Effect of domain interaction on apolipoprotein E levels in mouse brain - PubMed (original) (raw)

Comparative Study

Effect of domain interaction on apolipoprotein E levels in mouse brain

Gayathri Ramaswamy et al. J Neurosci. 2005.

Abstract

Apolipoprotein (apo) E4 is a risk factor for heart disease, Alzheimer's disease, and other forms of neurodegeneration, but the underlying mechanisms are unknown. Domain interaction, a structural property that distinguishes apoE4 from apoE2 and apoE3, results in more rapid turnover and lower plasma levels of apoE4. To determine whether domain interaction affects brain apoE levels, we analyzed brain homogenates from human apoE3 and apoE4 knock-in mice, wild-type mice, and Arg-61 apoE mice, in which domain interaction was introduced by gene targeting. As determined on Western blots, the hemibrain, cortex, hippocampus, and cerebellum of knock-in mice had 30-40% lower levels of apoE4 than apoE3, and Arg-61 mice had 25-50% lower apoE levels than wild-type mice. In the CSF, Arg-61 apoE level was 40% lower than the wild-type level. Arg-61 apoE mRNA levels were similar to or slightly higher than wild-type apoE mRNA levels. Thus, the lower Arg-61 apoE levels were not attributable to decreased mRNA levels. In culture medium from heterozygous Arg-61/wild-type and apoE4/apoE3 primary astrocytes, Arg-61 apoE and apoE4 levels were lower than wild-type apoE and apoE3, respectively, suggesting that primary astrocytes secrete lower amounts of Arg-61 apoE and apoE4. These results demonstrate that domain interaction is responsible for the lower levels of both human apoE4 and mouse Arg-61 apoE in mouse brain. Cells may recognize apoE4 and Arg-61 apoE as misfolded proteins and target them for degradation or accumulation. Thus, degradation/accumulation or lower levels of apoE4 may contribute to the association of apoE4 with Alzheimer's disease.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Specificity of mouse apoE antibody for wild-type (WT) and Arg-61 apoE. Purified mouse WT and Arg-61 apoE (250 and 500 ng) were subjected to SDS-PAGE and Western blotting with an antibody against full-length mouse apoE.

Figure 2.

Figure 2.

ApoE levels in human apoE knock-in mouse brains. The hemibrain, cortex, hippocampus, and cerebellum from 5-month-old female human apoE3 and apoE4 knock-in mice were subjected to SDS-PAGE and Western blotting with an antibody against full-length human apoE.

Figure 3.

Figure 3.

ApoE levels in wild-type (WT) and Arg-61 apoE mouse brains. The hemibrain, cortex, hippocampus, and cerebellum from 5-month-old WT and Arg-61 apoE homozygous female mice were subjected to SDS-PAGE and Western blotting with an antibody against fulllength WT mouse apoE.

Figure 4.

Figure 4.

ApoE levels in 1- and 2-year-old wild-type (WT) and Arg-61 apoE mouse brains. The hemibrains from 1- and 2-year-old WT and Arg-61 apoE homozygous female mice were subjected to SDS-PAGE and Western blotting with an antibody to full-length mouse apoE.

Figure 5.

Figure 5.

ApoE levels in CSF. CSF from 5-month-old heterozygous Arg-61/wild-type (WT) apoE heterozygous mice was subjected to isoelectric focusing and Western blotting.

Figure 6.

Figure 6.

ApoE mRNA levels in mouse brain. Total RNA from the hemibrain (A), cortex (B), and hippocampus (C) was extracted from 5-month-old male and female wild-type (WT) and Arg-61 apoE homozygous mice (n = 6 each) and analyzed for apoE mRNA levels by reverse transcription, followed by real-time PCR. The apoE mRNA levels were normalized to 18s mRNA. Error bars represent values ± SEM.

Figure 7.

Figure 7.

ApoE levels in conditioned medium from primary astrocytes. Primary astrocytes from 3-d-old Arg-61/wild-type (WT) apoE (A) and apoE4/apoE3 (B) heterozygous pups were cultured in serum-free medium for 72 h, and the conditioned medium was subjected to isoelectric focusing and Western blotting.

Similar articles

Cited by

References

    1. Bao F, Arai H, Matsushita S, Higuchi S, Sasaki H (1996) Expression of apolipoprotein E in normal and diverse neurodegenerative disease brain. NeuroReport 7: 1733-1739. - PubMed
    1. Beffert U, Cohn JS, Petit-Turcotte C, Tremblay M, Aumont N, Ramassamy C, Davignon J, Poirier J (1999) Apolipoprotein E and β-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. - PubMed
    1. Bertrand P, Poirier J, Oda T, Finch CE, Pasinetti GM (1995) Association of apolipoprotein E genotype with brain levels of apolipoprotein E and apolipoprotein J (clusterin) in Alzheimer disease. Mol Brain Res 33: 174-178. - PubMed
    1. Boschert U, Merlo-Pich E, Higgins G, Roses AD, Catsicas S (1999) Apolipoprotein E expression by neurons surviving excitotoxic stress. Neurobiol Dis 6: 508-514. - PubMed
    1. Boyles JK, Pitas RE, Wilson E, Mahley RW, Taylor JM (1985) Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system. J Clin Invest 76: 1501-1513. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources