High cholesterol-induced neuroinflammation and amyloid precursor protein processing correlate with...

*Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina, USA

�Department of Psychology, Arizona State University, Tempe, Arizona, USA

A number of findings suggest a link between abnormalcholesterol metabolism and Alzheimer’s disease (AD) path-ogenesis (Puglielli et al. 2003; Casserly and Topol 2004;Sambamurti et al. 2004). First, several AD-associated genes(i.e. ApoE, cyp46, and ABCA1) that show disease-specificpolymorphisms are otherwise normal participants in choles-terol metabolism (Roses 1996; Bjorkhem et al. 1998; Katzovet al. 2004). Second, clinical studies have indicated thatindividuals with increased cholesterol are more susceptible toAD and that elevated levels of low-density lipoprotein (LDL)cholesterol correlate well with AD incidence compared withasymptomatic cases (Puglielli et al. 2003). Third, animalstudies using New Zealand white rabbits (Sparks et al. 2000)and transgenic mouse models of AD (Refolo et al. 2000;Levin-Allerhand et al. 2002) have demonstrated that

diet-induced hypercholesterolemia could enhance brainbeta-amyloid (Ab) accumulation. Cholesterol has also beenshown to directly modulate the processing of the amyloid

Received February 22, 2008; revised manuscript received February 28,2008; accepted April 3, 2008.Address correspondence and reprint requests to Narayan R. Bhat,

PhD, Department of Neurosciences, Medical University of SouthCarolina, Charleston, SC 29425, USA. E-mail: [email protected] used: AD, Alzheimer’s disease; APP, amyloid precursor

protein; Ab, beta-amyloid; BACE1, b-site APP cleaving enzyme 1;COX2, cycloxygenase 2; CTF, C-terminal fragments; GAPDH, glycer-aldehyde 3-phosphate dehydrogenase; GFAP, glial fibrillary acidic pro-tein; hAPP, human APP; IL, interleukin; iNOS, inducible nitric oxidesynthase; LDL, low density lipoprotein; LDLR, low density lipoproteinreceptor; TNF, tumor necrosis factor; WM, working memory; wt, wild-type.

Abstract

Recent findings suggest that hypercholesterolemia may con-

tribute to the onset of Alzheimer’s disease-like dementia but

the underlying mechanisms remain unknown. In this study, we

evaluated the cognitive performance in rodent models of

hypercholesterolemia in relation to neuroinflammatory chan-

ges and amyloid precursor protein (APP) processing, the two

key parameters of Alzheimer’s disease pathogenesis. Groups

of normal C57BL/6 and low density lipoprotein receptor

(LDLR)-deficient mice were fed a high fat/cholesterol diet for

an 8-week period and tested for memory in a radial arm maze.

It was found that the C57BL/6 mice receiving a high fat diet

were deficient in handling an increasing working memory load

compared with counterparts receiving a control diet while the

hypercholesterolemic LDLR)/) mice showed impaired work-

ing memory regardless of diet. Immunohistochemical analysis

revealed the presence of activated microglia and astrocytes in

the hippocampi from high fat-fed C57BL/6 mice and LDLR)/)mice. Consistent with a neuroinflammatory response, the hy-

perlipidemic mice showed increased expression of cytokines/

mediators including tumor necrosis factor-a, interleukin-1b

and -6, nitric oxide synthase 2, and cycloxygenase 2. There

was also an induced expression of the key APP processing

enzyme i.e. b-site APP cleaving enzyme 1 in both high fat/

cholesterol-fed C57BL/6 and LDLR)/) mice accompanied by

an increased generation of C-terminal fragments of APP. Al-

though ELISA for beta-amyloid failed to record significant

changes in the non-transgenic mice, a threefold increase in

beta-amyloid 40 accumulation was apparent in a strain of

transgenic mice expressing wild-type human APP on high fat/

cholesterol diet. The findings link hypercholesterolemia with

cognitive dysfunction potentially mediated by increased

neuroinflammation and APP processing in a non-transgenic

mouse model.

Keywords: amyloid precursor protein processing, beta-site

APP cleaving enzyme 1, cholesterol, low density lipoprotein

receptor, neuroinflammation, working memory.

