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Human Molecular Genetics Advance Access originally published online on June 30, 2004
Human Molecular Genetics 2004 13(17):1959-1968; doi:10.1093/hmg/ddh199
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Human Molecular Genetics, Vol. 13, No. 17 © Oxford University Press 2004; all rights reserved

Independent effects of APOE on cholesterol metabolism and brain Aß levels in an Alzheimer disease mouse model

Karen M. Mann1,2, Fayanne E. Thorngate3, Yuko Katoh-Fukui4, Hiroki Hamanaka5, David L. Williams3, Shinobu Fujita6 and Bruce T. Lamb1,2,*

1Department of Genetics, Case Western Reserve University, Cleveland, OH 44106, USA, 2Center for Human Genetics, University Memory and Aging Center and Ireland Cancer Center, University Hospitals of Cleveland, Cleveland, OH 44106, USA, 3Department of Pharmacological Sciences, University Medical Center, Stony Brook University, Stony Brook, NY 11794, USA, 4Department of Developmental Biology, National Institute for Basic Biology, Myodaiji-cho, Okazaki 444-8585, Japan, 5Center for Neural Science, New York University, New York, NY 10003-6621, USA and 6Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo, Japan

Received March 30, 2004; Accepted June 15, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The APOE {varepsilon}4 allele is the most significant genetic risk factor associated with Alzheimer's disease to date. Epidemiological studies have demonstrated that inheritance of one or more {varepsilon}4 alleles affects both the age of onset and the severity of pathology development. Dosage of APOE {varepsilon}2 and {varepsilon}3 alleles, however, appear to be protective against the effects of {varepsilon}4. Although much of the biology of APOE in peripheral cholesterol metabolism is understood, its role in brain cholesterol metabolism and its impact on AD development is less defined. Several APOE transgenic models have been generated to study the effects of APOE alleles on APP processing and Aß pathology. However, these models have potential limitations that confound our understanding of the effects of apolipoprotein E (APOE) levels and cholesterol metabolism on disease development. To circumvent these limitations, we have taken a genomic-based approach to better understand the relationship between APOE alleles, cholesterol and Aß metabolism. We have characterized APOE knock-in mice, which express each human allele under the endogenous regulatory elements, on a defined C57BL6/J background. These mice have significantly different serum cholesterol levels and steady-state brain APOE levels, and yet have equivalent brain cholesterol levels. However, the presence of human APOE significantly increases brain Aß levels in a genomic-based model of AD, irrespective of genotype. These data indicate an independent role for APOE in cholesterol metabolism in the periphery relative to the CNS, and that the altered levels of cholesterol and APOE in these mice are insufficient to influence Aß metabolism in a mouse model of Alzheimer's disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Apolipoprotein E (APOE) functions as a lipid chaperone and facilitates cellular uptake of cholesterol and lipoproteins through receptor-mediated endocytosis. The APOE gene, located on human chromosome 19q13.2, is a polymorphic locus represented by three common alleles, {varepsilon}2, {varepsilon}3 and {varepsilon}4. These three alleles encode three APOE isoforms of 34 kDa that differ at amino acid positions 112 and 158. These two amino acid substitutions confer slightly different functions for the respective proteins. The E2 isoform, encoded by the {varepsilon}2 allele, contains a Cys at position 158, in comparison with the Arg found in the E3 and E4 isoforms. The substitution of Cys for Arg alters the positive ion potential within the receptor-binding domain, and thus decreases the affinity of the E2 protein for the LDL receptor to 1% of the normal binding capacity (1,2). Consequently, the level of circulating E2 protein is significantly higher than that of E3. In contrast, the level of E4 protein is significantly lower than that of E3, presumably a consequence of the isoform-specific differences in lipid binding and uptake. The ability of APOE to bind its receptors, which include LDLr, LRP, VLDLr and megalin, members of the LDL receptor family, dictates the efficiency with which these molecules are delivered to the appropriate cells and turned over.

APOE {varepsilon}4 was first shown through association studies to be a genetic risk factor for cardiovascular disease, presumably through its role in conferring high circulating cholesterol levels, mainly in the form of LDL, in individuals with one or more copies of the allele (3,4). Subsequently, APOE {varepsilon}4 was found to be associated with increased risk for late-onset Alzheimer's disease (LOAD) (5,6). Furthermore, the presence of one or more {varepsilon}4 alleles decreases the age of disease onset by as much as 10 years (7).

One hypothesis as to the link between APOE genotype, cardiovascular disease and AD involves the contribution of cholesterol homeostasis to both diseases and the role of APOE in this process. Several studies have implicated a role for cholesterol in the production of Aß, the primary constituent of senile plaques in the AD brain (8–12). High serum cholesterol levels in mice transgenic for APP, the precursor protein for the Aß peptide, correlate with increased brain Aß levels and a more severe plaque load (13,14). Retrospective epidemiological studies have demonstrated a decreased AD risk in human populations on statin therapy to lower cholesterol levels (reviewed in 15), although this effect may be indirect, as several studies have shown that statins do not alter the Aß levels in serum (16,17). Several studies have reported an association of elevated serum cholesterol levels with AD risk and levels of Aß (18,19), independent of APOE genotype. Other studies, however, have failed to demonstrate a correlation between serum cholesterol levels and AD development (20). APOE {varepsilon}4 may affect AD risk by conferring high cholesterol levels and thereby increasing Aß production. AD patients with an {varepsilon}4 allele have increased levels of Aß1–40 in their brains and CSF, and have more extensive plaque pathology (21,22). Interestingly, the {varepsilon}2 allele, which confers low cholesterol levels, is also considered to be protective against AD development (7).

Several APOE transgenic mouse models have been generated in efforts to gain insight into the role of APOE in AD (23–26). These models utilize various promoters to drive the expression of human APOE cDNAs in neurons and glia. This approach has several limitations concerning the regulation of APOE in both the brain and the periphery. First, most APOE transgenic studies have attempted to normalize APOE levels in the APOE {varepsilon}3 or {varepsilon}4 transgenic lines with respect to each other (23) or to endogenous levels (24). However, several human studies suggest that levels of APOE vary with genotype and may impact AD pathogenesis. Second, all APOE transgenic lines have been bred onto the Apoe-null background, which confers high circulating cholesterol levels. Given the role of Apoe in peripheral cholesterol metabolism and the growing evidence for cholesterol levels influencing brain Aß metabolism, it is unclear how the absence of Apoe affects AD pathology in these animals. Importantly, there has been no consensus about the impact of APOE genotype on AD pathogenesis from these studies, perhaps owing to the complications of different promoters and altered levels of APOE in the brain, combined with the complications of a mixed genetic background in these models.

