Elevated plasma cholesterol does not affect brain Ab in micelacking the low-density lipoprotein receptor

Gregory A. Elder,*,¶ Julie Y. Cho,*,**,1 Daniel F. English,*,**,1 Sonia Franciosi,*,**James Schmeidler,*,§ Miguel A. Gama Sosa,*,¶ Rita De Gasperi,*,¶ Edward A. Fisher,��Paul M. Mathews,��,§§ Vahram Haroutunian*,¶ and Joseph D. Buxbaum*,�,�,**

*Department of Psychiatry, Mount Sinai School of Medicine, New York, USA

�Department of Neuroscience, Mount Sinai School of Medicine, New York, USA

�Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, USA

§Department of Biomathematical Sciences, Mount Sinai School of Medicine, New York, USA

¶Research and Development Service, James J. Peters Department of Veterans Affairs Medical Center, Bronx, New York, USA

**Laboratory of Molecular Neuropsychiatry, Mount Sinai School of Medicine, New York, USA

��Marc and Ruti Bell Vascular Biology Program, Leon H. Charney Division of Cardiology/Department of Medicine,

New York University School of Medicine, New York, USA

��Center for Dementia Research, Nathan Kline Institute, Orangeburg, New York, USA

§§Department of Psychiatry, New York University School of Medicine, New York, USA

Abstract

Epidemiological studies support an association between vas-

cular risk factors, including hypercholesterolemia, and Alz-

heimer’s disease (AD). Recently, there has been much

interest in the possibility that hypercholesterolemia might

directly promote b-amyloid (Ab) production. Indeed, in vitro

studies have shown that increasing cellular cholesterol levels

enhances Ab production. However, studies in AD transgenic

mouse models have not consistently found that elevated

plasma cholesterol leads to increased Ab production or

deposition in vivo. In this study, we determined whether ele-

vated peripheral cholesterol influences Ab production in mice

with a null mutation of the low-density lipoprotein receptor

(LDLR). We show that dramatically elevated plasma choles-

terol levels, whether induced by high cholesterol, high fat, or

high fat/high cholesterol diets, did not affect either levels of

brain Ab40, Ab42, or APP, or the Ab42/40 or APP-CTF/APP

ratios, nor substantially alter brain cholesterol levels. ApoE

protein levels in brain were, however, elevated, in LDLR)/)mice by post-transcriptional mechanisms. Collectively, these

studies argue that plasma cholesterol levels do not normally

regulate production of brain Ab.

Keywords: Alzheimer’s disease, Apolipoprotein E, dietary

cholesterol and fat, low-density lipoprotein receptor, null

mutation.

J. Neurochem. (2007) 102, 1220–1231.

An array of epidemiological studies support an associationbetween risk factors associated with vascular disease andAlzheimer’s disease (AD). For example, many classicalvascular risk factors, including hypertension, diabetes mell-itus, and hypercholesterolemia have been suggested toincrease the risk of developing AD (Breteler 2000; Casserlyand Topol 2004; Shobab et al. 2005). In addition, recentstudies have shown an association between coronary arterydisease and the severity of AD-neuropathology (Beeri et al.2006). Hypercholesterolemia might promote dementia by

Received January 24, 2007; revised manuscript received March 15,2007; accepted March 16, 2007.Address correspondence and reprint requests to Dr Joseph D. Bux-

baum, Department of Psychiatry, Box 1668, Mount Sinai School ofMedicine, One Gustave Levy Place, New York, NY 10029, USA.E-mail: joseph.buxbaum@mssm.edu1Authors J.Y.C and D.F.E contributed equally to this work.Abbreviations used: AD, Alzheimer’s disease; ApoE, apolipoprotein

E; APP, amyloid precursor protein; Ab, Abeta; FAD, familial Alzhei-mer’s disease; LDLR, low-density lipoprotein receptor.

Journal of Neurochemistry, 2007, 102, 1220–1231 doi:10.1111/j.1471-4159.2007.04614.x

1220 Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 1220–1231� 2007 The Authors

causing ischemic vascular disease, and cerebrovasculardisease is commonly found in association with AD (Snow-don et al. 1997). Indeed, one effect of the e4 allele ofapolipoprotein E (ApoE) is to increase plasma cholesterol,low-density lipoprotein levels, and cardiovascular disease(Eichner et al. 2002). However, recently there has been muchinterest in the possibility that hypercholesterolemia mightdirectly impact Ab production. In vitro studies have suppor-ted such a view by demonstrating a close correlation betweencholesterol content of cells and processing of the amyloidprecursor protein (APP) with Ab production being promotedby high cellular levels of cholesterol and reduced bytreatments that lower intracellular cholesterol [reviewed inWolozin (2004b)]. In vivo support for a cholesterol influenceon APP processing can be found in the Niemann-Pick TypeC mouse model where, because of a defect in intracellulartransport, cholesterol is increased in a variety of neuronalcells and associated with increased Ab production (Burnset al. 2003).

Studies in rabbits (Sparks et al. 1994, 2002) as well astransgenic mice have also suggested that plasma cholesterolcan influence Ab production. In particular, studies by Refoloet al. (2000, 2001) found that a high cholesterol dietincreased Ab levels and plaque burdens while causingdecreased a-secretase cleavage of APP in a transgenic mousemodel of amyloid deposition. Moreover, treating these micewith a cholesterol-lowering drug decreased serum choles-terol, brain Ab production and plaque burden (Refolo et al.2001). Several additional studies in related mouse modelshave supported the notion that elevated peripheral cholesterollevels can increase brain Ab production or deposition (Levin-Allerhand et al. 2002; Petanceska et al. 2002; Shie et al.2002; Li et al. 2003; Hutter-Paier et al. 2004; Cao et al.2006). However, other studies have found that increasedplasma cholesterol may be associated with unchanged (Fryeret al. 2005) or even reduced (Howland et al. 1998; Georgeet al. 2004) Ab levels while lowering plasma cholesterolmay either not affect (Fagan et al. 2004) or even elevate(Park et al. 2003) brain Ab levels. Thus, the in vivo impact ofaltering plasma cholesterol on brain Ab remains unclear.