J. Neurochem. (2008) 106, 475–485.

JOURNAL OF NEUROCHEMISTRY | 2008 | 106 | 475–485 doi: 10.1111/j.1471-4159.2008.05415.x

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 106, 475–485 475

precursor protein (APP) in neuronal cell cultures (Bodovitzand Klein 1996; Simons et al. 1998; Frears et al. 1999;Ehehalt et al. 2003). Many such, but not all, observationshave suggested that cholesterol may play a prominent role inAD pathogenesis and that lowering cholesterol may benefitdisease prognosis. In fact, retrospective studies have dem-onstrated that cholesterol lowering drugs i.e. statins, couldlower the incidence of AD (Wolozin et al. 2000). However,the statin activity most likely involves mechanisms otherthan inhibiting cholesterol synthesis, a prominent one beingthe drug’s anti-inflammatory effects (Liao and Laufs 2005;Wolozin et al. 2006). In this regard, it is now clear thatatherosclerosis, AD as well as many other neurodegenerativediseases all have in common, an inflammatory component.While inflammation is central to atherosclerosis involvingchanges in the endothelium caused by oxidized lipids/lipoproteins, monocyte/macrophage accumulation, and ulti-mately resulting in sclerotic plaque formation (Steinberg2002), there is ample evidence for an activation of inflam-matory processes and the presence of cytokines and othermediators in pathologically vulnerable regions of the ADbrain (Akiyama et al. 2000; Wyss-Coray and Mucke 2002;Heneka and O’Banion 2007).

It is likely that a compromised cholesterol homeostasis,resulting either from dietary practice or genetic imposition,facilitates an exacerbated neuroinflammatory response inassociation with increased Ab generation leading to AD-associated degeneration. To test this idea, we administered ahigh fat/cholesterol diet to groups of 4-month-old C57BL/6and the hypercholesterolemic strain i.e. LDL receptor-deficient(LDLR)/)) mice for an 8-week period, followed by anassessment of working and reference memory function inrelation to neuroinflammatory and AD-related biochemicalchanges. Importantly, these evaluations were all performed inthe same animals, allowing correlational assessments betweenthe cognitive, physiological, and neurobiological variables.The results presented implicate a link between diet-inducedneuroinflammation and AD-associated cognitive impairment.

Materials and methods

Animals and treatmentNormal C57BL/6 mice, LDLR)/) mice (ldlr KO strain, B6.12957-

Ldlrtm1Her) and a transgenic line i.e. PDGFB-APPwt, were obtained

from Jackson Labs (Bar Harbor, ME, USA). The three strains of

mice (4-month old) were divided into two groups (each n = 6):

group 1, normal chow (5% fat and 0.05% cholesterol) and group 2, a

high fat (21%) and high cholesterol (1.25%) diet (Teklad, Madison,

WI, USA), resulting in a total of six treatment groups. The mice

were on these diets for 2 months. During the last 2 weeks of this

2-month diet, mice were tested for memory performance in an 8-arm

water maze (description below) before killing. Following behavioral

testing, to obtain tissue samples, the animals were anesthetized

with an i.p. injection of sodium pentobarbital (50 mg/kg) and

transcardially perfused with phosphate-buffered saline (4�C). Next,the brains were removed and divided sagittally. One hemibrain was

post-fixed in phosphate-buffered 4% p-formaldehyde, pH 7.4, at

4�C for 48 h for sectioning; the other was rapidly dissected for

hippocampus and cortical tissues, snapfrozen, and stored at )70�Cfor biochemical analyses. All animal procedures were carried out in

accordance with the USPHS policy on the Humane Care and Use of

Laboratory Animals.

ImmunohistochemistryThe fixed tissue was serially sectioned at 40 lm with a freezing

microtome (Microm HM400, Richard Allen Scientific, Kalamazoo,

MI, USA) and the sections used for immunohistochemical analysis

of activated glia i.e. microglia (stained with anti-CD45 antibodies)

and astrocytes (stained for glial fibrillary acidic protein (GFAP)). A

kit (Vector ABC Elite kit) from Vector Laboratories (Burlingame,

CA, USA) was used along with 3¢3¢ diaminobenzoic acid as

a chromogen to develop the reaction in the presence of H2O2.

For quantitative measurements of staining intensities, images of

stained areas were captured with a Nikon Eclipse E-600 microscope

(Melville, NY, USA) equipped with a CCD camera and analyzed

using NIH Image� Software (National Institutes of Health,

Bethesda, MD, USA). Measurements from four to six sections per

brain were averaged to obtain one value per subject.

Beta-amyloid ELISAKnown amounts of cortex samples were homogenized in 10

volumes of phosphate-buffered saline and centrifuged at

100 000 g for an hour to collect membranes. The membrane pellets

were then extracted in 10 volumes of a basic solution containing

0.2% diethylamine (Sigma-Aldrich, St Louis, MO, USA), 50 mM

NaCl, protease inhibitor cocktail (Roche Molecular Biochemicals,

Indianapolis, IN, USA), and 5 mM O-phenanthroline (Sigma-

Aldrich). The diethylamine extract (100 lL) was neutralized with

50 mM Tris–Hcl, pH 6.8, centrifuged at 100 000 g for 1 h and the

supernatant diluted in the ELISA buffer for further analysis. Ab40and 42 levels were measured by a Sandwich ELISA using kits from

IBL-America (Minneapolis, MN, USA). The antibodies used for the

Sandwich ELISA were specific for the Ab40 (1A10) and Ab42(affinity purified polyclonal) ends for capture and a monoclonal

antibody (12B2) against Ab residues 11–28 coupled to horseradish

peroxidase for detection. The target sequence is conserved in

rodents (except residue 13) and the antibody detects rodent Abpeptide as efficiently as human Ab as reported by the manufacturer.

Western blotFor western blot analysis of APP and C-terminal fragments (CTFs),

the homogenates representing equal amounts of protein were loaded

on a 10–20% gradient gel (Bio-Rad Criterion; Bio-Rad Laboratories,

Hercules, CA, USA). The antibody used i.e. 0443, was raised in

rabbits against the C-terminal 20 residues of APP (Pinnix et al.2001b). This antibody detects APP (from a variety of species fromfish

to humans) with an intact cytoplasmic domain, which includes full-

lengthAPP, CTFa (C-terminal 83 residues), andCTFb (C-terminal 99

residues). The antibody against the cytoplasmic domain of b-site APPcleaving enzyme 1(BACE1) used in this study has been described

before (Pinnix et al. 2001a) and detects a 62 kDa protein inmouse and

guinea pig brain extracts. Its specificity has been characterized by

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 106, 475–485� 2008 The Authors

476 | L. Thirumangalakudi et al.

western analysis and blocking experiments using cell extracts from

Chinese hamster ovary cells transfected with recombinant BACE

(Venugopal and Sambamurti, unpublished studies).

Quantitative RT-PCRTotal RNA was isolated using Trizol reagent (Invitrogen, Carlsbad,

CA, USA). The mRNAs were reverse transcribed into cDNAs using

M-MLV reverse transcriptase (Invitrogen). Specfic primers for the

mouse cytokines i.e. interleukin-1b (IL-1b), tumor necrosis factor-a(TNFa), IL-6, and glyceraldehyde 3-phosphate dehydrogenase

(GAPDH) were as described (Kitazawa et al. 2005). The primers

used for inducible nitric oxide synthase (iNOS) were: 5¢-CAGC-TGGGCTGTACAAACCTT; 5¢-CATTGGAAGTGAAGCGTTTCGand those for cycloxygenase 2 (COX2) were: 5¢-ACACTCTATCA-CTGGCACCC; 5¢-GAAGGGACACCCCTTCACAT. The primer

sets for mouse BACE was purchased from SuperArray (Frederick,

MD, USA). Real-time RT-PCR analysis was carried out on a

Bio-Rad iCycler system using a SYBR Green qPCR Supermix kit.

At the end of the runs, melting curves were obtained to make sure

that there are no primer-dimer artifacts. The products were verified

by agarose gel analysis. Threshold cycle values were calculated with

MyiQ software (Bio-Rad) and quantitative fold changes in mRNA

were determined as relative to GAPDH or 18S mRNA levels in each

treatment group.

Behavioral testingCognitive testing was performed using the water radial-arm maze.

The win-shift radial-arm maze utilizes water escape onto hidden

platforms as the reinforcer, thereby avoiding food deprivation

(Bimonte and Denenberg 2000; Bimonte et al. 2000). The 8 arm

maze had hidden escape platforms in the ends of 4 arms. Each

subject had different platform locations that were semi-randomly

determined and that remained fixed throughout the experiment. A

subject had 3 min to locate a platform. If the allotted time expired,

the subject was guided to the nearest available platform. Once a

platform was found, the animal remained on it for 15 s, and was

then returned to its heated home cage for 30 s until its next trial.

During the interval, the just-chosen platform was removed from the

maze. The animal was then placed again into the start alley and

allowed to locate another platform. A daily session consisted of this

sequence of events repeated until all four platforms were located.

Consequently, for each animal a daily session consisted of four trials

per session, with the number of platformed arms reduced by one on

each subsequent trial. Hence, the working memory (WM) system

was increasingly taxed as trials progressed. This version is similar to

the land version of the radial-arm maze in that animals had to avoid

arms that never contained a reinforcer (reference memory) and enter

only once into arms that contained a reinforcer (WM).

The following quantification and blocking procedures are based

upon previous studies using the water radial-arm maze (Hyde et al.1998, 2000; Bimonte and Denenberg 2000; Bimonte et al. 2000).Each subject was given one session a day for 15 consecutive days.

