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.2005 Dec 20;102(51):18694-9.
doi: 10.1073/pnas.0508254102. Epub 2005 Dec 12.

Lipid- and receptor-binding regions of apolipoprotein E4 fragments act in concert to cause mitochondrial dysfunction and neurotoxicity

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Lipid- and receptor-binding regions of apolipoprotein E4 fragments act in concert to cause mitochondrial dysfunction and neurotoxicity

Shengjun Chang et al. Proc Natl Acad Sci U S A..

Abstract

Apolipoprotein (apo) E4, a 299-aa protein and a major risk factor for Alzheimer's disease, can be cleaved to generate C-terminal-truncated fragments that cause neurotoxicity in vitro and neurodegeneration and behavioral deficits in transgenic mice. To investigate this neurotoxicity, we expressed apoE4 with C- or N-terminal truncations or mutations in transfected Neuro-2a cells. ApoE4 (1-272) was neurotoxic, but full-length apoE4(1-299) and apoE4(1-240) were not, suggesting that the lipid-binding region (amino acids 241-272) mediates the neurotoxicity and that amino acids 273-299 are protective. A quadruple mutation in the lipid-binding region (I250A, F257A, W264R, and V269A) abolished the neurotoxicity of apoE4(1-272), and single mutations in the region of amino acids 273-299 (L279Q, K282A, or Q284A) made full-length apoE4 neurotoxic. Immunofluorescence staining showed that apoE4(1-272) formed filamentous inclusions containing phosphorylated tau in some cells and interacted with mitochondria in others, leading to mitochondrial dysfunction as determined by MitoTracker staining and flow cytometry. ApoE4(241-272) did not cause mitochondrial dysfunction or neurotoxicity, suggesting that the lipid-binding region alone is insufficient for neurotoxicity. Truncation of N-terminal sequences (amino acids 1-170) containing the receptor-binding region (amino acids 135-150) and triple mutations within that region (R142A, K146A, and R147A) abolished the mitochondrial interaction and neurotoxicity of apoE4(1-272). Further analysis showed that the receptor-binding region is required for escape from the secretory pathway and that the lipid-binding region mediates mitochondrial interaction. Thus, the lipid- and receptor-binding regions in apoE4 fragments act together to cause mitochondrial dysfunction and neurotoxicity, which may be important in Alzheimer's disease pathogenesis.

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Figures

Fig. 1.
Fig. 1.
The lipid- and receptor-binding regions in apoE4 fragments act in concert to cause neurotoxicity, as determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (a) Survival of cells transfected with WT apoE4, apoE4(1-272), apoE4(1-240), or apoE4(1-191). (b) Survival of cells transfected with WT apoE4, apoE4(1-272), or apoE4(1-272) with four mutations (I250A, F257A, W264R, and V269A). (c) Survival of cells transfected with WT apoE4, apoE4(1-272), or apoE4 with single mutations (L279Q, K282A, or Q284A). (d) Survival of cells transfected with WT apoE4, apoE4(1-272), or apoE(241-272). (e) Survival of cells transfected with WT apoE4, apoE4(1-272), apoE4(87-272), apoE(127-272), or apoE(171-272). (f) Survival of cells transfected with WT apoE4, apoE4(1-272), or apoE4(1-272) with double (K146A and R147A) or triple (R142A, K146A, and R147A) mutations. Values are given as mean ± SD of three to six assays at 48 h after transfection.*,P < 0.05 vs. WT apoE4.
Fig. 2.
Fig. 2.
Intracellular distribution of various forms of apoE4 as determined by immunocytochemistry and confocal microscopy. (a) Cells transfected with WT apoE4, permeabilized with Tween 20, and stained with anti-apoE (green). (b) Cells transfected with apoE4(1-272), permeabilized with Tween 20, and stained with anti-apoE (green). (c) Cells cotransfected with apoE4(1-272) and DsRed2-Mito (red), permeabilized with STP-O, and stained with anti-apoE (green). Yellow in the merged image indicates colocalization of apoE4(1-272) with mitochondria.
Fig. 3.
Fig. 3.
The lipid and receptor-binding regions act in concert to cause mitochondrial mislocalization of apoE4 fragments. Cells transfected with WT apoE4 (a), apoE(171-272) (c), apoE4(1-240) (e), apoE4(1-272)-AARA with four mutations (I250A, F257A, W264R, and V269A) in the lipid-binding region (g), or apoE4(1-272)-3A with three mutations (R142A, K146A, and R147A) in the receptor-binding region (i) were permeabilized with 0.5% Tween 20 (a, c, e, g, andi) and stained with anti-apoE (green). Cells cotransfected with DsRed2-Mito (red) and various apoE4 constructs mentioned above were permeabilized with 500 units of STP-O (b, d, f, h, andj) and stained with anti-apoE (green). The cells were then analyzed by confocal microscopy for only green (a, c, e, g, andi) or both red and green (b, d, f, h, andj).
Fig. 4.
Fig. 4.
The receptor-binding region is required to escape the secretory pathway, and the lipid-binding region mediates mitochondrial interaction. Cells transfected with apoE(171-272) without signal peptide (a) or apoE4(1-191) without signal peptide (c) were permeabilized with 0.5% Tween 20 and stained with anti-apoE (green). Cells cotransfected with DsRed2-Mito (red) and either of those two apoE4 constructs were permeabilized with 500 units of STP-O (b andd) and stained with anti-apoE (green). The cells were analyzed as in Fig. 3.
Fig. 5.
Fig. 5.
The lipid- and receptor-binding regions in apoE4 fragments act in concert to cause mitochondrial dysfunction, as determined by MitoTracker Deep Red 633 staining and flow cytometry. (a) Effects of various forms of apoE4, expressed at similar levels, on mitochondrial function and integrity. (b) Effect on mitochondrial function and integrity depends on expression levels of apoE4 fragments, as measured by fluorescence intensity (FI) of GFP. Values are given as mean ± SD of three to six assays.*,P < 0.05 vs. WT apoE4, E4(171-272), and E4(1-272)-3A. E4(1-272)-3A, apoE4(1-272) with a triple mutation in the receptor-binding region.
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