One confounding variable in the studies in mice is thateach has utilized transgenic models in which familialAlzheimer’s disease (FAD)-associated mutations have beenover-expressed using exogenous transgenes. Thus, the FADmutation, the over-expression of the transgene, or a combi-nation of both might influence responses to elevated plasmacholesterol in these models. Whether altered plasma choles-terol levels affect endogenous brain Ab production in theabsence of an FAD mutation is unclear.

Hypercholesterolemic mice are widely used in athero-sclerosis research (Breslow 1996; Knowles and Maeda2000). Depending on the genetic modification and dietarymanipulation a wide range of serum cholesterol levels can beproduced. These models can thus be used to test the effects of

hypercholesterolemia on Ab production independent ofinfluences from an FAD mutation. In this study, weinvestigated how elevated peripheral cholesterol influencesAb production in mice with a null mutation of the low-density lipoprotein receptor (LDLR). We show that despitedramatic elevations in plasma cholesterol induced by thegenetic modification in combination with dietary manipula-tions, levels of brain Ab 40 or 42, the Ab42/40 ratio andbrain cholesterol levels are normal. ApoE protein levels inthe brain were, however, elevated in LDLR)/) micesupporting recent suggestions (Fryer et al. 2005) that LDLRmay have a more important role in lipoprotein metabolism inbrain than previously thought.

Experimental procedures

LDLR)/) mice

Male LDLR)/) mice were purchased from Jackson Labs (Ldlr KO

stock # 002207 strain name B6.12957-Ldlrtm1Her). These mice were

originally generated using a 129 embryonic stem cell line and have

been backcrossed for ten generations onto the C57Bl/6 background.

Age matched male C57Bl/6 wild type mice also obtained from

Jackson labs were used as controls. Animals were housed on 12 h

light/dark cycles. All protocols were approved by the Mount Sinai

School of Medicine Institutional Animal Care and Use Committee

and were in conformance with the National Institutes of Health

‘‘Guide for the Care and Use of Laboratory Animals.’’

Dietary manipulations

LDLR)/) and C57Bl/6 control mice were maintained on a standard

low cholesterol rodent chow diet containing 0.02% cholesterol and

6% fat (Lab Diet 5K52; Purina, St Louis, MO, USA) until 2 months

of age. At 2 months, mice were randomly assigned into the five

groups listed in Table 1. Those groups that received a high

cholesterol diet were fed D12102N base diet (Research Diets Inc.,

New Brunswick, NJ, USA) supplemented with 0.15% cholesterol

with a constant proportion of fat (4.3%) and other constituents for

16 weeks. Groups not assigned to the high cholesterol diet were

continued on the standard low cholesterol lab chow diet. After

13 weeks one group of LDLR)/) mice was switched from the low

Table 1 Experimental groups for manipulating plasma cholesterol

levels

Group Genotype Diet (Weeks)

Plasma

Cholesterol

Vascular

disease

I LDLR)/) Low (16) �240 mg/dL Minimal

II LDLR)/) High (16) >1000 mg/dL Severe

III LDLR)/) Low (13)/high (3) >1000 mg/dL Minimal

IV C57Bl/6 Low (16) �130 mg/dL None

V C57Bl/6 High (16) �130 mg/dL None

Shown are the experimental groups as well as the plasma cholesterol

levels that resulted after 16 weeks of the indicated dietary treatments.

Degree of vascular disease is estimated based on aortic root/arch

atherosclerosis (Teupser et al. 2003).

Plasma cholesterol does not affect brain Ab 1221

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 1220–1231

cholesterol to the high cholesterol diet. Mice were fed ad libitumwith free access to water. For studies involving the effect of dietary

fat, animals received a diet containing either 0.02% cholesterol/20%

palm oil (low cholesterol/high fat, D04050801, Research Diets Inc.)

or 0.5% cholesterol/20% palm oil (high cholesterol/high fat,

D04050802, Research Diets Inc.).

Measurement of serum and brain cholesterol

At the initiation of dietary manipulations and at the termination of

each study blood samples were taken from the retro-orbital sinus of

anesthetized mice and total plasma cholesterol levels were deter-

mined using the Infinity Cholesterol Reagent kit (Thermotrace,

Arlington, TX, USA) according to the manufacturer’s instructions.

For brain cholesterol levels frozen tissue from the anterior and

posterior cortices were homogenized in 6 mol/L urea, 100 mmol/L

Tris pH 7.4, 1 mmol/L dithiothreitol, 1 mmol/L EDTA, 0.5 mmol/L

phenylmethyl sulphonyl fluoride and 1% SDS and processed as

described in Levin-Allerhand et al. (2002). Brain cholesterol levels

were determined using the Inifinity Cholesterol Reagent (Thermo-

trace) and expressed as mg of cholesterol per mg of total protein

which was determined by the Markwell modified Lowry assay

(Markwell et al. 1978).

Tissue collection

Following blood collection mice were killed by cervical dislocation

while still under anesthesia. The brains were removed and divided

sagitally into halves. The tissue was regionally dissected into

anterior and posterior neocortex, hippocampus, thalamus, striatum,

cerebellum, and brainstem before being snap-frozen on dry ice and

stored at )80�C.