Day 1 was considered a training session because the animal had no

previous experience in the maze. Days 2–15 were testing sessions,

which were blocked into 2-day blocks for analyses. Errors were

quantified for each daily session using the orthogonal measures of

working and reference memory errors (Jarrard et al. 1984), as

performed previously in studies using the water escape radial-arm

maze (Bimonte et al. 2000; Hyde et al. 2000; Hunter et al. 2003).WM correct errors were the number of first and repeat entries into

any arm from which a platform had been removed during that

session. Reference memory errors were the number of first entries

into any arm that never contained a platform. WM incorrect errors

were the number of repeat entries into an arm that never contained a

platform in the past (thus, repeat entries into a reference memory

arm). Data were analyzed using repeated measures ANOVA, with

Genotype and Diet as between subjects variables and Block and Trial

as repeated measures (STATVIEW 5.0 SAS Institute, Cary, NC, USA).

Results

The high fat/high cholesterol diet did not alter weight gain atany point during the 2-month period. However, as shown inFig. 1, there were significant increases in plasma levels oftotal and LDL-associated cholesterol in both C57BL/6 mice(Fig. 1a) and in the LDLR-deficient mice (Fig. 1b). Whilethe high fat diet fed C57BL/6 mice showed an increase of150% over normal chow-fed controls for total cholesterol,there was a 10-fold increase in the case of the hypercholes-terolemic LDLR)/) mice. The excess cholesterol was almostexclusively associated with plasma LDL, the level of whichwas undetectable in the C57BL/6 mice on a normal diet.There was also a significant increase in plasma level oftriglycerides in LDLR-deficient mice. Interestingly, theLDLR)/) mice on a ‘normal’ diet contained significantlyhigher (over threefold) level of cholesterol compared withnormally fed C57BL/6 mice (Fig. 1c). Again, this increasewas reflected in a high level of LDL cholesterol.

The high-fat, high cholesterol diet impairs working memoryin normal miceDuring weeks 7 and 8 of the feeding protocol, groups ofC57BL/6 and LDLR)/) mice fed either the normal chow orthe high fat/high cholesterol diet for the entire 2-monthperiod, were cognitively tested on the working and referencememory water radial-arm maze as described under Materialsand methods. As shown in Fig. 2a, C57BL/6 mice receivingthe high fat diet were less able to successfully handle anincreasing WM load compared with counterparts receiving acontrol diet. Further, while a high fat diet impaired overallWM incorrect errors for C57BL/6 mice, LDLR)/) miceshowed impaired WM regardless of diet (Fig. 2b).

The high-fat, high cholesterol diet inducesneuroinflammation characterized by glial activation andcytokine expressionThe brain sections obtained from the two groups each ofC57BL/6 and LDLR)/) mice fed either a high fat/choles-terol diet or normal chow were processed for immunohisto-chemistry to detect signs of neuroinflammation. The sectionswere stained for CD45, a microglial marker and GFAP, amarker for reactive astrocytes. As shown in Fig. 3a, the

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 106, 475–485

Cholesterol-induced AD-like changes in mice | 477

hippocampi from high fat-fed C57BL/6 mice elicitedincreased immunostaining for CD45 compared with basaldiet fed C57BL/6 animals. Sections from LDLR)/) miceseemed to show increased CD45 staining irrespective of thediet indicating that the LDLRKO phenotype may predispose

the mice to increased neuroinflammatory response. High fatdiet also induced the expression of immunoreactive GFAP,indicating an activation of astrocytes in C57BL/6 mice(Fig. 3b). As in the case of CD45, there was an increasedbasal GFAP-immunoreactivity in the LDLR)/) mice thatwas further enhanced by high fat/cholesterol diet (Fig. 3b). Aquantitative analysis of these immunohistochemical data isshown in Fig. 3c.

Induction of brain expression of proinflammatory cytokinesin hypercholesterolemic miceReal-time qPCR was carried out to determine the relativeexpression levels of the cytokines i.e. IL-1b, IL-6, and TNFaas well as the two proinflammatory enzymes i.e. COX2 andiNOS, in the hippocampi dissected from the C57BL/6 andLDLR)/) fed either normal chow or the high-fat diet for8 weeks. GAPDH message was used to normalize the values.As shown in Fig. 4a, there was a marked increase in theexpression of the mediators tested. While the cytokines,TNFa and IL-1b showed increases of between 100- and 200-fold, there was a 30-fold increase in the expression of IL-6 inhigh fat fed C57BL/6 mice over normally fed controls. Therewas also a significant increase in the expression of the twoenzymes i.e. iNOS (35-fold) and COX2 (6-fold). Interest-

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Fig. 1 Plasma cholesterol and triglyceride levels in C57BL/6 and

LDLR)/) mice fed a normal chow versus a high fat/high cholesterol

diet. The plasma lipid profiles of different treatment groups were

determined in the GCRC facility at MUSC. (a) Total cholesterol, HDL-

cholesterol, LDL-cholesterol and triglyceride levels of plasma samples

from C57BL/6 mice fed either normal chow or a high fat diet. (b) Total

cholesterol, HDL-cholesterol, LDL-cholesterol, and triglyceride levels

of plasma from LDLR)/) on either normal chow or the high fat diet. (c)

A comparison of total cholesterol, HDL-cholesterol, LDL-cholesterol,

and triglyceride levels of plasma derived from normally fed C57BL/6

and LDLR)/) mice. *p < 0.05 versus the normally fed control group

and #represents significant (p < 0.05) difference of normally fed

LDLR)/) group from normally fed C57BL/6 (control) group.

