Reactivity of Apolipoprotein E4 and Amyloid β Peptide
2005; Elsevier BV; Volume: 281; Issue: 5 Linguagem: Inglês
10.1074/jbc.m506646200
ISSN1083-351X
AutoresZhong-Sheng Ji, Karin Müllendorff, Irene H. Cheng, R. Dennis Miranda, Yadong Huang, Robert W. Mahley,
Tópico(s)Computational Drug Discovery Methods
ResumoWe previously demonstrated that apolipoprotein E4 (apoE4) potentiates lysosomal leakage and apoptosis induced by amyloid β (Aβ) peptide in cultured Neuro-2a cells and hypothesized that the low pH of lysosomes accentuates the conversion of apoE4 to a molten globule, inducing reactive intermediates capable of destabilizing cellular membranes. Here we report that neutralizing lysosomal pH with bafilomycin or NH4Cl abolished the apoE4 potentiation of Aβ-induced lysosomal leakage and apoptosis in Neuro-2a cells. Consistent with these results, apoE4 at acidic pH bound more avidly to phospholipid vesicles and disrupted them to a greater extent than at pH 7.4. Comparison of "Arctic" mutant Aβ, which forms multimers, and GM6 mutant Aβ, which remains primarily monomeric, showed that aggregation is essential for apoE4 to potentiate Aβ-induced lysosomal leakage and apoptosis. Both apoE4 and Aβ1–42 had to be internalized to exert these effects. Blocking the low density lipoprotein receptor-related protein with small interfering RNA abolished the enhanced effects of apoE4 and Aβ on lysosomes and apoptosis. In cultured Neuro-2a cells, Aβ1–42 increased lysosome formation to a greater extent in apoE3- or apoE4-transfected cells than in Neo-transfected cells, as shown by immunostaining for lysosome-associated membrane protein 1. Similarly, in transgenic mice expressing apoE and amyloid precursor protein, hippocampal neurons displayed increased numbers of lysosomes. Thus, apoE4 and Aβ1–42 may work in concert in neurons to increase lysosome formation while increasing the susceptibility of lysosomal membranes to disruption, release of lysosomal enzymes into the cytosol, and neuronal degeneration. We previously demonstrated that apolipoprotein E4 (apoE4) potentiates lysosomal leakage and apoptosis induced by amyloid β (Aβ) peptide in cultured Neuro-2a cells and hypothesized that the low pH of lysosomes accentuates the conversion of apoE4 to a molten globule, inducing reactive intermediates capable of destabilizing cellular membranes. Here we report that neutralizing lysosomal pH with bafilomycin or NH4Cl abolished the apoE4 potentiation of Aβ-induced lysosomal leakage and apoptosis in Neuro-2a cells. Consistent with these results, apoE4 at acidic pH bound more avidly to phospholipid vesicles and disrupted them to a greater extent than at pH 7.4. Comparison of "Arctic" mutant Aβ, which forms multimers, and GM6 mutant Aβ, which remains primarily monomeric, showed that aggregation is essential for apoE4 to potentiate Aβ-induced lysosomal leakage and apoptosis. Both apoE4 and Aβ1–42 had to be internalized to exert these effects. Blocking the low density lipoprotein receptor-related protein with small interfering RNA abolished the enhanced effects of apoE4 and Aβ on lysosomes and apoptosis. In cultured Neuro-2a cells, Aβ1–42 increased lysosome formation to a greater extent in apoE3- or apoE4-transfected cells than in Neo-transfected cells, as shown by immunostaining for lysosome-associated membrane protein 1. Similarly, in transgenic mice expressing apoE and amyloid precursor protein, hippocampal neurons displayed increased numbers of lysosomes. Thus, apoE4 and Aβ1–42 may work in concert in neurons to increase lysosome formation while increasing the susceptibility of lysosomal membranes to disruption, release of lysosomal enzymes into the cytosol, and neuronal degeneration. Alzheimer disease (AD) 2The abbreviations used are: AD, Alzheimer