APOE knock-in (KI) mice provide unique model for understanding how each APOE allele behaves in the development of Alzheimer's disease. We have obtained these mice, previously published by Hamanaka et al. (27), in order to further characterize the behavior of human APOE alleles in a defined genetic background. We present APOE protein abundance, cholesterol and lipoprotein data from young animals expressing APOE {varepsilon}2, {varepsilon}3 and {varepsilon}4 on a defined C57BL/6J background. We demonstrate significant differences in protein abundance, which mirrors the human data, as well as significant differences in serum cholesterol levels in the absence of an effect on APP processing. The presence of APOE significantly increases levels of both Aß and cholesterol in brain, irrespective of allele. This genomic-based mouse model provides evidence that neither cholesterol levels nor APOE abundance alone is sufficient to influence Aß metabolism in young animals.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of genetically defined APOE KI mice
The APOE {varepsilon}4 allele is the most significant genetic risk factor for Alzheimer's disease identified to-date, Much work has been done in the mouse to understand how the different alleles of APOE affect the onset and development of pathology in an AD mouse model; however, these transgenic models have several limitations that confound the study of APOE gene regulation and gene function in mouse models of AD. To circumvent these limitations, we took advantage of APOE KI animals generated at the Mitsubishi Kagaku Institute of Life Science, Japan (27). The targeted allele placed the human cDNA for each APOE allele in-frame with the endogenous exon 1 sequence and part of exon 2. This allowed for control of human APOE expression by endogenous regulatory elements, while at the same time removing the mouse Apoe coding sequence. APOE {varepsilon}4 KI mice generated by this strategy were previously published (27). It was demonstrated that human APOE in these mice is expressed in serum and in astrocytes in the correct histological pattern. The APOE KI mice were originally maintained on a mixed C57BL/6;129P2 background and contained a neo selection cassette in the targeted allele. Work from our lab and others has demonstrated that genetic background can greatly influence phenotypic expression, particularly with regards to Aß generation and deposition (28). In addition, several lines of evidence have shown that the strong promoter of neo can influence the expression of both the targeted locus as well as the neighboring genes (29,30). For these reasons, the APOE KI lines were backcrossed onto the defined C57BL/6J (B6) inbred background and also mated to ZP3-cre transgenic mice congenic on the B6 background (31) to remove the neo cassette through cre-mediated loxP excision. The cre transgene was transferred onto the APOE KI background such that the expression of cre in the mouse oocytes of APOE KI females excised the floxed neo cassette in the targeted allele. These females were mated to B6 males, and the resulting offspring inherited the targeted allele lacking the neo tag, as demonstrated by both polymerase chain reaction (PCR) assay, using primers to neo, and by Southern blot (data not shown). Animals lacking the cre transgene were identified in subsequent progeny. Once each APOE KI line was backcrossed five generations (N5, incipient congenic, >98% homogeneous) (32), animals were intercrossed to obtain populations of APOE {varepsilon}2/{varepsilon}2, {varepsilon}3/{varepsilon}3 and {varepsilon}4/{varepsilon}4 mice (hereafter referred to as {varepsilon}2 KI, {varepsilon}3 KI and {varepsilon}4 KI, respectively). Animals were PCR genotyped on the basis of the presence of human APOE, using human-specific primers, and the absence of the endogenous Apoe locus, demonstrated by PCR using mouse Apoe-specific primers (data not shown). These mice were then characterized at 6–8 weeks of age for a variety of parameters in order to understand how each APOE allele behaves in the mouse with respect to steady-state protein levels and cholesterol metabolism, and how each impacts APP processing and Aß metabolism.

Protein abundance in APOE KI mice
The levels of steady-state APOE in brain, liver and serum of the three KI mouse lines were determined by western blot analysis using a human-specific APOE antibody. Parallel western blots of recombinant E2, E3 and E4 serial dilutions (6.25, 12.5, 25, 50 µg of protein) were run to determine the affinity of this antibody for each isoform. Standard curves of fluorescent units for each isoform revealed little difference in detection. E4 appeared to be detected slightly less well (data not shown). Levels of APOE protein in each KI animal were compared with a standard homogenate (or serum standard) from a homozygous {varepsilon}3 KI animal. Figure 1 illustrates the differences in APOE levels compared across lines as the ratio of steady-state levels of each sample relative to the standard. APOE {varepsilon}2 KI animals had an average 2-fold higher steady-state level of APOE in both brain and fasted liver when compared with {varepsilon}3 KI and {varepsilon}4 KI animals (Fig. 1A and B, P<0.001). All three lines had significantly different levels of APOE in the brain. Levels of steady-state APOE in brain were confirmed by a human-specific APOE ELISA, using a standard curve of recombinant E2, E3 or E4 to quantitate levels of each isoform. {varepsilon}2 KI animals demonstrated a 2-fold increase in the level of brain APOE by this method as well (P<0.001), although E3 and E4 were not significantly different. The difference shown by western blotting in the {varepsilon}3 and {varepsilon}4 lines may be explained by the slightly lower affinity of the antibody for recombinant E4. The level of APOE in fasted serum of {varepsilon}2 KI animals was also significantly higher than those found in {varepsilon}3 KI and {varepsilon}4 KI animals by western blotting (Fig. 1D, P<0.01). The relationship between genotype and APOE levels in the KI mice recapitulates that observed in human serum (33–35).



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  		Figure 1. Analysis of APOE abundance in APOE KI animals. Relative levels of APOE in brain, fasted liver and fasted serum in the APOE KI lines were assayed in both males and females 6–8 weeks of age. Protein extracts of both brain and fasted liver were made in 1% CHAPS with protease inhibitors. APOE levels in brain (A, n=8), fasted liver (B, n=8) and fasted serum (D, n=6) were assayed by western blot analysis using a human-specific APOE antibody (Calbiochem). APOE quantitation is expressed as a ratio of APOE abundance in each sample relative to that in a standard of the same total protein concentration. {varepsilon}2 KI APOE levels are significantly increased in both brain (A) and fasted liver (B) (P<0.001, one-factor ANOVA with post-Tukey) when compared with {varepsilon}3 KI and {varepsilon}4 KI levels. {varepsilon}2 KI APOE levels are also significantly increased in fasted serum (D) (P<0.01, one-factor ANOVA followed by Dunn's post-test). {varepsilon}3 KI levels in brain are significantly increased over {varepsilon}4 KI levels (P<0.01, one-factor ANOVA with post-Tukey). A representative western blot of APOE abundance in brain and fasted liver (C) demonstrates the relative 4-fold increase in APOE in liver versus brain and the 2-fold higher levels in {varepsilon}2 KI homogenates compared with {varepsilon}3 KI and {varepsilon}4 KI homogenates. APOE brain levels were also measured by human-specific ELISA (E); {varepsilon}2 KI levels are significantly increased over {varepsilon}3 KI and {varepsilon}4 KI levels (P<0.001, one-factor ANOVA with post-Tukey).

 
Cholesterol metabolism in APOE KI mice
To examine how each allele affects cholesterol metabolism in the mouse, serum cholesterol and triglyceride levels were measured after a 6 h fast using an enzymatic assay. Analysis of cholesterol levels in more than 20 animals of each genotype and of mixed sex demonstrated an inverse relationship between genotype and phenotype relative to the human data. The {varepsilon}2 KI animals had both the highest cholesterol (mean 234 mg/dl) and triglycerides (mean 115 mg/dl). In contrast, the {varepsilon}4 KI animals had the lowest cholesterol (mean 67 mg/dl), whereas the {varepsilon}3 KI animals did not significantly differ from B6 wild-type animals (Table 1). Triglyceride levels in the {varepsilon}3 KI, {varepsilon}4 KI and B6 animals were statistically indistinguishable.


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  		Table 1. Fasted serum cholesterol and triglyceride levels by APOE genotype
 
To further understand how the different APOE isoforms participate in peripheral cholesterol metabolism in the mouse, we generated lipoprotein profiles by FPLC for each KI line. The distribution of APOE and cholesterol was analyzed for each profile. Fasted serum from three animals of the same sex was pooled for analysis of each genotype; no sex difference was apparent (data not shown). The profiles for each genotype are shown in Figure 2. APOE {varepsilon}3 KI and {varepsilon}4 KI profiles do not differ from that of B6 wild-type animals, in that the majority of circulating cholesterol is found in the HDL fraction, whereas the VLDL and LDL/IDL fractions are minimal. In contrast, the {varepsilon}2 KI profiles demonstrated that, although the levels of HDL cholesterol remained equal to that found in the other lines, the cholesterol distribution extended to the LDL/IDL fraction and had the highest level in the VLDL fraction. Thus, the increased total cholesterol levels in the {varepsilon}2 KI mice were due to an increase in VLDL and LDL/IDL cholesterol.