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Fig. 2 Behavioral analysis of working memory. Mean (±SE) number of

working memory incorrect errors for each trial committed on the water

radial maze for normally fed and high fat fed C57BL/6 mice were

determined as described under Materials and methods. (b) Working

memory incorrect ± SE for each treatment group i.e. C57BL/6 and

LDLR)/) mice fed either normal or high fat diet. *p < 0.05 versus the

normally (basal diet) fed control group and #represents significant

(p < 0.05) difference of normally fed LDLR)/) group from normally fed

C57BL/6 (control) group.



ingly, LDLR)/) mice fed a normal diet had markedlyelevated levels of all these mediators relative to normally fedC57BL/6 mice (Fig. 4b). Feeding of these mice with the highfat diet did not result in a significant increase in their levelsover already elevated basal levels (Fig. 4c).

BACE1 expression, APP processing, and Ab generation inresponse to high fat/cholesterol feedingWe next analyzed parameters associated with APP process-ing including the expression of BACE1, a key enzymeinvolved in this process. Figure 5a shows the qPCR data forthe expression of BACE1 mRNA in response to cholesterol-enriched diet. There was over fourfold increase in the levelsof BACE1 message in high fat fed C57BL/6 mice overnormally fed mice while the basal level of BACE1 expres-sion in the LDLR)/) mice was about threefold higher thanthe C57BL/6 mice. The LDLR)/) showed a further increasewhen fed a high cholesterol diet. Figure 5b shows theexpression of the enzyme protein as determined by westernblot, again confirming the induction of the enzyme as a resultof cholesterol feeding in both C57BL/6 and LDLR)/) mice.

The protein extracts were also used for western analysis ofAPP processing using the antibody 0443. Figure 6(a and b)represent western blot analysis showing increased levels ofCTFs in high cholesterol-fed C57BL/6 and LDLR)/) mice,respectively, compared with normal chow-fed counterparts.We also analyzed APP processing products in brain samplesderived from wild-type (wt) human APP (hAPP) mice fednormal chow and high fat diet. As shown in Fig. 6c, theincreased CTF bands seem to contain both a and b forms inthe high fat fed samples. Figure 6d gives quantitative dataobtained from a densitometric analysis of the western blots ofall the samples.

We next determined the changes in the levels of Ab40 and42 by ELISA. Despite the observed increases in theproduction of CTFs (above), the ELISA assay failed todetect significant changes in Ab levels in high fat fedC57BL/6 mice (data not shown). However, LDLR)/) micefed a high fat diet had an appreciable increase in the level ofAb40 (Fig. 7a) but not Ab42 (not shown). As the transgenicmice expressing hAPP produce Ab that can be easilydetected by the available antibodies, we tested the effects of

C57 LDLR-/- LDLR-/-

Normal

C57

Normal

High fat

CD45

High fat

GFAP

(a) (b)

(c)

Fig. 3 Immunohistochemical analysis of high fat/cholesterol diet-in-

duced glial activation. (a) CD45 staining of microglia in the hippo-

campus of C57BL/6 and LDLR)/) mice fed normal chow or the high

fat/cholesterol diet. (b) GFAP-immunoreactive astrocytes in the hip-

pocampus of C57BL/6 and LDLR)/) mice fed normal chow or the high

fat/cholesterol diet. (c) Quantitation of CD45 and GFAP stainings of

C57BL/6 (left panel) and LDLR)/) (right panel) mice fed normal and

high fat diet. The images (four sections per animal) were captured and

quantified using the NIH Image� software package. *p < 0.05 versus

the normally (basal diet) fed control group and **p < 0.01 versus the

normally (basal diet) fed control group.



the high cholesterol diet on the production of Ab in the wthAPP mice, the brain extracts from which showed higherlevels of CTFs, as above (Fig. 6c). As shown in Fig. 7b, the

levels of Ab were much higher than in the normal mice.Moreover, there was a significant, threefold increase in theproduction of Ab40 in response to high fat/cholesterol

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Fig. 4 Quantitative PCR analysis of high fat/cholesterol-induced pro-

inflammatory mediator expression. (a) Relative levels of cytokine/

mediator mRNAs in C57BL/6 mice fed either normal or high fat diet. (b)

Relative cytokine/mediator mRNA levels in normally fed C57BL/6 and

LDLR)/) mice. *p < 0.05 versus the normally fed control group and

#represents significant (p < 0.05) difference of normally fed LDLR)/)group from normally fed C57BL/6 (control) group. (c) Relative levels of

cytokine/mediator mRNAs in LDLR)/) mice fed either normal or high

fat diet.