Quantification of Ab40 and Ab42 peptide levels

Ab levels were determined by sandwich ELISAwhich is described in

detail elsewhere (Schmidt et al. 2005a,b). Combined anterior and

posterior cortical samples obtained as described above were homo-

genized at 10% weight/volume in sucrose buffer (250 mmol/L

sucrose, 20 mmol/L Tris base, 1 mmol/L EDTA, 1 mmol/L EGTA)

followed by diethylamine extraction (0.4% diethylamine/100 mmol/

L NaCl). For capture, Ab carboxy-terminal monoclonal antibodies

were used which recognize exclusively either Abx-40 (JRF/cAb40/10) or Abx-42 (JRF/cAb42/26). Bound Ab was detected with

horseradish peroxidase-conjugated JRF/rAb1-15/2 which recognizesthe Ab amino-terminus. Standard curves were constructed using

synthetic Ab1-40 and Ab1-42 peptide standards (American Peptide

Co. Sunnyvale, CA, USA). Results are reported as the mean ± SEM

in fmol Ab per gram wet brain weight.

Determination of ApoE RNA levels by quantitative PCR

(qPCR)

Total RNA was isolated using the Trizol reagent (Invitrogen, Grand

Island, NY, USA). cDNAwas synthesized from total RNA (100 ng–

1 lg) in a 100 lL reaction using the ABI High Capacity cDNA

Archive Kit (Applied Biosystems, Foster City, CA, USA) according

to the protocols supplied by the manufacturer. The reaction was

performed at 25�C for 10 min followed by 37�C for 120 min. ApoE

RNA was amplified from aliquots of cDNA (4–6 ng/lL) in 20 lLreactions using the ABI ‘‘Assay on Demand’’ primer/probe mixtures

(Applied Biosystems) according to protocols supplied by the

manufacturer. 18S ribosomal RNA was used as a normalization

control. Amplifications were performed and analyzed using an ABI

Prism 7900HT thermal cycler and ABI SDS 2.2.1 Software.

Western blotting

Samples were homogenized in 1% SDS in neutral Tris using

protease inhibitors followed by boiling for 5 min. Protein samples

were separated on Bis–Tris gels (Invitrogen) and transferred to

nitrocellulose (for ApoE; GE Osmonics, Minnetonka, MN, USA) or

polyvinylidene difluoride (for APP; Millipore Corporation, Bill-

erica, MA, USA) membranes. Western blotting for ApoE was

performed with a mouse monoclonal antibody generated against

human ApoE (BD Biosciences, San Jose, CA, USA). Western

blotting for a-tubulin was performed with a rabbit anti-a-tubulinantibody (Abcam, Cambridge MA, USA). Western blotting for APP

and APP fragments was performed using antibody 369 which

recognizes the C-terminus of APP (Buxbaum et al. 1990). Bandswere visualized with ECL SuperSignal (Pierce Biotechnologies,

Rockford, IL, USA) and quantitation was performed on a Bio Rad

ChemiDoc XRS Scanner (Bio Rad Laboratories, Hercules, CA,

USA). ApoE and full-length APP (holoAPP) were normalized to the

a-tubulin signal, while levels of APP C-terminal fragments (CTFs)

were normalized to levels of holoAPP.

Statistical procedures

All data are presented as mean ± the standard error of the mean

(SEM). Statistical comparisons were made using unpaired t-tests orusing one- or two-way repeated or non-repeated measures ANOVA

with Tukey post hoc tests. Correlation coefficients were calculated

using the method of Pearson. Statistical tests were performed using

the program GraphPad Prism 4.0 (GraphPad Software Inc., San

Diego CA, USA).

Results

Experimental design for manipulation of plasma

cholesterol in LDLR)/) mice

Wild type mice fed a high cholesterol diet develop at mostmild elevations in plasma cholesterol levels. Indeed, evenLDLR)/) mice fed a standard low cholesterol rodent chowdiet (�4–6% fat, 0.04% or less cholesterol) have onlymoderately increased plasma cholesterol levels and a slightlyincreased susceptibility to atherosclerosis (Ishibashi et al.1993, 1994; Powell-Braxton et al. 1998). However, byincreasing dietary cholesterol to 0.15% or greater (Teupseret al. 2003) or by feeding LDLR)/) mice a high fat Westerndiet (�20% fat) (Palinski et al. 1995) plasma cholesterolincreases to over 1000 mg/dL and within 16 weeks ofdietary modification the animals develop extensive athero-sclerotic lesions that mimic the essential features of humanatherosclerosis (Breslow 1996; Knowles and Maeda 2000).

Prior studies on how dietary cholesterol affects Abproduction including those performed on the LDLR)/)background (Cao et al. 2006; Fryer et al. 2005) have utilizedstandard lab chow diets that elevate plasma cholesterol only

1222 G. A. Elder et al.

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modestly. We reasoned that if plasma cholesterol levelsregulate brain Ab production, then Ab levels should bemarkedly elevated in LDLR)/) mice fed a high cholesteroldiet. To test this hypothesis we designed a study consisting ofthe five groups shown in Table 1. In Groups I and II,LDLR)/) mice were fed either low (standard lab chow) orhigh cholesterol (0.15%) diets for 16 weeks. These twogroups were thus expected to have either moderately high(Group I) or extremely elevated (Group II) plasma choles-terol levels (Teupser et al. 2003). However, besides havinghigh plasma cholesterol after 16 weeks exposure to a highcholesterol diet, LDLR)/) mice develop significant vasculardisease that could affect Ab production independent ofcholesterol levels. Therefore, to control for concurrentvascular disease a third group of LDLR)/) mice were feda low cholesterol diet for 13 weeks followed by the highcholesterol diet for the last 3 weeks (Group III). This groupprovides an approximate control for the effect of vasculardisease since plasma cholesterol levels reach a new steadystate within a few weeks after the switch to a high cholesteroldiet while several months of exposure to a high fat/highcholesterol diet is required to produce significant vascularpathology (Ishibashi et al. 1994; Palinski et al. 1995;Shimada et al. 1996; Teupser et al. 2003). At 16 weeksthese animals were therefore age matched with group II, but

would have hypercholesterolemia in a setting of only limitedvascular disease. To provide controls for the effects of thehigh cholesterol diet in wild type mice, C57Bl/6 mice werefed low (Group IV) or high (Group V) cholesterol diets for16 weeks.

Elevated peripheral cholesterol does not alter levels

of endogenous murine Ab 40 or 42 in brain

Plasma cholesterol levels at the beginning and the end ofthe 16 week study are shown in Fig. 1a. Before dietarymanipulations plasma cholesterol was �130 mg/dL inC57Bl/6 mice and was moderately elevated (�240 mg/dL)in LDLR)/) mice (one-way ANOVA, F4,36 = 16.73, p <0.0001; Tukey post hoc test p < 0.0001). Despite 16 weekson the high cholesterol diet plasma cholesterol in C57Bl/6mice was unchanged from baseline. By contrast, plasmacholesterol increased to over 1000 mg/dL in LDLR)/) micefed a high cholesterol diet for 16 weeks (LDLR-H) as well asthe switch group (LDLR-S) that received the high cholesteroldiet for the last 3 weeks (Fig. 1). A two-way ANOVA revealedeffects of both diet (F1,36 = 101.0, p < 0.0001) and genotype(F4,36 = 49.20, p < 0.0001) and a significant interactionreflecting that the effects of diet and genotype occurred onlyin combination (F4,36 = 33.44, p < 0.0001). Post-plasmacholesterol levels in the C57Bl/6 and LDLR groups fed the

(a) (b)

(c) (d)

Fig. 1 Levels of plasma cholesterol and brain Ab in LDLR)/) and

C57Bl/6 wild type mice fed a high cholesterol diet. C57Bl/6 mice were

fed either low (C57Bl-L) or high (C57Bl-H) cholesterol diets while

LDLR)/) mice were fed low (LDLR-L), high (LDLR-H) or the switch

(LDLR-S) diet described in the text and Table 1. Plasma cholesterol

levels (a) prior to (Pre) and at the end of the 16-week period (Post) are

shown (C57Bl6-L, n = 8; C57Bl6-H, n = 9; LDLR-L, n = 8; LDLR-S,

n = 8; LDLR-H, n = 8). Results are discussed in the text. Levels of

brain Ab 40 (b), Ab 42 (c) and the ratio of Ab42/40 (d) were determined

by ELISA on pooled samples of anterior and posterior cortex from the

animals studied in panel (a). There were no significant effects of

genotype or diet.

Plasma cholesterol does not affect brain Ab 1223

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 1220–1231

low and high cholesterol diets did not differ betweenthe C57Bl/6 groups but were elevated in LDLR-H versuseither LDLR-L or the C57Bl/6 groups with an effect ofboth genotype (F1,29 = 62.76, p < 0.0001) and diet (F1,29 =30.06, p < 0.0001) as well as a significant interaction effect(F1,29 = 25.47, p < 0.0001). Because the LDLR-S grouplacked a comparable C57Bl/6 switch group, it was comparedwith the other LDLR groups using a one-way ANOVA

(F2,22 = 20.34, p < 0.001), which found that plasma choles-terol was higher in the LDLR-S versus the LDLR-L(p < 0.001) while the LDLR-S and LDLR-H were notsignificantly different.

We determined whether elevated plasma cholesterolaffected levels of endogenous murine brain Ab40 or 42using ELISA assays. Despite the dramatically elevatedplasma cholesterol in the LDLR-H and LDLR-S groups,there was no significant difference in brain Ab 40 (Fig. 1b)or 42 (Fig. 1c) between any of the groups. In AD there maybe a shunting of Ab production towards the longer moreamyloidogenic forms including Ab42 (Hardy 1997). How-ever, we found no difference in the ratio of Ab42/40 betweenany of the groups (Fig. 1d).

Since some variation in plasma cholesterol levels occurredwithin all the groups we examined the relationship of plasmacholesterol to brain Ab levels. As shown in Fig. 2 there wasno significant correlation between plasma cholesterol andbrain Ab 40 (Fig. 2a) or Ab 42 (Fig. 2b) levels when animalsin the five groups were combined. To determine whether any

correlation might exist in animals with the highest plasmacholesterols we compared plasma cholesterols and brain Ablevels in the LDLR-S (Fig. 2c) and LDLR-H (Fig. 2d)groups. In both, any trend was towards a slightly negativecorrelation between plasma cholesterol and brain Ab 40 or42 levels, although none of the trends reached statisticalsignificance. Therefore, despite dramatic elevations inplasma cholesterol in LDLR)/) mice fed a high cholesteroldiet, elevated peripheral cholesterol does not significantlyaffect levels of soluble Ab 40 or 42 in brain.

Elevated plasma cholesterol does not alter total brain

cholesterol levels in LDLR)/) mice

In vitro cellular levels of cholesterol appear to modulate Abproduction (Wolozin 2004b). For plasma cholesterol tomodulate brain Ab production in an analogous mannerin vivo, elevated plasma cholesterol must lead to increasedbrain cholesterol. However, when we measured braincholesterol in the five groups, we found it to be similar inall groups (Fig. 3a). In addition, we found no correlationbetween plasma and brain cholesterol in individual animalsfrom the five groups combined (Fig. 3b) or within any of theindividual groups themselves (data not shown). Thus, even inthe face of dramatically elevated plasma cholesterol, braincholesterol levels are unchanged in LDLR)/) mice fed ahigh cholesterol diet, offering one explanation for whyelevated peripheral cholesterol does not appear to beaffecting brain Ab levels in these mice.

(a) (b)

(c) (d)

Fig. 2 Relationship of plasma cholesterol to brain Ab levels in

LDLR)/) and C57Bl/6 wild type mice fed low or high cholesterol diets.