disease; apo, apolipoprotein; Aβ, amyloid β; APP, amyloid precursor protein; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine; LRP, low density lipoprotein receptor-related protein; MEM, minimum essential medium; NSE, neuron-specific enolase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; siRNA, small interfering RNA. is a debilitating neurodegenerative disease. With no effective treatment available, the incidence of AD is likely to increase as the population continues to age. Although in most cases the exact cause is unknown, two proteins have been implicated in its pathogenesis: apolipoprotein E4 (apoE4) and amyloid β (Aβ). ApoE has important functions in lipid transport in the blood and in the redistribution of lipids among cells in the brain (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3388) Google Scholar). One of its three common isoforms (2Mahley R.W. Rall Jr., S.C. Annu. 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Reagents—Heparinase I, Lucifer Yellow, bafilomycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were from Sigma. 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) was from Avanti (Alabaster, AL), Aβ1–42 from Bachem (Torrance, CA), and LysoSensor DND-160 was from Molecular Probes, Inc. (Eugene, OR). Rabbit anti-LRP antibody was a gift from Dr. E. Koo (University of California, San Diego, CA). LAMP1 (lysosome-associated membrane protein 1) monoclonal antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Recombinant human apoE3 and apoE4 (provided by Karl H. Weisgraber) were prepared as described (52Morrow J.A. Hatters D.M. Lu B. Höchtl P. Oberg K.A. Rupp B. Weisgraber K.H. J. Biol. Chem. 2002; 277: 50380-50385Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). 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They were chemically synthesized by Dharmacon (Lafayette, CO), according to the following sequences: si-LRP6600, sense 5′-UGGCAUCUCAGUAGACUAUUU-3′ and antisense 5′-AUAGUCUACUGAGAUGCCAUU-3′; si-LRP12348, sense 5′-UGUGUACUGGACCGAUUCAUU-3′ and antisense 5′-UGAAUCGGUCCAGUACACAUU-3′. For transfection, Neuro-2a cells were seeded in 6-well plates and allowed to reach 60% confluence. After 24 h, cells were transfected with siRNA (1.5 μg/ml) with Lipofectamine (Invitrogen) according to the manufacturer's instructions. The transfection complex was diluted in Opti-MEM (final volume, 1 ml), which was replaced after 4 h. Aβ1–42 (20 μm) was added 48 h after transfection. Cells were lysed for immunoblotting at 72 h. After blocking of LRP expression with siRNA, other cells were homogenized, and the cytosol was isolated by centrifugation and assayed for hexosaminidase activity. Some cells were also examined for apoptosis by measuring cellular DNA fragmentation with cell death detection enzyme-linked immunosorbent assay kits (Roche Applied Science). For LAMP1 immunocytochemistry, cells were fixed in ethyl alcohol. Measurement of Lysosomal Membrane Stability—The stability of lysosomal membranes was assessed by measuring the leakage of the lysosomal enzyme β-hexosaminidase or the concentrated lysosomal fluorescent dye Lucifer Yellow into the cytosol of Neuro-2a cells. The cells were incubated with Lucifer Yellow (100 μg/ml) for 16 h, washed three times, and incubated with Aβ1–42 at 37 °C for 8–40 h. After incubation, the cells were examined by confocal microscopy (Radiance 2000 KR3; Bio-Rad) for Lucifer Yellow in the cytosol. The percentage of cells showing diffuse fluorescence indicative of lysosomal leakage was determined by counting 10–20 cells in each of 12–20 fields at ×60 magnification. β-Hexosaminidase was measured as described (63Kaback M.M. Shapiro L.J. Hirsch P. Roy C. Prog. Clin. Biol. Res. 1977; 18: 267-279PubMed Google Scholar). Alteration of Lysosomal pH—The cells were prelabeled with LysoSensor DND-160 (2 μm) for 16 h at 37 °C. LysoSensor DND-160 accumulates in lysosomes and, at the low pH in lysosomes, is robustly fluorescent with an emission peak of 540 nm and an excitation wavelength of 365 nm. The labeled cells were incubated with bafilomycin or NH4Cl to raise the pH of endosomes and lysosomes (64Kane M.D. Schwarz R.D. St. Pierre L. Watson M.D. Emmerling M.R. Boxer P.A. Walker G.K. J. Neurochem. 