BACE-1

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Fig. 5 High fat/cholesterol-induced expression of BACE1. (a) Total

RNA was isolated from the corteces of C57BL/6 and LDLR)/) mice

fed the two diet regimen and analyzed by qPCR for BACE1 mRNA.

*p < 0.05 versus the normally fed control group and #represents sig-

nificant (p < 0.05) difference of normally fed LDLR)/) group from

normally fed C57BL/6 (control) group. (b) Corresponding tissue ex-

tracts were analyzed by immunoblot for BACE1 protein levels using

BACE1-specific antibodies. The line graphs below show the relative

BACE1 protein levels.



feeding although Ab42 did not show a further increase overthe basal levels.

Discussion

In this study, we have examined the influence of hypercho-lesterolemia on the cognitive performance in mice in relationto neuroinflammatory changes and APP processing, the twocommon traits associated with Alzheimer’s dementia. Thestudies employed normal C57BL/6 mice fed a high fat, highcholesterol diet as well as mice with a targeted disruption ofthe LDLR gene (Ldlr)/)) that develop moderate hypercho-lesterolemia that can be greatly exacerbated by cholesterolfeeding (Ishibashi et al. 1993). We found that both high fat/high cholesterol fed C57BL/6 mice and the LDLR)/) miceexperience significant memory deficits in an 8-arm radialwater maze test. In the case of LDLR)/) mice, the memorydeficit occurred regardless of diet most likely due to basallyelevated cholesterol levels. However, the findings with theLDLR)/) mice must be viewed with the caveat that althoughtheir genetic background is the same as that of the controlstrain, i.e. C57BL/6 used for comparison, it may not beidentical as could have been with the use of littermatecontrols. In C57BL/6 mice, the atherogenic diet induced aneuroinflammatory response characterized by an activation of

glia (i.e. microglia and astrocytes) and increased expression ofa number of inflammatory cytokines/mediators. In contrast,the CNS of LDLR)/) mice on a ‘normal’ diet, showed basalactivation of glia and an up-regulation of the expression ofinflammatory markers. Feeding of high fat/cholesterol diet tothese mice resulted in an increased glial activation but thiseffect was not accompanied by a further induction of theexpression of inflammatory mediators. The reason for thisdiscrepancy is not clear but may suggest a dissociation ofreactive phenotype from mediator expression or may reflectdifferential transcriptional versus post-transcriptional mecha-nisms of marker/mediator expression under the two condi-tions. Nonetheless, the elevated pro-inflammatory responseand behavioral deficits observed in C57BL/6 mice on a highcholesterol diet the LDLR-deficient mice correlated well withthe high plasma cholesterol status of these mice. There wasalso an induction of the expression of BACE1, an enzymeresponsible for the amyloidogenic processing of APP. APPprocessing itself showed changes with an increased produc-tion of CTFs in both C57BL/6 and LDLR-deficient mice fed ahigh cholesterol diet. There was also an appreciable (but notreaching significance with n = 6) increase in Ab40 but notAb42 levels in hypercholesterolemic LDLR)/) mice and asignificant increase in Ab40 levels in an hAPPwt transgenicstrain fed high cholesterol. The main findings of the presentstudies are summarized in Table 1.

Mol wt Std, kDa APP/CTF std

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APP

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Wt-hAPP

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** **

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37

20

25

10

*

Fig. 6 Western blot analysis of APP processing. Representative blots

of APP/CTFs in tissue samples from C57BL/6, (a) LDLR)/) (b) and

APPTg (c) mice fed either a normal chow or the high fat/cholesterol

diet. Densitometric analysis of CTF relative to APP (d). All three

strains of high fat/cholesterol fed mice showed significantly higher

CTF : APP ratio compared with normal chow fed counterpart animals;

**p < 0.01 and *p < 0.05.

0.0

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25.0

Aβ40 Aβ42

Control

(a)

(b)

High fat

Wt-hAPP

00.40.81.21.62.02.42.8

Aβ4

0, n

g/g

tissu

e

Normal High fat

LDLR–/–

*

Aβ4

0/42

, ng/

g tis

sue

Fig. 7 Ab generation in LDLR)/) mice (a) and hAPPtg mice (b) fed

either the normal chow or the high fat/cholesterol diet. The tissue

extracts were prepared as described under Materials and methods

and analyzed for Ab species using a sensitive ELISA. The levels of

Ab42 were very low in the LDLR)/) mice. The levels of Ab40 as well

as 42 were very low in the normal C57BL/6 mice; *p < 0.05 versus the

normally fed control group.