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  		Figure 2. FPLC analysis of lipoprotein profiles of APOE KI animals. Fasted serum from three animals of the same sex and APOE line was pooled and fractionated by FPLC on a Superose 6 column. FPLC profiles were generated for both males and females of each line in two separate experiments; representative results are shown in this figure. The VLDL peak at around 20 min for (A) B6 with endogenous Apoe (Apoewt) (B) {varepsilon}4 KI and (C) {varepsilon}3 KI profiles contains little cholesterol when compared with the VLDL fraction in (D) from the {varepsilon}2 KI profile. The large peak at 40 min represents the HDL fraction; the cholesterol levels for all four groups are relatively equal in this fraction. (E) The distribution of APOE in the lipoprotein fractions was analyzed by western blot analysis using a human-specific APOE antibody (Calbiochem). Five fractions for each lipoprotein peak were pooled and equal volumes of the combined fractions were loaded in each lane; a representative western blot of APOE abundance is shown. The absence of APOE in the VLDL fractions of Apoewt, {varepsilon}3 KI and {varepsilon}4 KI is distinct compared with the abundance of APOE in the {varepsilon}2 KI profile. The majority of APOE in the {varepsilon}4 KI profile is found in the HDL fraction in contrast to the {varepsilon}3 KI and Apoewt profiles, which show the majority of APOE and Apoe in the LDL/IDL fraction.

 
The distribution of APOE protein in each lipoprotein fraction was determined by western blot analysis. Five fractions for each lipoprotein class, including the peak fraction, were pooled and run under the same conditions as for the APOE quantitation in tissues. The resulting data are shown in Figure 2E. No APOE was detected in the VLDL fractions for {varepsilon}3 KI, {varepsilon}4 KI and non-transgenic control profiles, which reflected the low levels of cholesterol in this fraction. Levels of APOE were highest in the LDL/IDL fraction, followed by the HDL fraction. {varepsilon}4 KI profiles demonstrated the highest levels of APOE in the HDL fraction. This may reflect preferential binding of E4 for larger lipoproteins; mouse plasma is enriched in a large, Apoe-rich HDL that is absent in normal human plasma (36). In contrast to these profiles, high levels of APOE were observed in all fractions of the {varepsilon}2 KI profile, which was consistent with the high levels of cholesterol found to be distributed in all fractions.

APP processing, Aß metabolism and brain cholesterol in APOE KI mice
To examine the effects of the different APOE alleles on APP biochemistry, APOE KI mice were crossed to the R1.40 APP transgenic line congenic on the B6 background. The R1.40 line is a YAC-based transgenic model of AD that over-expresses APPSwe in the proper spatial and temporal manner (37). Homozygous R1.40 transgenic animals display Aß deposition at 13.5 months of age. APOE KI animals were crossed to R1.40 transgenic animals to generate animals homozygous for each APOE allele and hemizygous for APP. Brains from 28-day-old APOE/APOE; R1.40/– animals (subsequently denoted as APOE;R1.40 or {varepsilon}2;R1.40, etc.) were utilized to examine the levels of APP processing intermediates and Aß.

The levels of APP holo protein and C-terminal fragments (CTFs) were determined using 369, an APP C-terminal antibody. No differences in holo APP were observed between lines (Fig. 3A and B). In addition, the relative amount of APP CTFs (expressed as the ratio of CTFß/total CTFs) was determined for each APOE;R1.40 line and R1.40 animals with endogenous Apoe (Fig. 3C). Despite significant differences in serum cholesterol among the three APOE;R1.40 lines (Fig. 4A), there was no difference in the ratios of CTFß/total CTFs. Likewise, the APP CTF ratios were unchanged in the presence of APOE or Apoe in animals of 28 days of age.



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  		Figure 3. APP processing in APOE;R1.40 animals. APP processing products were analyzed in 6 M urea homogenates of half-brains from APOE;R1.40 animals at 28 days of age. Western blotting with an APP antibody (369) was used to assay levels of both holo APP and CTFs in each APOE;R1.40 line (n=5 for each line), as well as in R1.40 animals with endogenous Apoe (n=3). A representative western blot is shown in (A). APP quantitation (B) is expressed as a ratio of APP abundance in each sample relative to endogenous levels in a wild-type extract of the same total protein concentration. Analysis of holo APP levels demonstrated no difference between lines (P>0.05, one-factor ANOVA). Relative ratios of CTFß to total APP CTFs (C) were unaffected by APOE genotype (P>0.05, one-factor ANOVA; four or five animals per group).

 


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  		Figure 4. Total cholesterol and brain Aß1–40 levels in APOE;R1.40 animals. Measurements of total cholesterol in both serum and brain, and brain Aß levels were obtained from animals of 28 days of age. (A) Serum cholesterol was assayed for APOE;R1.40 and R1.40 animals with endogenous Apoe by colorimetric assay (Thermotrace). The results confirmed that the APP transgene does not affect serum cholesterol metabolism. {varepsilon}2;R1.40 animals have 2-fold higher levels of total cholesterol than {varepsilon}3;R1.40, {varepsilon}4;R1.40 and B6.R1.40 animals (P<0.001, one-factor ANOVA followed by post-Tukey). (B) Levels of total brain cholesterol were measured in 6 M urea homogenates of half-brains by colorimetic assay (Thermotrace). The presence of human APOE{varepsilon}2 or {varepsilon}4 significantly increases brain cholesterol in R1.40 animals (P<0.05, one-factor ANOVA). E3;R1.40 brain cholesterol levels were also elevated compared to B6.R1.40 animals, although this was not significant (P>0.05, one-factor ANOVA). (C) Levels of Aß1–40 in brain, measured in 5 M guanidine extracts of half-brains by ELISA (Biosource), are not significantly different between APOE;R1.40 lines (P>0.05, one-factor ANOVA; n=8 for each line). All APOE;R1.40 lines have significantly increased Aß1–40 levels when compared with B6.R1.40 animals (n=11; P<0.01 for E2 versus B6; P<0.001 for E3 versus B6; P<0.05 for E4 versus B6, one-factor ANOVA with post-Tukey). {varepsilon}2;R1.40 is increased by 30.7%, {varepsilon}3;R1.40 by 35.0% and {varepsilon}4;R1.40 by 25.7%.

 
Levels of serum cholesterol in each APOE;R1.40 line at the N8 backcross generation were determined as previously mentioned. Similar to our results in the absence of the R1.40 transgene, the {varepsilon}2.R1.40 mice had significantly higher cholesterol levels compared with all other lines. However, the {varepsilon}4.R1.40 animals had slightly higher levels of cholesterol relative to {varepsilon}3.R1.40 animals and R1.40 animals with endogenous Apoe (Fig. 4A). This difference compared with the data in Table 1 for the N5 generation most likely illustrates a genetic background effect on cholesterol metabolism in {varepsilon}4 KI mice.

Levels of steady-state Aß1–40 were assessed by ELISA in half-brain extracts. Given that increased serum cholesterol, conferred by diet, effectively increases Aß1–40 in the brain (14), the expectation was that the {varepsilon}2.R1.40 animals, with serum cholesterol levels nearly twice those of {varepsilon}3.R1.40 and {varepsilon}4.R1.40 animals, would have the greatest amount of Aß1–40. However, no significant difference in Aß1–40 levels between the three APOE;R1.40 lines was observed (Fig. 4C). These data suggest that there is a fundamental distinction between genetic and dietary control of cholesterol levels and their effects on Aß1–40 metabolism. Interestingly, all three APOE;R1.40 lines demonstrated significantly increased levels of Aß1–40 relative to B6.R1.40 animals with endogenous Apoe. Thus, the presence of human APOE in the R1.40 line led to increased Aß1–40 steady-state levels at 28 days of age.