Shown are plasma cholesterol levels plotted against brain Ab 40 (a) or

42 (b) from all animals in the five groups studied in Fig. 1 combined.

Plasma cholesterol vs. brain Ab 40 or 42 is also shown for mice fed the

switch (LDLR-S, c) and high cholesterol (LDLR-H, d) diets. Pearson

correlation coefficient (r) and p-values are indicated. None of the

relationships reached statistical significance.

1224 G. A. Elder et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 1220–1231� 2007 The Authors

No change in brain Ab 40 or 42 levels in LDLR)/) mice

fed high fat or high fat/high cholesterol diets

In humans hypercholesterolemia generally occurs in a settingof high intake of dietary fat. Interestingly, high dietary fatconsumption has also been suggested to increase the risk ofAD (Kalmijn et al. 1997). LDLR)/) mice also develophypercholesterolemia when fed a high fat diet (Ishibashiet al. 1994; Palinski et al. 1995). Therefore, to determinewhether modulating plasma cholesterol by increasing dietaryfat or fat in combination with cholesterol might affectbrain Ab levels we designed an additional study in whichLDLR)/) mice were fed either high fat (20% provided aspalm oil) or high fat/high cholesterol (0.5%) diets for16 weeks. As controls an additional group of LDLR)/) micewere fed standard lab chow and C57Bl/6 mice were fedeither standard lab chow or the high fat/high cholesterol diet.

Results are shown in Fig. 4. Pre- and post-plasmacholesterol levels were unchanged from baseline in theC57Bl6-LL, and C57Bl6-HH groups. Both high fat and highfat/high cholesterol diets led to substantial elevations inplasma cholesterol in LDLR)/) mice (Fig. 4a) reaching over900 mg/dL in LDLR)/) mice fed the exclusively high fatdiet and over 1400 mg/dL in mice fed the high fat/highcholesterol diet with a repeated measures two-way ANOVA

revealing effects of both diet (F1,20 = 77.26, p < 0.0001) andgroup (F4,20 = 21.32, p < 0.0001) and a significant interac-tion between diet and group (F4,20 = 11.56, p < 0.0001).Post-plasma cholesterol levels did not differ between theC57Bl/6 groups, but were higher in LDLR-HH versus eitherLDLR-LL or the C57Bl6-HH groups with an effect ofboth genotype (F1,19 = 39.04, p < 0.0001) and diet(F1,19 = 11.39, p = 0.0032) and a significant interactioneffect (F1,19 = 8.607, p = 0.0085). Because the LDLR-LHgroup lacked a comparable C57Bl/6 group, the LDLR-LHgroup was compared with the other LDLR groups using aone-way ANOVA (F2,12 = 5.451, p = 0.02) with post hoc tests

showing that plasma cholesterol was unchanged in theLDLR-LH, compared with the LDLR-LL and LDLR-HHgroups, but higher in LDLR-HH than LDLR-LL (p < 0.05).

However, despite these increases in plasma cholesterol atthe end of the 16-week treatment, brain Ab 40 (Fig. 4b) and42 (Fig. 4c) levels as well as the ratio of Ab 42/40 (Fig. 4d)were unchanged in mice fed either high fat or high fat/highcholesterol diets. In addition, we found no correlationbetween plasma cholesterol levels and brain Ab 40 (Fig. 4e)or 42 (Fig. 4f) and no increase in brain cholesterol levels(Fig. 5a). Only comparing plasma to brain cholesterol levelsin the combined groups revealed a significant correlation(r = 0.5181, p = 0.004) between plasma and brain choles-terol levels (Fig. 5b). However, it is notable that despite a20-fold range of plasma cholesterol levels, brain cholesterolincreased by less than 0.5-fold over this span. Thus, whetherinduced by manipulating dietary cholesterol, fat, or both,elevated plasma cholesterol levels do not modulate Ab levelsand influence brain cholesterol levels at most minimally.

No change in levels of full length APP or in the ratio

of APP CTFs to full length APP in LDLR)/) mice

fed high cholesterol, high fat or high fat/high

cholesterol diets

As an additional test of whether altered dietary cholesterol orfat might alter APP metabolism we determined levels of fulllength APP as well as the ratio of APP CTFs to full lengthAPP. We saw no effect of high cholesterol, high fat or highcholesterol/high fat diets on levels of full length APP (datanot shown) or on the ratio of APP CTFs to full-length APP(Fig. 5c; no significant effects under either dietary conditionby two way ANOVA when comparing all groups excludingLDLR-S, or by one way ANOVA when comparing LDLRgroups only). Thus, the substrates for Ab production are notaffected by the manipulation of dietary fat or cholesterol,alone or in combination.

(a) (b)

Fig. 3 Levels of brain cholesterol and relationship of plasma cho-

lesterol to brain cholesterol in LDLR)/) and C57Bl/6 wild type mice

fed low or high cholesterol diets. Brain cholesterol levels (a) were

determined on pooled samples of anterior and posterior cortex (C57Bl-

L, n = 4; C57Bl-H, n = 5; LDLR-L, n = 4; LDLR-S, n = 4; LDLR-H,

n = 3). The five groups were compared using two-way and one-way

ANOVAs as in Fig. 1. No significant effects of genotype or diet were

found. In (b) plasma cholesterol is plotted vs. brain cholesterol in the

mice from the five groups in (a) combined. Pearson correlation coef-

ficient (r) and p-value are indicated.

