoreactivity in the CNS. Neurobiol. Aging 17, 29,120–29,129. Sparks D. L., Liu H., Gross D. R., and Scheff S. W. (1995) Increased density of cortical apolipoprotein E immunoreactive neurons in rabbit brain after dietary administration of cholesterol. Neurosci. Lett. 187, 142–144.


Petanceska et al. Srivastava R. (1996a) Regulation of the apolipoprotein E by dietary lipids occurs by transcriptional and post-transcriptional mechanisms. Mol. Cell. Sci. 155, 153–162. Srivastava R. A., Bhasin N., and Srivastava N. (1996b) Apolipoprotein E gene expression in various tissues of mouse and regulation by estrogen. Biochem. Mol. Biol. Intern. 38, 91–101. Stone D. J., Rozovsky I., Morgan T. E., Anderson C. P., Hajian H., and Finch C. E. (1997) Astrocytes and microglia respond to estrogen with increased ApoE mRNA in vivo and in vitro. Exp. Neurol. 143, 313–318. Wahrle S., Das P., Nyborg A. C., McLendon C., et al. (2002) Cholesterol-dependent gamma-secretase activity in buoyant cholesterol-rich membrane microdomains. Neurobiol. Dis. 9, 11–23. Wang J. C., Kwon J. M., and Shah P. (2000) Effect of APOE genotype and promoter polymorphism on risk of Alzheimer’s disease. Neurology 55, 1644–1649. Werb Z. and Chin J. R. (1983) Endotoxin suppresses expression of apolipoprotein E by mouse macrophages in vivo and in culture. A biochemical and genetic study. J. Biol. Chem. 258, 10,642–10,648. Wolozin B., Kellman W., Ruosseau P., Celesia G. G., and Siegel G. (2000) Decreased prevalence of Alzheimer’s disease associated with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Arch. Neurol. 57, 1439–1443. Wong L. and Rubenstein D. (1997) The levels of apolipoprotein in hypercholesterolemic rat serum. J. Biochem. 56, 161–166. Yaffe K., Barrett-Connor E., Lin F., and Grady D. (2002) Serum lipoprotein levels, statin use, and cognitive function in older women. Arch. Neurol. 59, 378–384. Yoshitake T., Kiyohara Y., Kato I., Ohmura T., Iwamoto H., Nakayama K., et al. (1995) Incidence and risk factors of vascular dementia and Alzheimer’s disease in a defined elderly Japanese population: the Hisayama Study. Neurology 6, 1161–1168. Xu Q., Li Y., Cyras C., Sanan D. A., and Cordell B. (2000) Isolation and characterization of apolipoproteins from murine microglia. Identification of a low density lipoprotein-like apolipoprotein J-rich but E-poor spherical particle. J. Biol. Chem. 275, 31,770–31,777.

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