Levels of total brain cholesterol were measured by enzymatic assay and normalized to the concentration of protein extracted (mg cholesterol/mg protein). The average levels of brain cholesterol in {varepsilon}2.R1.40, {varepsilon}3.R1.40 and {varepsilon}4.R1.40 animals were higher than the average brain cholesterol in R1.40 animals with endogenous Apoe (Fig. 4B; P<0.05 for {varepsilon}2.R1.40 versus R1.40 and {varepsilon}4.R1.40 versus R1.40). This increase in brain cholesterol in APOE;R1.40 animals appeared to coincide with increased Aß1–40 levels, suggesting a link between APOE, brain cholesterol and Aß metabolism. Despite high levels of serum cholesterol, {varepsilon}2.R1.40 animals had brain cholesterol levels indistinguishable from those in {varepsilon}3.R1.40 and {varepsilon}4.R1.40 animals. These cholesterol data lend further evidence to the hypothesis that peripheral and brain cholesterol levels are independently regulated.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The current study examines the effects of the three common human APOE alleles on APOE protein levels, cholesterol metabolism, and APP processing and Aß metabolism. We show that APOE KI mice on the defined C57BL/6J background have significantly different levels of APOE in both serum and tissues, as well as significantly different cholesterol levels. APOE increases Aß levels in a genomic-based APP transgenic mouse in the absence of changes in APP processing. Unlike previously reported APOE transgenic models, which express normalized levels of APOE only in the brain, our genomic-based approach allows us to address two hypotheses surrounding the role of APOE {varepsilon}4 as a risk factor for AD. APOE genotype and AD risk in humans correlate with both serum cholesterol levels and steady-state levels of APOE protein. APOE {varepsilon}4, the most significant genetic risk factor for AD, is associated with both high circulating cholesterol levels and reduced steady-state APOE levels. In contrast, APOE {varepsilon}2 is protective against AD and incidentally confers both low serum cholesterol levels and high levels of APOE. Thus, APOE may affect AD risk through its involvement in cholesterol metabolism, shown through diet studies in the mouse to directly affect Aß production. Alternatively, brain APOE levels may influence the clearance efficiency of the Aß peptide and its subsequent deposition.

APOE abundance
Much is known about APOE biology in humans, particularly its role in lipid metabolism. APOE is primarily synthesized in the liver and brain; additional sites include the adrenal gland and kidney (38,39). APOE genotype has been shown to correlate with APOE abundance in the periphery (33,40–42); the {varepsilon}2 allele confers high APOE, whereas the {varepsilon}4 allele confers low APOE, with respect to {varepsilon}3 levels. The effect of APOE alleles on APOE abundance in the brain and CSF is less clear owing to conflicting reports, with many studies concentrating on the abundance of APOE in AD brains relative to controls, irrespective of APOE genotype (35,43,44).

We demonstrate by both western blot analysis and ELISA that the steady-state levels of APOE in tissues and serum from the three KI lines are genotype-dependent and reflect the human data for serum APOE levels. {varepsilon}2 KI animals have significantly higher levels of APOE in all tissues examined (P<0.001). Our findings, particularly the data from brain, illustrate the innate differences in the behavior of the three human APOE alleles in the mouse, arguing for the relevance of a genomic-based APOE model in understanding APOE function. The differences in brain APOE levels in our KI mice may have important implications for how APOE influences pathology development in an AD mouse model. A recent report by Sullivan et al. (45), showing that brain APOE levels are genotype-independent in a different set of APOE KI animals contradicts these data. However, their reported APOE lines contain a neo cassette in the targeted allele that may affect protein expression. An additional difference is the inclusion of human APOE intron 1 in the targeted allele of their mice, which may contain additional endo genous regulatory elements. Further comparisons between their mice and ours will be necessary to determine the innate differences between the two models.

APOE and cholesterol metabolism in mice
In addition to protein levels, we show that APOE genotype also impacts cholesterol levels in the periphery. The {varepsilon}4 KI animals have fasted cholesterol levels that are slightly below those of B6 wild-type animals, whereas the {varepsilon}2 KI animals have 2-fold higher cholesterol levels. The {varepsilon}3 KI and B6 wild-type cholesterol levels do not significantly differ. These data recapitulate what has been shown in other APOE KI models (46–48), but are opposite from the findings in humans, where the {varepsilon}4 allele is associated with high and {varepsilon}2 with low cholesterol levels. These data highlight the innate differences in mouse and human cholesterol metabolism, which include the utilization of different lipoproteins to transport cholesterol. Our data also contrast with the initial report of the {varepsilon}4 KI mice by Hamanaka et al. (27), showing a 2.5-fold increase in levels of circulating cholesterol relative to wild-type controls. This discrepancy in the data may be attributed to several factors. The first, and perhaps most notable, is the difference in genetic background between these two studies. Several lines of evidence demonstrate a strong genetic component to cholesterol metabolism in the mouse (46–48). This complex trait involves genes that work both in an additive manner as well as epistatically (49). Our analysis of cholesterol in these KI mice over subsequent backcrosses further argues for background effects, as demonstrated by comparing the cholesterol levels in the {varepsilon}4.N5 animals and the {varepsilon}4.R1.40 animals at the N8 backcross generation (Table 1 versus Fig. 4). Second, the KI mice that we report no longer transmit the neo cassette in the targeted allele. Although there is no direct evidence to suggest that the presence of neo affects the function of APOE or cholesterol metabolism in these mice, this cannot be ruled out. Finally, the differences in diet or methodology between the two studies may also impact the reported cholesterol levels.

Further characterization of lipoprotein profiles from our reported {varepsilon}3 and {varepsilon}4 KI mice demonstrated that the majority of cholesterol distributed to the HDL fraction, similar to B6 wild-type animals. These data suggest that the presence of human APOE {varepsilon}3 or {varepsilon}4 does not disrupt mouse cholesterol metabolism, an observation also made by Maeda and colleagues (50–52). Comparison of these profiles with that published by Hamanaka et al. highlights a further distinction between their {varepsilon}4 KI model and that characterized here. In contrast to our data, Hamanaka et al. showed cholesterol distributed to all lipoprotein classes in the {varepsilon}4 KI mice, including VLDL and LDL. This profile is actually most similar to that of the {varepsilon}2 KI animals presented here. These animals have significantly higher cholesterol levels and appear to transport the excess circulating cholesterol in the VLDL and LDL fractions, suggesting an impaired cholesterol metabolism. The noted increase in triglyceride levels in these {varepsilon}2 KI animals also suggests the development of type III hyperlipoproteinemia, a complex disorder characterized by high cholesterol and triglyceride levels, which affects ~10% of humans homozygous for the {varepsilon}2 allele (53). It has been suggested by Sullivan et al. and others (51,54) that this condition manifests itself in {varepsilon}2 KI mice owing to the presence of additional genetic factors in the mouse genome that interact with the {varepsilon}2 allele.

Qualitative western blot analysis of {varepsilon}3 and {varepsilon}4 KI FPLC profiles demonstrated that the behavior of E3 in mice maintained on a low cholesterol diet follows that of endogenous Apoe, found mainly in the IDL/LDL, with some in the HDL fraction; E4 appears to be enriched in the HDL fraction. This observation may reflect another difference between mouse and human cholesterol metabolism. The E4 isoform has a greater affinity for large lipoproteins, which in humans are chylomicrons and VLDL. E2 and E3, on the other hand, prefer smaller lipoproteins and are primarily found associated with HDL, the smallest lipoprotein class. Importantly, mouse plasma is enriched in a large, Apoe-rich HDL that is absent in normal human plasma (36). It may be that E4 has a preference for these particles over the other two isoforms. Our mice do not show an enrichment of APOE in VLDL, as observed by Maeda and colleagues. This discrepancy with our data may be explained by differences in genetic background or diet. Further comparisons between these two sets of APOE KI models, including levels of protein, will be required to thoroughly explain these results.

Cholesterol and APP processing
In order to identify the effects of each APOE allele on APP processing and Aß production, particularly with regard to cholesterol metabolism and APOE abundance, we crossed the APOE KI animals to R1.40 APPSwe transgenic animals. Several studies have established a link between cholesterol and APP processing (10,55,56). Refolo et al. (13) demonstrated that cholesterol influences the cleavage of APP by beta-secretase in vivo, with hypercholesterolemia increasing CTFß levels. However, neither levels of holo APP nor CTFs were altered in the APOE;R1.40 animals at 28 days of age. The lack of an effect on APP processing in the {varepsilon}2 KI animals was unexpected, considering their high serum cholesterol levels.

Analysis of Aß1–40 levels in these animals provided further evidence that serum cholesterol alone is not enough to affect APP processing at this young age. Given the strong evidence for high cholesterol levels increasing Aß production (13) we predicted that the {varepsilon}2 KI animals, with a 2-fold increase in total serum cholesterol, would demonstrate much higher levels of Aß than the {varepsilon}3 KI and {varepsilon}4 KI mice. However, similar to the CTF data, the levels of Aß1–40 in brain were indistinguishable across lines. There are several possible explanations for these results. First, the data we present here come from 28-day-old animals, an age just post-weaning and prior to sexual maturity. It is possible that the ability of cholesterol to affect APP processing and Aß metabolism is time- or age-dependent. Successful diet studies have chronically fed mice high cholesterol diets for several weeks (13,14). Chronic exposure to high serum cholesterol levels for several weeks or months may be required to affect Aß metabolism in the APOE;R140 animals. Assaying levels of Aß1–40 in older animals will be useful to further understand the age component of cholesterol effects and APOE on APP processing.

Second, we show that peripheral cholesterol metabolism does not affect CNS cholesterol regulation. Despite the differences in serum cholesterol in the three APOE;R1.40 lines described in this manuscript, brain cholesterol levels are indistinguishable in these animals. This finding is supported by a large body of evidence demonstrating that cholesterol metabolism in the periphery is fundamentally different from and independent of that in the central nervous system, involving different lipoproteins and apolipoproteins (57–59). This argues against an effect of peripheral cholesterol on processes in the brain.

Importantly, E2;R1.40 and E4;R1.40 animals demonstrate significantly increased brain cholesterol levels over R1.40 animals with endogenous Apoe; brain cholesterol levels in the E3.R140 animals were also greater than those of R1.40, although the trend was not statistically significant. These data correspond with significant increases in brain Aß1–40 levels in the APOE;R1.40 animals, suggesting that brain cholesterol levels in particular may have a significant impact on Aß production. Notably, one diet study increased brain cholesterol in addition to Aß levels (14). Our data argue for the independent roles of APOE in peripheral and CNS cholesterol metabolism, and furthermore suggest a distinction between APOE and endogenous Apoe effects on both murine brain cholesterol metabolism and steady-state Aß levels.

APOE and Alzheimer's disease
This manuscript highlights the first report of an APOE mouse model that utilizes endogenous regulation to study native APOE levels and cholesterol metabolism in relation to Alzheimer's disease. Importantly, the correlation between APOE genotype, APOE steady-state levels and cholesterol metabolism observed in humans is uncoupled in the KI mice. This allows us to further dissect two aspects of APOE function that may impact the development of AD, cholesterol metabolism and Aß clearance. APOE abundance in the brains of {varepsilon}4 KI animals is low, but these animals have normal serum cholesterol levels. These animals allow us to study the effects of low APOE levels in the absence of high cholesterol, which is often observed in humans who inherit an {varepsilon}4 allele. Likewise, the {varepsilon}2 KI animals have both high serum cholesterol and high brain APOE levels. These animals are useful to determine the effects of high cholesterol independent of low APOE levels. Future experiments will assess the effects of both age and diet in the APOE;R1.40 lines on cholesterol metabolism, particularly in brain, and APOE abundance, as well as Aß metabolism and amyloid deposition.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mice
The original stocks of APOE {varepsilon}2, {varepsilon}3 and {varepsilon}4 KI animals were produced at the Mitsubishi Kagaku Institute of Life Sciences; the {varepsilon}4 KI animals were previously published (27). APOE characterization: male KI animals for each line were backcrossed two generations onto the C57BL/6J background. Male progeny of the N2 generation were then crossed to Zp3-cre transgenic animals congenic on the C57BL6/J background (31). Female progeny from this cross (N3 generation) were selected for on the basis of the presence of both the human APOE and the Zp3-cre transgene. An additional backcross of these animals yielded progeny of the N4 generation that were selected for on the basis of the presence of APOE and the absence of the neo cassette in the targeted allele. Males from this cross were backcrossed to N5 before animals were intercrossed to obtain homozygous KI animals of each allele. These animals were used for the characterization studies of human APOE in the mouse, and no longer carried the ZP3-cre transgene. For analysis of APP processing and Aß metabolism, APOE KI animals backcrossed to the N7 generation were crossed to B6.R140 APP transgenic mice congenic on the C57BL6/J background, and subsequent progeny intercrossed to obtain mice homozygous for APOE and hemizygous for the APP transgene. All mice were maintained on a normal chow diet (LabDiets 5021; 9.5% fat) and in accordance with IACUC regulations.

Cholesterol and triglyceride measurements
Serum for cholesterol and triglyceride measurements was obtained from animals fasted 6 h by retro-ortbital sinus bleed. Measurements were obtained using the Infinity Cholesterol reagent and the Infinity Triglyceride reagent, respectively (Thermotrace, Arlington, TX, USA) according to the manufacturer's instructions. Brain cholesterol from hemispheres was extracted by homogenization in 6 M urea buffer (6 M urea, 100 mM Tris pH 7.4, 1 mM DTT, 1 mM EDTA, 0.5 M AEBSF and 1% SDS) as reported by Levin-Allerhand et al. (14). Brain cholesterol was measured using the Inifinity Cholesterol reagent (Thermotrace) after diluting the homogenates 1 : 1 in PBS. Brain cholesterol concentrations are expressed as mg cholesterol relative to mg protein measured by BCA assay (Pierce, Rockford, IL, USA).

FPLC
For analysis of lipoprotein profiles and cholesterol distribution in the APOE KI animals, fasted serum from three animals of each genotype and of the same sex was pooled before fractionation on a Superose 6 column (Pharmacia, Kalamazoo, MI, USA) as previously described (60).

Western blot analysis
For analysis of APOE levels from tissues, brain or liver homogenates were prepared in 1% CHAPS with protease inhibitors (1 µM pepstatin, 4.5 µg/ml leupeptin, 30 µg/ml aprotinin and 1 mM AEBSF). Brain and fasted liver homogenates were run on 4–12% Bis–Tris gradient gels (Invitrogen, Carlsbad, CA, USA) under reducing conditions according to the manufacturer's instructions. Equal concentrations of protein, determined by BCA assay, were loaded on every gel. A linear standard curve of a serially diluted control {varepsilon}3 KI of known concentration was run on every gel to normalize across gels (R2≥0.90). Protein was transferred to Immobilon-p membranes (Millipore, Bedford, MA, USA) and probed with an anti-human polyclonal APOE antibody (Calbiochem, LaJolla, CA, USA), followed by an HRP-conjugated secondary antibody and detected using ECL (Pierce) on film. This APOE antibody has been used extensively to detect human APOE in mouse by immunohistochemistry, western blotting and ELISA (26,46,51,52). APOE protein levels in serum and tissue were quantitated by calculating the intensity of the sample relative to that of the standard of the same concentration. ImageQuant software was used in the analysis of protein abundance.