Plasma cholesterol does not affect brain Ab 1225

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 1220–1231

ApoE protein levels are increased in LDLR)/) mice and

are regulated by post-transcriptional mechanisms

Low-density lipoprotein receptor is expressed in brain(Hofmann et al. 1987). Yet, LDLR deficient mice show noobvious defects in brain and other lipoprotein receptors havegenerally been thought to be more important than LDLR inbrain development and function (Herz and Bock 2002).However, recently ApoE levels have been found elevated inLDLR)/) mice fed a standard lab chow diet and it has beenfurther suggested that LDLR is an important receptor forApoE in brain (Fryer et al. 2005).

We also compared by western blotting levels of ApoEin the cortex of LDLR)/) and C57Bl/6 mice fed a

standard lab chow diet. ApoE levels (Figs 6a and b) wereincreased nearly 2.5-fold in LDLR)/) mice compared withC57Bl/6 wild type mice (p = 0.04, unpaired t-test). PlasmaApoE is elevated in LDLR)/)mice and ApoE levels risein the presence of hypercholesterolemia (Ishibashi et al.1994). To determine whether alterations in plasma choles-terol might affect brain ApoE levels in LDLR)/) micewe also compared ApoE levels in LDLR)/) mice feda standard lab chow diet to the LDLR)/) groups fed highcholesterol or fat modified diets. ApoE levels were,however, similar in both the high cholesterol fedgroups (Fig. 6d), as well as in LDLR)/) mice fed highfat or high fat/high cholesterol diets (data not shown),

(a) (b)

(c) (d)

(e) (f)

Fig. 4 Plasma cholesterol and brain Ab levels in LDLR)/) and

C57Bl/6 wild type mice fed high fat or high fat/high cholesterol diets.

C57Bl/6 mice were fed either low cholesterol/low fat (C57Bl-LL) or low

cholesterol/high fat (C57Bl-LH) diets while LDLR)/) mice were fed low

cholesterol/low fat (LDLR-LL), low cholesterol/high fat (LDLR-LH) or

high cholesterol/high fat (LDLR-HH) diets. In (a) baseline plasma

cholesterols at the initiation of dietary treatment (Pre) and at the end of

the study (Post) are shown for the animals that were used for Ab

analysis in panels b–d are shown (n = 5/group). Results are discussed

in the text. Levels of brain Ab 40 (b), Ab 42 (c) and the ratio of Ab 42/40

(d) were determined by ELISA on pooled samples of anterior and

posterior cortex from the animals shown in panel (a). No significant

effects of genotype or diet were found. In (e) and (f) plasma cholesterol

levels are plotted against brain Ab 40 (e) or 42 (f) from animals in the

five groups studied in panels a-d combined. Pearson correlation

coefficient (r) and p-values are indicated. None of the relationships

reached statistical significance.

1226 G. A. Elder et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 1220–1231� 2007 The Authors

suggesting that the effect on brain ApoE is caused bylack of the LDLR rather than altered plasma cholesterollevels.

To determine if ApoERNA levels were altered in LDLR)/)mice we performed qPCR. As shown in Fig. 6c, ApoE RNAlevels were actually decreased more than two-fold inLDLR)/) mice compared with C57Bl/6 mice (p = 0.03)while ApoE RNA levels in the LDLR)/) groups fed a highcholesterol diet were similar to LDLR)/) mice fed standardlab chow (Fig. 6e). Thus, the effect of LDLR on ApoE proteinlevels appears to be mediated at a post-transcriptional level,with elevated ApoE protein levels possibly exerting a negativefeedback effect on ApoE transcription. Collectively thesestudies, like a previous study (Fryer et al. 2005), argue that theLDLR receptor may play an important role in lipoproteinmetabolism in adult brain although its disruption alone doesnot itself influence brain Ab levels.

Discussion

Raising levels of cellular cholesterol in vitro increases Abproduction and reduces a-secretase cleavage of APP (Wolo-zin 2004b). Conversely, treatments that lower intracellularcholesterol in vitro increase a-secretase cleavage and reduceAb production. Whether plasma cholesterol levels signifi-cantly impact Ab production in brain is less clear. Inparticular, studies in transgenic mouse models that exhibitAD-like amyloid deposition have produced conflictingresults (Howland et al. 1998; Levin-Allerhand et al. 2002;Petanceska et al. 2002; Shie et al. 2002; Li et al. 2003; Parket al. 2003; Fagan et al. 2004; George et al. 2004; Hutter-Paier et al. 2004; Cao et al. 2006; Fryer et al. 2005).Likewise, in humans, no firm conclusions can be reached yetconcerning serum cholesterol as an AD risk factor withstudies suggesting an increased, unchanged or evendecreased risk associated with higher serum cholesterollevels (Panza et al. 2006; Sjogren et al. 2006). Suggestionsthat statins may provide protection against developing ADare also now more controversial (Wolozin 2004a).

Nearly all prior studies in mouse amyloid models haverelied on transgenic lines generated by pronuclear injectionthat express human APP transgenes containing FAD muta-tions, sometimes in combination with presenilin-1 FADmutations. Because of the over-expression nature of suchmodels, results are potentially affected by the FAD mutationand its over-expression, in addition to any dietary manipu-lations. Furthermore, deposition of human Ab occurs in thesemice. Ab levels in the absence of deposition reflect a balancebetween formation and degradation or clearance. However,where deposition as plaque amyloid is a major component,Ab levels also reflect the deposition rate of soluble Ab andthe rate of degradation or clearance of deposited Ab. Thus inthese models the process is inherently more complex than Abmetabolism in normal murine brain, since effects of choles-terol or statins may occur at any of these four steps, whichcould also include regulation of expression of Ab-bindingproteins.