1999; 72: 1939-1947Crossref PubMed Scopus (20) Google Scholar, 65Schrader-Fischer G. Paganetti P.A. Brain Res. 1996; 716: 91-100Crossref PubMed Scopus (65) Google Scholar, 66Yu Z. Persson H.L. Eaton J.W. Brunk U.T. Free Radic. Biol. Med. 2003; 34: 1243-1252Crossref PubMed Scopus (198) Google Scholar). As the pH of the lysosomes approaches neutral levels, the fluorescence intensity of the LysoSensor DND-160 is greatly reduced (67Lin H.-J. Herman P. Kang J.S. Lakowicz J.R. Anal. Biochem. 2001; 294: 118-125Crossref PubMed Scopus (124) Google Scholar). Fluorescence intensity was monitored with a BD FACS Vantage SE flow cytometer (BD Biosciences). Turbidity Clearance of DMPC—DMPC was dissolved in benzene, lyophilized, resuspended in 10 mm Tris-HCl, 5.5% KBr, 1 mm EDTA (pH 7.4 or 4.0), and preincubated in water at 23.9 °C. DMPC multilamellar vesicles (0.5 ml, 500 μg/ml) were added to a cuvette, and 250 μg of apoE was added to the solution. The decrease in turbidity was monitored and recorded with a Hitachi F-2000 spectrofluorometer; excitation and emission were set at 600 nm. Aβ Preparation—Except where noted, Aβ1–42 was solubilized in Me2SO and water and incubated at 37 °C for 24–48 h, as described (51Ji Z.-S. Miranda R.D. Newhouse Y.M. Weisgraber K.H. Huang Y. Mahley R.W. J. Biol. Chem. 2002; 277: 21821-21828Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In studies that compared Arctic and GM6 Aβ, the Aβ was prepared as described for Aβ-derived diffusible ligands (34Lambert M.P. Barlow A.K. Chromy B.A. Edwards C. Freed R. Liosatos M. Morgan T.E. Rozovsky I. Trommer B. Viola K.L. Wals P. Zhang C. Finch C.E. Krafft G.A. Klein W.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6448-6453Crossref PubMed Scopus (3128) Google Scholar). Briefly, each form of Aβ was dissolved in cold hexafluoroisopropanol, incubated at room temperature for 1 h, and centrifuged to remove hexafluoroisopropanol. The Aβ was dried, resuspended in a small amount of Me2SO, diluted in water, and incubated at 4 °C for 24 h. The Aβ solution was centrifuged at 14,000 × g for 10 min, the supernatant was collected, and the protein concentration was determined. To assess Aβ aggregation, each Aβ sample (100 ng) was separated on Tris-Tricine (Bio-Rad) gels and transferred to a nitrocellulose membrane. The membrane was incubated with anti-Aβ monoclonal antibody (3D6). Aβ was visualized by horseradish peroxidase-conjugated secondary antibody and ECL reagent (Amersham Biosciences). Internalization of 125I-Aβ in Neuro-2a Cells—Neo-, apoE3-, and apoE4-transfected cells were grown to about 90% confluence in MEM containing 10% fetal bovine serum, washed with serum-free medium, and incubated with 125I-Aβ (3 μg/ml) at 37 °C for 24 h. The cells were then placed on ice. After five washes with phosphate-buffered saline containing 0.2% bovine serum albumin and two with phosphate-buffered saline, the cells were washed with 10 mm suramin at 4 °C for 30 min, and radioactivity was counted with a γ-counter. Wild type and mutant Aβ were iodinated with Iodogen (Pierce). Aβ (0.5 mg) was incubated with Iodogen beads on ice for 15 min, and the 125I-labeled Aβ was dialyzed against phosphate-buffered saline. Nonspecific cell association was determined by adding a 50-fold excess of unlabeled Aβ. DNA Fragmentation Assay—Apoptotic cell death was measured with a cellular DNA fragmentation assay as described (51Ji Z.