The levels of APOE from unfractionated serum were determined by loading equal concentrations of protein from fasted serum samples (diluted in PBS) onto Bis–Tris gels (under non-reducing conditions) and following the same analytical paradigm as for tissues. APOE in serum fractionated by FPLC was analyzed qualitatively by combining equal volumes from the peak fraction, and 2 volumes above and below for each lipoprotein class. APOE was detected using the same human-specific APOE antibody and visualized by ECL using film.

The relative levels of holo APP were determined using brain homogenates prepared in 6 M urea (as mentioned earlier). Equal concentrations of protein from every sample were loaded onto 7% Tris–acetate gels (Invitrogen) and run according to the manufacturer's instructions. A linear standard curve of a serially diluted homogenate from a B6 non-transgenic animal was run on every gel to normalize across gels (R2≥0.90). Protein was transferred as mentioned earlier and the membranes were probed with a C-terminal antibody to human APP (369, gift of Sam Gandy, Thomas Jefferson University). The intensity of the APP protein band was determined by ECL detected using a Fluor-S Max imaging machine (BioRad, Hercules, CA, USA) and quantitated by Quantity-one software (BioRad). APP CTFs were determined from the same extracts run on 4–12% Bis–Tris gradient gels (as mentioned earlier) using the 369 antibody. Quantitation was carried out on film as mentioned earlier.

ELISA
Levels of human APOE in brain were measured by a human-specific APOE ELISA (MBL International) according to the manufacturer's instructions. Homogenates made in 1% CHAPS (as mentioned earlier) were run in duplicate and detected using the Pan–APOE antibody. Standard curves of recombinant E2, E3 and E4 were run in duplicate on the same plate. Levels of APOE were determined using the standard curve for each respective isoform.

Levels of Aß1–40 were measured by a human-specific Aß1–40 ELISA (Biosource International) according to the manufacturer's instructions. Half-brains were homogenized in 5 M guanidine–100 mM Tris pH 8.0 and incubated at room temperature for 3.5–4 h to ensure complete protein extraction. Samples were diluted in standard/sample buffer and centrifuged before loaded in triplicate onto the ELISA plate.

Statistics
Statistical analysis was carried out using Prism Graphpad software (San Diego, CA, USA). The specific test used for each analysis is indicated in the appropriate figure legend.


   ACKNOWLEDGEMENTS
 
The authors acknowledge Minesuke Yokoyama, Rika Migishima, Yoshiko Motegi, Yoko Nakahara, Aya Takeshita and Mariko Kobayashi who were all involved in the initial generation of the APOE {varepsilon}2, {varepsilon}3 and {varepsilon}4 KI animals. We thank Sam Gandy (Thomas Jefferson University) for providing the 369 APP antibody and Barbara Knowles (Jackson Labs) for the Zp3-Cre animals. We also thank the Ireland Cancer Center of the University Hospitals, Cleveland, Ohio for the use of their equipment. This work was supported by in part by NIH training grant GM08613 (K.M.M.); NIH grant R01 AG14451, Alzheimer's Association grant IIRG-02-3750 and an American Health Assistance Founding Grant (B.T.L.); the University Memory and Aging Center (P50 AG08012); the Ireland Cancer Center (CA 43703) and NIH grant R01 HL32868 (D.L.W.).


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Genetics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4955, USA. Tel: +1 2163682979; Fax: +1 2163683432; Email: btl{at}cwru.edu


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Weisgraber, K.H., Innerarity, T.L. and Mahley, R.W. (1982) Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to cysteine–arginine interchange at a single site. J. Biol. Chem., 257, 2518–2521.[Free Full Text]

  2. Dong, L.M., Parkin, S., Trakhanov, S.D., Rupp, B., Simmons, T., Arnold, K.S., Newhouse, Y.M., Innerarity, T.L. and Weisgraber, K.H. (1996) Novel mechanism for defective receptor binding of apolipoprotein E2 in type III hyperlipoproteinemia. Nat. Struct. Biol., 3, 718–722.[CrossRef][Web of Science][Medline]

  3. Menzel, H.J., Kladetzky, R.G. and Assmann, G. (1983) Apolipoprotein E polymorphism and coronary artery disease. Arteriosclerosis, 3, 310–315.[Abstract/Free Full Text]

  4. Stengard, J.H., Zerba, K.E., Pekkanen, J., Ehnholm, C., Nissinen, A. and Sing, C.F. (1995) Apolipoprotein E polymorphism predicts death from coronary heart disease in a longitudinal study of elderly Finnish men. Circulation, 91, 265–269.[Abstract/Free Full Text]

  5. Corder, E.H., Saunders, A.M., Strittmatter, W.J., Schmechel, D.E., Gaskell, P.C., Small, G.W., Roses, A.D., Haines, J.L. and Pericak-Vance, M.A. (1993) Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. Science, 261, 921–923.[Abstract/Free Full Text]

  6. Strittmatter, W.J., Saunders, A.M., Schmechel, D., Pericak-Vance, M., Enghild, J., Salvesen, G.S. and Roses, A.D. (1993) Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc. Natl Acad. Sci. USA, 90, 1977–1981.[Abstract/Free Full Text]

  7. Corder, E.H., Saunders, A.M., Risch, N.J., Strittmatter, W.J., Schmechel, D.E., Gaskell, P.C., Jr, Rimmler, J.B., Locke, P.A., Conneally, P.M., Schmader, K.E. et al. (1994) Protective effect of apolipoprotein E type 2 allele for late onset Alzheimer disease. Nat. Genet., 7, 180–184.[CrossRef][Web of Science][Medline]

  8. Bodovitz, S. and Klein, W.L. (1996) Cholesterol modulates alpha-secretase cleavage of amyloid precursor protein. J. Biol. Chem., 271, 4436–4440.[Abstract/Free Full Text]

  9. Frears, E.R., Stephens, D.J., Walters, C.E., Davies, H. and Austen, B.M. (1999) The role of cholesterol in the biosynthesis of beta-amyloid. Neuroreport, 10, 1699–1705.[Web of Science][Medline]

  10. Simons, M., Keller, P., De Strooper, B., Beyreuther, K., Dotti, C.G. and Simons, K. (1998) Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons. Proc. Natl Acad. Sci. USA, 95, 6460–6464.[Abstract/Free Full Text]

  11. Runz, H., Rietdorf, J., Tomic, I., de Bernard, M., Beyreuther, K., Pepperkok, R. and Hartmann, T. (2002) Inhibition of intracellular cholesterol transport alters presenilin localization and amyloid precursor protein processing in neuronal cells. J. Neurosci., 22, 1679–1689.[Abstract/Free Full Text]

  12. Wahrle, S., Das, P., Nyborg, A.C., McLendon, C., Shoji, M., Kawarabayashi, T., Younkin, L.H., Younkin, S.G. and Golde, T.E. (2002) Cholesterol-dependent gamma-secretase activity in buoyant cholesterol-rich membrane microdomains. Neurobiol. Dis., 9, 11–23.[CrossRef][Web of Science][Medline]

  13. Refolo, L.M., Malester, B., LaFrancois, J., Bryant-Thomas, T., Wang, R., Tint, G.S., Sambamurti, K., Duff, K. and Pappolla, M.A. (2000) Hypercholesterolemia accelerates the Alzheimer's amyloid pathology in a transgenic mouse model. Neurobiol. Dis., 7, 321–331.[CrossRef][Web of Science][Medline]

  14. 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.[Medline]

  15. Austen, B., Christodoulou, G. and Terry, J.E. (2002) Relation between cholesterol levels, statins and Alzheimer's disease in the human population. J. Nutr. Health Aging, 6, 377–382.[Medline]

  16. Ishii, K., Tokuda, T., Matsushima, T., Miya, F., Shoji, S., Ikeda, S. and Tamaoka, A. (2003) Pravastatin at 10 mg/day does not decrease plasma levels of either amyloid-beta (Abeta) 40 or Abeta 42 in humans. Neurosci. Lett., 350, 161–164.[CrossRef][Web of Science][Medline]

  17. Tokuda, T., Tamaoka, A., Matsuno, S., Sakurai, S., Shimada, H., Morita, H. and Ikeda, S. (2001) Plasma levels of amyloid beta proteins did not differ between subjects taking statins and those not taking statins. Ann. Neurol., 49, 546–547.[CrossRef][Web of Science][Medline]

  18. Pappolla, M.A., Bryant-Thomas, T.K., Herbert, D., Pacheco, J., Fabra Garcia, M., Manjon, M., Girones, X., Henry, T.L., Matsubara, E., Zambon, D. et al. (2003) Mild hypercholesterolemia is an early risk factor for the development of Alzheimer amyloid pathology. Neurology, 61, 199–205.[Abstract/Free Full Text]

  19. Kuo, Y.M., Emmerling, M.R., Bisgaier, C.L., Essenburg, A.D., Lampert, H.C., Drumm, D. and Roher, A.E. (1998) Elevated low-density lipoprotein in Alzheimer's disease correlates with brain abeta 1–42 levels. Biochem. Biophys. Res. Commun., 252, 711–715.[CrossRef][Web of Science][Medline]

  20. Tan, Z.S., Seshadri, S., Beiser, A., Wilson, P.W., Kiel, D.P., Tocco, M., D'Agostino, R.B. and Wolf, P.A. (2003) Plasma total cholesterol level as a risk factor for Alzheimer disease: the Framingham Study. Arch. Intern. Med., 163, 1053–1057.[Abstract/Free Full Text]

  21. Mann, D.M., Iwatsubo, T., Pickering-Brown, S.M., Owen, F., Saido, T.C. and Perry, R.H. (1997) Preferential deposition of amyloid beta protein (Abeta) in the form Abeta40 in Alzheimer's disease is associated with a gene dosage effect of the apolipoprotein E E4 allele. Neurosci. Lett., 221, 81–84.[CrossRef][Web of Science][Medline]

  22. Morishima-Kawashima, M., Oshima, N., Ogata, H., Yamaguchi, H., Yoshimura, M., Sugihara, S. and Ihara, Y. (2000) Effect of apolipoprotein E allele epsilon4 on the initial phase of amyloid beta-protein accumulation in the human brain. Am. J. Pathol., 157, 2093–2099.[Abstract/Free Full Text]

  23. Carter, D.B., Dunn, E., McKinley, D.D., Stratman, N.C., Boyle, T.P., Kuiper, S.L., Oostveen, J.A., Weaver, R.J., Boller, J.A. and Gurney, M.E. (2001) Human apolipoprotein E4 accelerates beta-amyloid deposition in APPsw transgenic mouse brain. Ann. Neurol., 50, 468–475.[CrossRef][Web of Science][Medline]

  24. Holtzman, D.M., Bales, K.R., Wu, S., Bhat, P., Parsadanian, M., Fagan, A.M., Chang, L.K., Sun, Y. and Paul, S.M. (1999) Expression of human apolipoprotein E reduces amyloid-beta deposition in a mouse model of Alzheimer's disease. J. Clin. Invest., 103, R15–R21.[Medline]

  25. Lesuisse, C., Xu, G., Anderson, J., Wong, M., Jankowsky, J., Holtz, G., Gonzalez, V., Wong, P.C., Price, D.L., Tang, F. et al. (2001) Hyper-expression of human apolipoprotein E4 in astroglia and neurons does not enhance amyloid deposition in transgenic mice. Hum. Mol. Genet., 10, 2525–2537.[Abstract/Free Full Text]

  26. Sun, Y., Wu, S., Bu, G., Onifade, M.K., Patel, S.N., LaDu, M.J., Fagan, A.M. and Holtzman, D.M. (1998) Glial fibrillary acidic protein-apolipoprotein E (apoE) transgenic mice: astrocyte-specific expression and differing biological effects of astrocyte-secreted apoE3 and apoE4 lipoproteins. J. Neurosci., 18, 3261–3272.[Abstract/Free Full Text]

  27. Hamanaka, H., Katoh-Fukui, Y., Suzuki, K., Kobayashi, M., Suzuki, R., Motegi, Y., Nakahara, Y., Takeshita, A., Kawai, M., Ishiguro, K. et al. (2000) Altered cholesterol metabolism in human apolipoprotein E4 knock-in mice. Hum. Mol. Genet., 9, 353–361.[Abstract/Free Full Text]

  28. Lehman, E.J., Kulnane, L.S., Gao, Y., Petriello, M.C., Pimpis, K.M., Younkin, L., Dolios, G., Wang, R., Younkin, S.G. and Lamb, B.T. (2003) Genetic background regulates beta-amyloid precursor protein processing and beta-amyloid deposition in the mouse. Hum. Mol. Genet., 12, 2949–2956.[Abstract/Free Full Text]

  29. Xu, X., Li, C., Garrett-Beal, L., Larson, D., Wynshaw-Boris, A. and Deng, C.X. (2001) Direct removal in the mouse of a floxed neo gene from a three-loxP conditional knockout allele by two novel approaches. Genesis, 30, 1–6.[CrossRef][Web of Science][Medline]

  30. Hirotsune, S., Fleck, M.W., Gambello, M.J., Bix, G.J., Chen, A., Clark, G.D., Ledbetter, D.H., McBain, C.J. and Wynshaw-Boris, A. (1998) Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality. Nat. Genet., 19, 333–339.[CrossRef][Web of Science][Medline]

  31. de Vries, W.N., Binns, L.T., Fancher, K.S., Dean, J., Moore, R., Kemler, R. and Knowles, B.B. (2000) Expression of Cre recombinase in mouse oocytes: a means to study maternal effect genes. Genesis, 26, 110–112.[CrossRef][Web of Science][Medline]

  32. Silver, L.M. (1995) Mouse genetics. In Mouse Genetics: Concepts and Applications. Oxford University Press, New York, pp. 46–49.

  33. Sing, C.F. and Davignon, J. (1985) Role of the apolipoprotein E polymorphism in determining normal plasma lipid and lipoprotein variation. Am. J. Hum. Genet., 37, 268–285.[Web of Science][Medline]

  34. Beffert, U., Cohn, J.S., Petit-Turcotte, C., Tremblay, M., Aumont, N., Ramassamy, C., Davignon, J. and Poirier, J. (1999) Apolipoprotein E and beta-amyloid levels in the hippocampus and frontal cortex of Alzheimer's disease subjects are disease-related and apolipoprotein E genotype dependent. Brain Res., 843, 87–94.[CrossRef][Web of Science][Medline]

  35. Bertrand, P., Poirier, J., Oda, T., Finch, C.E. and Pasinetti, G.M. (1995) Association of apolipoprotein E genotype with brain levels of apolipoprotein E and apolipoprotein J (clusterin) in Alzheimer disease. Brain Res. Mol. Brain Res., 33, 174–178.[Medline]

  36. de Silva, H.V., Mas-Oliva, J., Taylor, J.M. and Mahley, R.W. (1994) Identification of apolipoprotein B-100 low density lipoproteins, apolipoprotein B-48 remnants, and apolipoprotein E-rich high density lipoproteins in the mouse. J. Lipid Res., 35, 1297–1310.[Abstract]