(a)

(b)

(c)

Fig. 5 Levels of brain cholesterol and relationship of plasma cho-

lesterol to brain cholesterol in LDLR)/) and C57Bl/6 wild type mice

fed high fat or high fat/high cholesterol diets. Brain cholesterol levels

(a) were determined on pooled samples of anterior and posterior

cortex (C57Bl-LL, n = 5; C57Bl-HH, n = 5; LDLR-LL, n = 9, LDLR-LH,

n = 5; LDLR-HH, n = 5). The five groups were compared using two-

way and one-way ANOVAs as in Fig. 4. No significant effects of geno-

type or diet were found. In (b) plasma cholesterol is plotted versus

brain cholesterol in the mice from the five groups in (a) combined.

Pearson correlation coefficient (r) and p-value are indicated. In (c)

levels of full length APP and APP CTFs were determined by Western

blotting on combined anterior and posterior cortical samples (C57Bl6-

LL, n = 9; C57Bl6-HL, n = 5; C57Bl-HH, n = 3; LDLR-LL, n = 8;

LDLR-HL, n = 8; LDLR-S, n = 4; LDLR-LH, n = 4; LDLR-HH, n = 4).

The ratio of APP-CTFs to full length APP is shown. There were no

significant differences between the groups.

Plasma cholesterol does not affect brain Ab 1227

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 1220–1231

Whether hypercholesterolemia impacts Ab production innormal mouse brain has been difficult to assess as plasmacholesterols in mice typically change little in response toincreased dietary cholesterol or fat. Indeed, none of thedietary manipulations utilized here whether involving highcholesterol, high fat or combinations of the two, elevatedplasma cholesterol in C57Bl/6 mice. To overcome this limi-tation, in addition to wild type mice we studied LDLR)/)mice in which plasma cholesterol can be dramaticallyincreased by manipulating dietary cholesterol or fat (Palinskiet al. 1995; Teupser et al. 2003) and where effects on brainAb could be studied without interference from an FADmutation. Indeed administration of diets containing 0.15–0.5% cholesterol, elevated plasma cholesterol in LDLR)/)mice to over 1000 mg/dL in mice fed a high cholesterol dietand over 1400 mg/dL in mice fed a high fat/high cholesteroldiet, levels far above those seen when FAD mutant lines withan intact LDLR receptor were administered 1.25–5% dietarycholesterol in combination with elevated fat (Refolo et al.2000; Shie et al. 2002). However, despite these elevations inplasma cholesterol there was no change in levels of solubleAb 40 or 42 in brain. In addition, no change occurred in theratio of Ab 42/40 and there was no correlation betweenplasma cholesterol levels and brain Ab. Moreover, we saw

no effect of altering plasma cholesterol levels with highcholesterol or high cholesterol/high fat diets on the levels offull length APP or the ratio of APP C-terminal fragments tofull-length APP.

In vitro studies presume that cholesterol is exerting a directeffect on Ab production by modulating cellular cholesterol.For plasma cholesterol to modulate brain Ab productionin vivo in an analogous manner, plasma cholesterol mustinfluence brain cholesterol levels. The brain, however, differsfrom peripheral tissues in that it normally receives little if anycholesterol from circulating plasma lipoproteins (Edmondet al. 1991; Jurevics and Morell 1995; Turley et al. 1996,1997; Quan et al. 2003). Instead nearly all brain cholesterolis synthesized within the CNS and indeed the brain seems tobe a net exporter of cholesterol to the systemic circulation[reviewed in Dietschy and Turley (2001)].

Indeed in LDLR)/) mice peripheral cholesterol seems tohave little to no influence on brain cholesterol levels as wefound no changes in total brain cholesterol in LDLR)/)mice fed high cholesterol, high fat or high fat/high choles-terol diets. In addition, in mice fed the exclusively highcholesterol diet there was no correlation between levels ofplasma and brain cholesterol. One limitation of these studiesis that 70% of brain cholesterol is contained in myelin

(a) (b)

(d) (e)

(c)

Fig. 6 Expression of ApoE in brain of LDLR)/) mice. In (a) western

blotting for ApoE in LDLR)/) and C57Bl/6 mice fed standard lab chow

diets is shown. Lanes were loaded with 42 lg of protein. Lower panel

shows blotting for a-tubulin as a loading control. Quantitation of ApoE

protein levels (n = 9 animals/group) is shown in (b). In (c) ApoE RNA

levels were determined by qPCR (C57Bl/6, n = 4; LDLR)/), n = 3). In

(d) and (e) ApoE protein (LDLR-S, n = 4; LDLR-H, n = 3) and RNA

(LDLR-S, n = 5; LDLR-H, n = 4) levels in the LDLR-S and LDLR-H

groups were compared to LDLR-L. Asterisks indicate values that are

statistically significant (p < 0.05, unpaired t-test). No significant dif-

ferences were found between the LDLR groups (d and e) using a one-

way ANOVA.

1228 G. A. Elder et al.

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membranes (Dietschy and Turley 2001) which turnoverrelatively slowly making an effect on the non-myelin poolmore difficult to detect in total brain homogenates and in factin the combined pool of animals that included mice fed ahigh fat/high cholesterol diet we did find a correlationbetween plasma and brain cholesterol levels. We suspect thatthese results reflect the higher plasma cholesterol levels thatcan be attained by feeding LDLR)/) mice a diet high in bothfat and cholesterol, compared to high cholesterol alone, withat the highest levels plasma cholesterol exerting an effect onbrain cholesterol. However, it is of note that despite a 20-foldrange of plasma cholesterol levels, brain cholesterolincreased by less than 0.5-fold in those animals with thehighest peripheral cholesterol values. Other studies have alsofound brain cholesterol unchanged in LDLR)/) mice fedstandard lab chow (Fryer et al. 2005) or high cholesterol/high fat diets (Quan et al. 2003). It is noteworthy that evenwhen plasma cholesterol levels were raised to such an extentas to increase brain cholesterol levels, brain Ab levels werenot affected significantly.

