Introduction of human apolipoprotein E4 "domain interaction" into mouse apolipoprotein E - PubMed (original) (raw)

Introduction of human apolipoprotein E4 "domain interaction" into mouse apolipoprotein E

R L Raffai et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Human apolipoprotein E4 (apoE4) binds preferentially to lower density lipoproteins, including very low density lipoproteins, and is associated with increased risk of atherosclerosis and neurodegenerative disorders, including Alzheimer's disease. This binding preference is the result of the presence of Arg-112, which causes Arg-61 in the amino-terminal domain to interact with Glu-255 in the carboxyl-terminal domain. ApoE2 and apoE3, which have Cys-112, bind preferentially to high density lipoproteins (HDL) and do not display apoE4 domain interaction. Mouse apoE, like apoE4, contains the equivalent of Arg-112 and Glu-255, but lacks the critical Arg-61 equivalent (it contains Thr-61). Thus, mouse apoE does not display apoE4 domain interaction and, as a result, behaves like human apoE3, including preferential binding to HDL. To assess the potential role of apoE4 domain interaction in atherosclerosis and neurodegeneration, we sought to introduce apoE4 domain interaction into mouse apoE. Replacing Thr-61 in mouse apoE with arginine converted the binding preference from HDL to very low density lipoproteins in vitro, suggesting that apoE4 domain interaction could be introduced into mouse apoE in vivo. Using gene targeting in embryonic stem cells, we created mice expressing Arg-61 apoE. Heterozygous Arg-61/wild-type apoE mice displayed two phenotypes found in human apoE4/E3 heterozygotes: preferential binding to lower density lipoproteins and reduced abundance of Arg-61 apoE in the plasma, reflecting its more rapid catabolism. These findings demonstrate the successful introduction of apoE4 domain interaction into mouse apoE in vivo. The Arg-61 apoE mouse model will allow the effects of apoE4 domain interaction in lipoprotein metabolism, atherosclerosis, and neurodegeneration to be determined.

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Figures

Figure 1

Figure 1

Generation and characterization of an Arg-61 Apoe allele. (A) The sequence-replacement, gene-targeting strategy. A 7-kb Eco_RI-BstB_1 fragment containing exons 1, 2, and 3 and 5′ flanking sequences was inserted upstream of a neo cassette flanked by_loxP sites. A 1.3-kb fragment containing exon 4 and 3′ flanking sequences was amplified by PCR and cloned into the_Asc_1 site downstream of the neo cassette. Homologous recombination between the gene-targeting vector and the_Apoe locus in ES cells introduced a G-for-C change in the second position of codon 61 at the 3′ end of exon 3 and introduced a unique _Dde_I restriction site. A neo cassette flanked by loxP sites was placed in close proximity to the novel Arg-61 codon in intron 3 to monitor the correct integration of the point mutation. Targeted ES cell clones and mice were identified by digestion of genomic DNA with Eco_RI and Southern blotting with an Apoe exon 4 probe, which revealed an expanded 10.3-kb fragment vs. a wt 8.3-kb fragment. Cre-mediated excision of the neo cassette produces a mutant Apoe allele (designated Arg-61_Apoe).

Figure 2

Figure 2

Plasma lipoprotein-binding preferences of human apoE and mouse apoE. Recombinant mouse apoE and human apoE isoforms were radiolabeled, incubated with human plasma, and fractionated into various lipoprotein classes by size-exclusion FPLC.

Figure 3

Figure 3

Comparison of apoE mRNA levels in various tissues and organs from wt (+) mice and Arg-61-targeted mice crossed with Cre-deleter mice (R). (A) Total RNA from various tissues and organs was isolated from wt and Agr-61 mice and subjected to Northern blot analysis with an_Apoe_ exon 4 probe. Tissues and organs included: B, brain; Li, liver; Lu, lung; M, muscle; Si, small intestine; T, testis; H, heart; St, stomach; K, kidney; Sk, skin; Sp, spleen; and E, eye. (B) A control mouse β-actin hybridization is shown.

Figure 4

Figure 4

IEF and Western blot of mouse plasma and CSF apoE. Mouse apoE isoforms in plasma and CSF were monitored by IEF and Western blot detection. Mice were fasted for 4 h before bleeding.

Figure 5

Figure 5

IEF and Western blot of apoE secreted from primary hepatocytes into the culture medium. Primary hepatocytes were isolated from wt and Arg-61/wt heterozygous mice and placed into culture for 2 days, and the medium was collected. Mouse wt and Arg-61 apoE in medium were assessed by IEF and Western blot detection.

Figure 6

Figure 6

Distribution of wt and Arg-61 apoE in plasma from a heterozygous, gene-targeted mouse on a high-cholesterol diet. Arg-61/wt apoE heterozygous mice were fed the Paigen diet for 6 days to increase VLDL levels. Nonfasted plasma was fractionated by size-exclusion FPLC. (Upper) Fractions corresponding to the different lipoprotein classes were resolved by IEF, and apoE was detected by Western blotting.

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