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. 2008 Dec;118(12):4002-13.
doi: 10.1172/JCI36663. Epub 2008 Nov 13.

apoE isoform-specific disruption of amyloid beta peptide clearance from mouse brain

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Free PMC article

apoE isoform-specific disruption of amyloid beta peptide clearance from mouse brain

Rashid Deane et al. J Clin Invest. .
Free PMC article

Abstract

Neurotoxic amyloid beta peptide (Abeta) accumulates in the brains of individuals with Alzheimer disease (AD). The APOE4 allele is a major risk factor for sporadic AD and has been associated with increased brain parenchymal and vascular amyloid burden. How apoE isoforms influence Abeta accumulation in the brain has, however, remained unclear. Here, we have shown that apoE disrupts Abeta clearance across the mouse blood-brain barrier (BBB) in an isoform-specific manner (specifically, apoE4 had a greater disruptive effect than either apoE3 or apoE2). Abeta binding to apoE4 redirected the rapid clearance of free Abeta40/42 from the LDL receptor-related protein 1 (LRP1) to the VLDL receptor (VLDLR), which internalized apoE4 and Abeta-apoE4 complexes at the BBB more slowly than LRP1. In contrast, apoE2 and apoE3 as well as Abeta-apoE2 and Abeta-apoE3 complexes were cleared at the BBB via both VLDLR and LRP1 at a substantially faster rate than Abeta-apoE4 complexes. Astrocyte-secreted lipo-apoE2, lipo-apoE3, and lipo-apoE4 as well as their complexes with Abeta were cleared at the BBB by mechanisms similar to those of their respective lipid-poor isoforms but at 2- to 3-fold slower rates. Thus, apoE isoforms differentially regulate Abeta clearance from the brain, and this might contribute to the effects of APOE genotype on the disease process in both individuals with AD and animal models of AD.