Plasma cholesterol might not influence brain cholesterol inLDLR)/) mice if LDLR were required for cholesteroltransport across the blood-brain barrier and indeed one studyhas suggested that LDLR may transport low-density lipo-proteins into brain endothelial cells (Dehouck et al. 1997).This explanation seems unlikely, however, since braincholesterol levels are also unchanged in ApoE)/), SrbI1)/),ApoAI)/) and ABCA1)/) mice (Quan et al. 2003) all ofwhich have either substantially elevated (ApoE)/), SrbI)/))or reduced (ApoAI)/), ABCA1)/)) plasma cholesterol butan intact LDLR. Thus even in the presence of a functionalLDLR, plasma cholesterol levels do not appear to influencebrain cholesterol.

Given the minimal impact that high plasma cholesterollevels have on brain cholesterol in LDLR)/) mice, it iscurious that in several of the transgenic mouse amyloidmodels small but detectible increases in brain cholesterolhave been seen, despite modest elevations in plasmacholesterol (Howland et al. 1998; Refolo et al. 2000; Georgeet al. 2004). Such effects do not appear to be limited tohypercholesterolemic states as ApoAI)/) mice have dra-matically lowered plasma cholesterol but normal braincholesterol (Quan et al. 2003). However, when PDAPP micewere bred onto the ApoAI)/) background brain cholesterolfell in the presence of the PDAPP transgene (Fagan et al.2004). Thus, one effect of the FAD transgenes may be toloosen the normal tight separation between central andperipheral cholesterol compartments, permitting CNS cho-lesterol to become more like the periphery, and indeedTg2576 mice have abnormalities in the blood–brain barrierthat precede the deposition of amyloid plaques (Ujiie et al.2003). These abnormalities could allow plasma choles-terol (or other systemic factors that may modulate Abformation, degradation, deposition, or clearance) to influence

brain Ab levels. If these findings are applicable to humans,hypercholesterolemia may not be a true disease risk factor.However, once the earliest stages of AD pathology areestablished higher plasma cholesterol might acceleratedisease progression.

Although brain Ab and cholesterol levels were not alteredin LDLR)/) mice, ApoE levels were elevated in brain. Lowlevels of LDLR RNA are found in brain (Hofmann et al.1987) with expression being widely distributed (Swansonet al. 1988) and likely found in neurons, astrocytes andendothelial cells (Hofmann et al. 1987; Pitas et al. 1987;Swanson et al. 1988; Lucarelli et al. 2002). However, LDLRdeficient mice show no obvious defects in brain. Indeed, weinitially chose to study LDLR)/) mice as LDLR has beengenerally thought to be relatively unimportant in braindevelopment and function relative to other LDLR familymembers such as LRP, ApoER2, or VLDLR (Herz and Bock2002). However, recently it has been reported that LDLR)/)mice have defects in spatial memory (Mulder et al. 2004)and that LDLR is up-regulated in brain following ischemicinjury (Kamada et al. 2005) suggesting that LDLR may playa more important role in brain function than previouslyappreciated.

Fryer et al. (2005) recently reported that brain ApoE levelsare elevated in LDLR)/) mice fed a standard lab chow dietand suggested that LDLR may be a major receptor for ApoEin brain. We also found that brain ApoE levels wereincreased nearly 2.5-fold in LDLR)/) mice fed a lab chowdiet. Because ApoE binds to LDLR (Wilson et al. 1991)these elevations are most readily explained by there beingfewer ApoE receptors in the brains of LDLR)/) mice.Plasma ApoE levels are increased in LDLR)/) mice andincrease further in response to hypercholesterolemia (Ishiba-shi et al. 1994). However, the failure of brain ApoE levels tobe affected by modulating dietary cholesterol or fat arguesthat the LDLR effect on brain ApoE is most likely due to thelack of LDLR in brain rather than altered plasma lipoproteinlevels. Interestingly, ApoE RNA levels were decreased morethan two-fold in LDLR)/) mice suggesting that the absenceof LDLR modulates ApoE protein levels at a post-transcrip-tional level and that elevated ApoE protein may negativelyregulate ApoE transcription in brain.

ApoE levels and isoforms clearly modulate Ab depositioninto plaque amyloid in PDAPP mice (Bales et al. 1997;Holtzman et al. 2000a,b). In particular, the absence of ApoElargely prevents deposition, despite brain Ab levels asmeasured by ELISA being unchanged (Bales et al. 1997). Itis interesting in this context to compare our results to tworecent studies in which either Tg2576 or PDAPP mice werebred onto the LDLR)/) background (Cao et al. 2006; Fryeret al. 2005). In both studies mice were maintained on astandard lab chow diet and thus plasma cholesterols wereonly modestly elevated in comparison to the plasmacholesterols in the present study. With PDAPP mice Ab 40

Plasma cholesterol does not affect brain Ab 1229

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 1220–1231

and 42 levels were unchanged in 3-month old animals and noclear effect on amyloid deposition was observed in 10-monthold animals (Fryer et al. 2005). By contrast Cao et al. (2006)found two-fold increases in plaque deposition in Tg2576mice on an LDLR)/) background at 13 months of age. Ablevels were not measured biochemically in this study. Thus,the LDLR effect on Ab production, degradation, clearance,and deposition appears model-dependent although interest-ingly both studies found that brain ApoE levels wereelevated.

Collectively, the present studies argue that plasma choles-terol levels do not normally regulate levels of brain Ab, andseen in the context of other studies in AD related transgenicmodels, suggests that any effect of hypercholesterolemia ismore likely as a disease accelerator rather than a diseaseinitiator. These results also provide further support to thenotion that LDLR plays a significant role in lipoproteinmetabolism in adult brain.

Acknowledgements

This work was supported by National Institutes of Health grants

AG002219, AG010491 AG017617 and HL084312. We acknow-

ledge the support of the Mount Sinai Quantitative PCR Shared

Resource Facility.

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