The most significant and novel finding of our studies is theoccurrence of diet-induced cognitive impairment in thenormal mice. This represents an advance over previousreports indicating cholesterol-induced memory deficits in thetransgenic mice expressing human mutant APP (Li et al.2003). There have also been reports of hypercholesterolemia-induced memory dysfunction in the LDLR)/) mice (Mulderet al. 2004; Buga et al. 2006) similar to our findings and anaggravation of learning deficits in association with amyloiddeposition in the APPTg with LDLR-deficiency (Cao et al.2006). Another important aspect of our findings pertains toneuroinflammatory changes in the hypercholesterolemicmice that show a close correlation with behavioral changessuggesting that cholesterol-induced neuroinflammation mayplay a causal role in memory dysfunction observed in ourmodel. There is now convincing evidence that inflammatorycascades mainly mediated by activated microglia play asignificant role in AD-like changes (Akiyama et al. 2000;Wyss-Coray and Mucke 2002; Heneka and O’Banion 2007).In AD and AD models, a common trigger of inflammation isthought to be an activation of glia by plaque-associated Ab,and hence, neuroinflammation represents a secondary con-sequence of AD-associated amyloidogenesis (Hardy andSelkoe 2002). Although elevated membrane cholesterolmight induce production of extracellular Ab (see below)potentially contributing to enhanced microglial activation,the mechanisms underlying cholesterol-induced neuroinflam-mation may be different and likely involve neurovascularchanges. A damaged or dysfunctional cerebrovasculatureunder an hypercholesterolemic condition may trigger anactivation of perivascular microglia involved in barrier andscavenger functions in the brain (Perry et al. 2003). Thevascular pathology may also promote systemic macrophageinvasion into the brain parenchyma. It is possible that someof the CD45-immunoreactive cells in our analysis mightrepresent invading macrophages. The recruitment of localimmune cells to the inflamed brain arterioles could theninitiate a cascade of events leading to an altered amyloidprocessing, neurodegeneration and synaptic/cognitive dys-function. Although cognitive studies have not been feasible,the rabbit model of hypercholesterolemia does providesupporting evidence for the vascular mechanism of neuro-

inflammation including increased expression of VCAM1(Streit and Sparks 1997; Sparks et al. 2000). There is alsoevidence for an activation of microglia (Streit and Sparks1997) as well as an occurrence of increased Ab immunore-activity (intraneuronal and occasional extracellular) in thehippocampus and cortical regions (Sparks et al. 1994). Arecent study by the same group found that microglialactivation was restricted to the hippocampus indicating itsoccurrence independent of increased amyloid production(Xue et al. 2007). Instead, it was thought that high levels ofserum cholesterol would induce vascular changes similar toearly inflammatory lesions of atherosclerosis but, in addition,would induce blood brain barrier permeability and localizedneuroinflammatory changes observed. Another study by(Ghribi et al. 2006b) with the rabbit model shows choles-terol-induced Ab and iron deposition in the cortex, more sothan in the hippocampus, in association with BBB disruption.The connection between cerebrovascular (including region-specific) changes and neuroinflammation in our modelremains to be further explored. It is possible that an inflamedvasculature might have contributed substantially to theincreased levels of the cytokines/mediator observed. Insupport of the occurrence of vascular inflammation in ADitself, brain microvessels isolated from AD brain do containhigh levels of cytokines and chemokines (Grammas et al.2006) thereby supporting a cerebrovascular contribution toAD pathogenesis via neuroinflammatory processes (Zlokovic2005). Of related interest, a prominent role played byinflammation in inducing behavioral deficits in a model ofcerebral microvascular amyloid has been emphasized in astudy using minocycline (Fan et al. 2007). The anti-inflam-matory drug was able to correct/reverse cognitive deficitsalong with reduction in the number of activated microgliaand cytokine (i.e. IL-6) levels but without altering theaccumulation and distribution of Ab.