  37. Lamb, B.T., Call, L.M., Slunt, H.H., Bardel, K.A., Lawler, A.M., Eckman, C.B., Younkin, S.G., Holtz, G., Wagner, S.L., Price, D.L. et al. (1997) Altered metabolism of familial Alzheimer's disease-linked amyloid precursor protein variants in yeast artificial chromosome transgenic mice. Hum. Mol. Genet., 6, 1535–1541.[Abstract/Free Full Text]

  38. Elshourbagy, N.A., Liao, W.S., Mahley, R.W. and Taylor, J.M. (1985) Apolipoprotein E mRNA is abundant in the brain and adrenals, as well as in the liver, and is present in other peripheral tissues of rats and marmosets. Proc. Natl Acad. Sci. USA, 82, 203–207.[Abstract/Free Full Text]

  39. Blue, M.L., Williams, D.L., Zucker, S., Khan, S.A. and Blum, C.B. (1983) Apolipoprotein E synthesis in human kidney, adrenal gland, and liver. Proc. Natl Acad. Sci. USA, 80, 283–287.[Abstract/Free Full Text]

  40. Gregg, R.E., Zech, L.A., Schaefer, E.J. and Brewer, H.B., Jr (1984) Apolipoprotein E metabolism in normolipoproteinemic human subjects. J. Lipid Res., 25, 1167–1176.[Abstract]

  41. Gregg, R.E., Zech, L.A., Schaefer, E.J., Stark, D., Wilson, D. and Brewer, H.B., Jr (1986) Abnormal in vivo metabolism of apolipoprotein E4 in humans. J. Clin. Invest., 78, 815–821.[Web of Science][Medline]

  42. Larson, I.A., Ordovas, J.M., DeLuca, C., Barnard, J.R., Feussner, G. and Schaefer, E.J. (2000) Association of apolipoprotein (Apo)E genotype with plasma apo E levels. Atherosclerosis, 148, 327–335.[CrossRef][Web of Science][Medline]

  43. Pirttila, T., Soininen, H., Heinonen, O., Lehtimaki, T., Bogdanovic, N., Paljarvi, L., Kosunen, O., Winblad, B., Riekkinen, P., Sr, Wisniewski, H.M. et al. (1996) Apolipoprotein E (apoE) levels in brains from Alzheimer disease patients and controls. Brain Res., 722, 71–77.[CrossRef][Web of Science][Medline]

  44. Artiga, M.J., Bullido, M.J., Frank, A., Sastre, I., Recuero, M., Garcia, M.A., Lendon, C.L., Han, S.W., Morris, J.C., Vazquez, J. et al. (1998) Risk for Alzheimer's disease correlates with transcriptional activity of the APOE gene. Hum. Mol. Genet., 7, 1887–1892.[Abstract/Free Full Text]

  45. Sullivan, P.M., Mace, B.E., Maeda, N. and Schmechel, D.E. (2004) Marked regional differences of brain human apolipoprotein e expression in targeted replacement mice. Neuroscience, 124, 725–733.[CrossRef][Web of Science][Medline]

  46. Paigen, B., Morrow, A., Brandon, C., Mitchell, D. and Holmes, P. (1985) Variation in susceptibility to atherosclerosis among inbred strains of mice. Atherosclerosis, 57, 65–73.[CrossRef][Web of Science][Medline]

  47. Lyons, M.A., Wittenburg, H., Li, R., Walsh, K.A., Churchill, G.A., Carey, M.C. and Paigen, B. (2003) Quantitative trait loci that determine lipoprotein cholesterol levels in DBA/2J and CAST/Ei inbred mice. J. Lipid Res., 44, 953–967.[Abstract/Free Full Text]

  48. Ordovas, J.M. and Schaefer, E.J. (1999) Genes, variation of cholesterol and fat intake and serum lipids. Curr. Opin. Lipidol., 10, 15–22.[CrossRef][Web of Science][Medline]

  49. Ishimori, N., Li, R., Kelmenson, P.M., Korstanje, R., Walsh, K.A., Churchill, G.A., Forsman-Semb, K. and Paigen, B. (2004) Quantitative trait loci analysis for plasma HDL-cholesterol concentrations and atherosclerosis susceptibility between inbred mouse strains C57BL/6J and 129S1/SvImJ. Arterioscler. Thromb. Vasc. Biol., 24, 161–166.[Abstract/Free Full Text]

  50. Sullivan, P.M., Mezdour, H., Aratani, Y., Knouff, C., Najib, J., Reddick, R.L., Quarfordt, S.H. and Maeda, N. (1997) Targeted replacement of the mouse apolipoprotein E gene with the common human APOE3 allele enhances diet-induced hypercholesterolemia and atherosclerosis. J. Biol. Chem., 272, 17972–17980.[Abstract/Free Full Text]

  51. Sullivan, P.M., Mezdour, H., Quarfordt, S.H. and Maeda, N. (1998) Type III hyperlipoproteinemia and spontaneous atherosclerosis in mice resulting from gene replacement of mouse Apoe with human Apoe*2. J. Clin. Invest., 102, 130–135.[Web of Science][Medline]

  52. Knouff, C., Hinsdale, M.E., Mezdour, H., Altenburg, M.K., Watanabe, M., Quarfordt, S.H., Sullivan, P.M. and Maeda, N. (1999) Apo E structure determines VLDL clearance and atherosclerosis risk in mice. J. Clin. Invest., 103, 1579–1586.[Web of Science][Medline]

  53. Mahley, R.W., Huang, Y. and Rall, S.C., Jr (1999) Pathogenesis of type III hyperlipoproteinemia (dysbetalipoproteinemia). Questions, quandaries, and paradoxes. J. Lipid Res., 40, 1933–1949.[Abstract/Free Full Text]

  54. Huang, Y., Rall, S.C., Jr and Mahley, R.W. (1997) Genetic factors precipitating type III hyperlipoproteinemia in hypolipidemic transgenic mice expressing human apolipoprotein E2. Arterioscler. Thromb. Vasc. Biol., 17, 2817–2824.[Abstract/Free Full Text]

  55. 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.[Abstract/Free Full Text]

  56. Kojro, E., Gimpl, G., Lammich, S., Marz, W. and Fahrenholz, F. (2001) Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the alpha-secretase ADAM 10. Proc. Natl Acad. Sci. USA, 98, 5815–5820.[Abstract/Free Full Text]

  57. Koch, S., Donarski, N., Goetze, K., Kreckel, M., Stuerenburg, H.J., Buhmann, C. and Beisiegel, U. (2001) Characterization of four lipoprotein classes in human cerebrospinal fluid. J. Lipid Res., 42, 1143–1151.[Abstract/Free Full Text]

  58. Pitas, R.E., Boyles, J.K., Lee, S.H., Hui, D. and Weisgraber, K.H. (1987) Lipoproteins and their receptors in the central nervous system. Characterization of the lipoproteins in cerebrospinal fluid and identification of apolipoprotein B,E(LDL) receptors in the brain. J. Biol. Chem., 262, 14352–14360.[Abstract/Free Full Text]

  59. Roheim, P.S., Carey, M., Forte, T. and Vega, G.L. (1979) Apolipoproteins in human cerebrospinal fluid. Proc. Natl Acad. Sci. USA, 76, 4646–4649.[Abstract/Free Full Text]

  60. Thorngate, F.E., Rudel, L.L., Walzem, R.L. and Williams, D.L. (2000) Low levels of extrahepatic nonmacrophage ApoE inhibit atherosclerosis without correcting hypercholesterolemia in ApoE-deficient mice. Arterioscler. Thromb. Vasc. Biol., 20, 1939–1945.[Abstract/Free Full Text]


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D. R. Riddell, H. Zhou, K. Atchison, H. K. Warwick, P. J. Atkinson, J. Jefferson, L. Xu, S. Aschmies, Y. Kirksey, Y. Hu, et al.
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