-S. Miranda R.D. Newhouse Y.M. Weisgraber K.H. Huang Y. Mahley R.W. J. Biol. Chem. 2002; 277: 21821-21828Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). LAMP1 Immunoblotting and Immunostaining—Neo- and apoE-transfected cells were incubated with or without Aβ for 0, 8, or 16 h. The cells were then lysed for LAMP1 immunoblotting or fixed in 100% cold ethyl alcohol for 15 min for immunostaining. Proteins (40 μg) from lysed cells were separated on SDS gels and transferred to nitrocellulose membranes, and the LAMP1 immunoreactive bands were scanned. For immunostaining, the cells were treated with Triton X-100 (0.1%) and Tween 20 (0.005%) in 10 ml of 10 mm Tris-HCl and 150 mm NaCl buffer. Nonspecific reactivity was blocked with 10% goat serum and incubated with rat anti-LAMP1 antibody (1 μg of IgG/ml). Fluorescein-labeled goat anti-rat IgG was used as the second antibody. LAMP1 quantitation, as determined by immunoactivity, was measured with a BioQuant image analysis system (R&M Biometrics, Nashville, TN). Briefly, the pixel number (intensity) of immunofluorescence in each cell from Neo- and apoE-transfected cells was measured. The mean pixel number of immunofluorescence was obtained from 10 cells randomly chosen for each condition. LAMP1 immunocytochemistry was also performed on brain sections of Apoe-/- mice and transgenic mice expressing apoE3 or apoE4 under control of the neuron-specific enolase (NSE) promoter (68Buttini M. Orth M. Bellosta S. Akeefe H. Pitas R.E. Wyss-Coray T. Mucke L. Mahley R.W. J. Neurosci. 1999; 19: 4867-4880Crossref PubMed Google Scholar), APP (69Mucke L. Masliah E. Yu G.-Q. Mallory M. Rockenstein E.M. Tatsuno G. Hu K. Kholodenko D. Johnson-Wood K. McConlogue L. J. Neurosci. 2000; 20: 4050-4058Crossref PubMed Google Scholar), or APP and either apoE3 or apoE4 (70Raber J. Wong D. Buttini M. Orth M. Bellosta S. Pitas R.E. Mahley R.W. Mucke L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10914-10919Crossref PubMed Scopus (304) Google Scholar, 71Buttini M. Akeefe H. Lin C. Mahley R.W. Pitas R.E. Wyss-Coray T. Mucke L. Neuroscience. 2000; 97: 207-210Crossref PubMed Scopus (103) Google Scholar). All of the mice were on an Apoe-/- background and studied at ∼12 months of age. The construct for the APP mice was platelet-derived growth factor-APP with the Swedish and Indiana mutations (K670N/M671L and V717F, respectively) (69Mucke L. Masliah E. Yu G.-Q. Mallory M. Rockenstein E.M. Tatsuno G. Hu K. Kholodenko D. Johnson-Wood K. McConlogue L. J. Neurosci. 2000; 20: 4050-4058Crossref PubMed Google Scholar). Immunocytochemistry was performed on 50-μm free floating vibratome sections of brains fixed in paraformaldehyde. The staining procedure is described above. The sections were examined under a BX-60 fluorescence microscope (Olympus, Melville, NY). The intensity of LAMP1 immunofluorescence in the CA3 region of the hippocampus was quantitated with a BioQuant image analysis system, which measures the area of the CA3 region (183,302–197,677 μm2) and determines the mean number of pixels of LAMP1 immunofluorescence in each area. Co-culture of ApoE-transfected C6 Cells and Neuro-2a Cells—Neo-transfected Neuro-2a cells were grown in 6-well culture plates or chamber slides. ApoE-transfected and nontransfected C6 astrocytic cells were grown in separate cell culture inserts with pores (0.3 μm in diameter) in the bottom, which allows free exchange of culture medium through the pores (BD Biosciences). The inserts were placed in culture dishes containing Neuro-2a cells. Aβ1–42 (20 μm) was added to the Neuro-2a cells, and incubation was continued for 24 h. The cells were fixed in cold 10
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