Our results show that in addition to several proinflamma-tory cytokines, BACE1 expression is induced in hypercho-lesterolemic mice. This finding is in line with a recent reportindicating an up-regulation of BACE1 expression in therabbit model (Ghribi et al. 2006a). It is known that, theregulation of BACE1 and other processing enzymes canoccur not only at the level of their activities but also in the

Table 1 Summary of the relative effects of a high fat/high cholesterol diet on biochemical and behavioral changes in C57BL/6 and LDLR)/) mice

Genotype Diet

Plasma

cholesterol Gliosis

Mediator

expression

BACE1

expression

APP

processing

Behavior

number

of errors

C57BL/6 Normal Normal Minimal Minimal Low Low Low

High fat Increased Increased Increased Increased Increased Increased

LDLR)/) Normal Elevated Increased Increased Higher Higher Higher

High fat Very high Further

increase

No further

increase

Further

increase

Further

increase

No further

increase



expression of these enzymes, especially, in response toinflammation and oxidative stress. The expression ofBACE1, in particular, is subject to regulation by cytokinessuch as TNFa, IL-1b, and interferon-c (Sastre et al. 2006),oxidants (Tamagno et al. 2005), and hypoxia (Sun et al.2006). There is also evidence for induced expression andactivity of the secretases in the brain of late-onset sporadicAD (Li et al. 2004), perhaps due to the presence of suchinducing factors. Such findings suggest that an inflammatoryenvironment can not only have a direct neuro/synaptotoxiceffect but also induce APP processing and amyloid gener-ation. Most likely, BACE1 expression in the brains ofhypercholesterolemic mice that we observed is the result ofcholesterol-induced inflammation and oxidative stress.

An obvious consequence of an up-regulation of BACE1 isincreased APP processing. However, although our westernblot data do indicate the generation of CTFs, the predominantform accumulating is CTFa and not CTFb. Our observationof increased APP processing in the LDLR)/) mice contrastswith a recent study by (Elder et al. 2007) who found that ahigh plasma cholesterol level did not affect brain APPprocessing. The reason for this discrepancy is unclear butlikely relates to different reactivities of the antibodies usedfor the western analysis of APP. Increased APP processingactivity we observed could be due to cholesterol’s directinfluence on the activities of the APP processing enzymes(reviewed in (Sambamurti et al. 2004). Alternatively, cho-lesterol may affect both b- and c-secretase activitiesindirectly via regulation of isoprenoid synthesis (Cole et al.2005; Zhou et al. 2008). Although it is observed that thebrains of AD do not contain increased levels of cholesterol(Lutjohann and von Bergmann 2003), there is enoughevidence to indicate functional interactions between choles-terol and APP metabolism including recent studies showingthat APPlnd/Swe mice treated with an acyl-CoA: cholesterolO-acyltransferase inhibitor display decreased Ab levels(Hutter-Paier et al. 2004) and that APP metabolism itselfregulates cholesterol homeostasis via regulation of theexpression of LDL receptor-related protein-1 (Liu et al.2007). These interactions may play out along altered intra/subcellular distribution, transport, and metabolism of cho-lesterol rather than alterations of the cholesterol content inthe central compartment per se. In fact, the studies by Elderet al. (2007) showed no increase in brain cholesterol levels inthe hypercholesterolemic LDLR)/) mice. An alternativeexplanation for plasma cholesterol affecting brain functionmay be found in its metabolism to oxysterol derivatives thatare able to pass the BBB. According to Bjorkhem (2006), theflux of 27-OH cholesterol, an oxysterol that correlates withplasma cholesterol levels, into the brain may represent themissing link between hypercholesterolemia and AD. Theyhave found evidence for elevated plasma and brain levels ofthis oxysterol in AD, potentially affecting amyloidogenesis.Future studies with our model would include an analysis of

the levels of oxysterols including 27-hydroxy- and 24-hydroxy cholesterol, a ‘brain-specific’ oxysterol that, via itsdiffusion out of the brain, functions to maintain cholesterolhomeostasis within the brain (Bjorkhem et al. 1998, 2006). Itwill also be interesting to investigate effects of cholesteroland a high dietary fat or lipid regimen separately as the latteris also known to significantly impact upon cognition (Grant1999; Cooper 2003; Winocur and Greenwood 2005).

In conclusion, the present study has demonstrated theadverse effects of high cholesterol on memory performancein a non-familial Alzheimer’s disease mouse model.Although our results demonstrate an important in vivo rolefor elevated cholesterol levels in the induction of APPprocessing potentially contributing to AD-like dementia, it ismore likely that increased neuroinflammatory changesobserved might play a primary role in cognitive dysfunctionin response to hypercholesterolemia, a possibility amenableto a direct validation with the use of specific anti-inflamma-tory agents in future studies.

Acknowledgement

We thank Ms Kari Werness and Jacob Pugh for carrying out the

behavioral testing and Ms Hong Zhu for help with immunohisto-

logical analysis. The studies were supported by grants #

R01NS051575 (NRB) and R01AG023055 (KS) and the work

utilized a facility constructed with support from the National

Institutes of Health, Grant Number C06 RR015455 from the

Extramural Research Facilities Program of the National Center for

Research Resources.

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