Figures

Figure 1. apoE isoform–specific clearance across the mouse BBB in vivo.
(A) Time-disappearance curves of 14C-inulin (reference molecule, black) and 125I-labeled human lipid-poor apoE4 (dark green), apoE3 (light green), apoE2 (yellow green), Aβ42 (dark blue), and Aβ40 (light blue) after microinfusion of tracers mixture into brain ISF in the caudate nucleus. Test tracers were studied at 40 nM. The percentage recovery in brain was calculated using Equation 1 (see Methods). TCA-precipitable 125I-radioactivity was used. Each point represents a single experiment. (B) Time-dependent efflux across the BBB of 125I-labeled Aβ40, Aβ42, lipid-poor apoE2, apoE3, and apoE4 (yellow green, light green, dark green) and lipo-apoE2 (brown), lipo-apoE3 (red), and lipo-apoE4 (orange) was calculated from data in Figure 1A and Equation 4 (see Methods). The ISF bulk flow for studied test tracers was calculated using Equation 2 (see Methods). (C) Relative contributions of transport across the BBB (black bars), ISF flow (white bars), and degradation (dark gray bars) to clearance of apoE isoforms from brain and their retention in the brain (light gray bars) were studied at 40 nM concentrations and calculated from fractional coefficients given in Supplemental Table 1. Mean ± SEM; n = 11–24 mice per group for multiple-time series. *P < 0.05, lipid-poor apoE4 versus lipid-poor apoE3 or apoE2; P < 0.05, lipo-apoE4, lipo-apoE3, and lipo-apoE2 versus corresponding lipid-poor apoE4, apoE2 and apoE3. P < 0.05, lipo-apoE4 versus lipo-apoE3 or lipo-apoE2. (D and E) Time-appearance curves of 14C-inulin and 125I-labeled lipid-poor apoE4, apoE3, and apoE2 (TCA-precipitable 125I-radioactivity) in the CSF (D) and plasma (E) from experiments as in A. ID, injected dose. §P < 0.05, apoE2, apoE3, and apoE4 versus inulin; P < 0.05, apoE4 versus apoE2 or apoE3. Mean ± SEM; n = 3–5 mice per group.
Figure 2. apoE isoform–specific clearance across the mouse BBB in vivo depends on differential contributions of VLDLR-mediated and LRP1-mediated transport.
(A) 125I-labeled lipo-apoE2, lipo-apoE3, and lipo-apoE4 (TCA-precipitable 125I-radioactivity) BBB clearance at 90 minutes in the presence and absence of receptor-specific blocking antibodies against VLDLR, LRP1, and LDLR and excess unlabeled ligands at 0.5 μM. (B) Western blot analysis of VLDLR, LDLR, and LRP1 in brain microvessels isolated from control, VLDLR–/–, and LDLR–/– mice. β-actin was used as a loading control. The lanes were run on the same gel but were noncontiguous. Representative blots from 3 mice per group are shown. (C and D) 125I-labeled lipo-apoE2, lipo-apoE3, and lipo-apoE4 (TCA-precipitable 125I-radioactivity) BBB clearance at 90 minutes in VLDLR–/– (C) and LDLR–/– mice (D) in the presence and absence of receptor-specific antibodies against VLDLR, LRP1, or LDLR. Values are mean ± SEM; n = 3–5 mice per group.
Figure 3. apoE isoforms disrupt Aβ clearance across the mouse BBB in vivo (apoE4>apoE3 or apoE2) by redirecting differentially redirecting transport of Aβ-apoE complexes from LRP1 to VLDLR.
125I-labeled apoE-Aβ complexes (40 nM) and 14C-inulin were microinfused into brain ISF and clearance determined at 90 minutes. 125I-label was either on Aβ40 and Aβ42 or on apoE2 and apoE4. (A) FPLC purification of apoE2-Aβ40. Upper panel shows dot blots of Aβ40-apoE2 and free Aβ peaks with Aβ-specific (6E10) and apoE-specific (3D12) antibodies. (B and C) BBB clearance of Aβ40 (B) and Aβ42 (C) with and without an LRP1-specific blocking antibody and of their complexes with lipid-poor and lipo-apoE2 and lipid-poor and lipo-apoE4, as indicated. (D) Clearance of Aβ40 and Aβ42 by transport across the BBB (black bars), ISF flow (white bars) and degradation (light gray bars) and retention in the brain (dark gray bars) studied from different 125I-Aβ40-apoE and Aβ42-apoE complexes at 40 nM and compared with free Aβ40 or Aβ42. 125I-label was on Aβ. Clearance and retention were calculated from fractional coefficients using Equations 2, 5, and 6 (see Methods). Mean ± SEM, n = 5–6 mice per group in a single time-point series. *P < 0.05, Aβ40-apoE2 and Aβ40-apoE4 versus Aβ40 and Aβ42–lipo-apoE2, Aβ42–lipo-apoE3, and Aβ42–lipo-apoE4 versus Aβ42; P < 0.05, Aβ40-apoE4 versus Aβ40-apoE2 and Aβ42–lipo-apoE4 versus Aβ42–lipo-apoE3 or Aβ42–lipo-apoE2; P < 0.05, Aβ40–lipo-apoE2 and Aβ40–lipo-apoE4 versus Aβ40-apoE2 and Aβ40-apoE4; §P < 0.05, Aβ40–lipo-apoE4 versus Aβ40–lipo-apoE3 or Aβ40–lipo-apoE2. (E) BBB clearance of 125I-Aβ40–lipo-apoE2 and 125I-Aβ40–lipo-apoE3 in control mice with and without blocking antibodies to VLDLR, LRP1, and LDLR. (F and G) BBB clearance of 125I-Aβ40–lipo-apoE2 (F) and 125I-Aβ42–lipo-apoE4 complexes (G) in control (white bars), VLDLR–/– (gray bars), and RAP–/– (black bars) mice with and without blocking antibodies to LRP1, VLDLR, and/or LDLR. Mean ± SEM; n = 4–6 mice per group.
Figure 4. Isoform-specific lipid-poor apoE clearance at the abluminal surface of mouse brain capillaries in vitro is regulated by differential internalization rates of VLDLR and LRP1.
(A) Specific binding of 125I-labeled lipid-poor apoE2, apoE3, and apoE4 (2 nM, TCA-precipitable 125I-radioactivity) by brain microvessels studied for a period of 30 minutes at 4°C with and without excess of unlabeled ligand at 0.5 μM. (BD) Time-dependent internalization of lipid-poor 125I-apoE2 (B), 125I-apoE3 (C), and 125I-apoE4 (D) on the abluminal surface of brain microvessels in the presence of receptor-specific blocking antibodies to LRP1 and VLDLR and excess of unlabeled ligand at 0.5 μM.
Figure 5. Isoform-specific lipo-apoE clearance at the abluminal surface of mouse brain capillaries in vitro is regulated by differential internalization rates of VLDLR and LRP1.
(A) Binding of 125I-labeled lipo-apoE2 and lipo-apoE4 (2 nM, TCA-precipitable 125I-radioactivity) to isolated brain microvessels. (B and C) Time-dependent internalization of 125I-labeled lipo-apoE2 (B) and lipo-apoE4 (C) in the presence of receptor-specific blocking antibodies against VLDLR and LRP1 and excess of unlabeled ligand at 0.5 μM. (D) Binding of 125I-labeled lipo-apoE2 to brain microvessels from control, VLDLR–/–, and LDLR–/– mice. (EG) Internalization of 125I-labeled lipo-apoE2 (E), lipo-apoE3 (F), and lipo-apoE4 (G) at the abluminal surface of brain microvessels from control (white bars) and VLDLR–/– (black bars) mice studied for a period of 30 minutes. Means ± SEM, n = 3 experiments per group.
Figure 6. apoE isoform–specific inhibition (apoE4>apoE3 and apoE2) of Aβ internalization at the abluminal surface of mouse brain capillaries in vitro is mediated by VLDLR.
(A) Specific binding of 125I-labeled Aβ40 and Aβ42 complexes with apoE2 and apoE4 at 4°C in the absence and presence of receptor-specific blocking antibodies to VLDLR, LDLR, or LRP1 and excess unlabeled ligand at 0.5 μM. (B) Internalization of 125I-Aβ40 in the absence and presence of receptor-specific blocking antibodies against LRP1 and of 125I-labeled Aβ40–lipo-apoE2, Aβ40–lipo-apoE3, and Aβ40–lipo-apoE4 complexes for a period of 30 minutes. (C) Internalization of 125I-labeled Aβ40, Aβ40–lipo-apoE2, Aβ40–lipo-apoE3, and Aβ40–lipo-apoE4 in the absence and presence of receptor-specific blocking antibodies against LRP1 and VLDLR. Means ± SEM; n = 3–5 experiments per group.

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