Amyloid β-induced Changes in Nitric Oxide Production and Mitochondrial Activity Lead to Apoptosis
2004; Elsevier BV; Volume: 279; Issue: 48 Linguagem: Inglês
10.1074/jbc.m405600200
ISSN1083-351X
AutoresUta Keil, Astrid Bonert, Celio A. Marques, Isabel Scherping, Jörg Weyermann, Joanna B. Strosznajder, F. Müller‐Spahn, Christian Haass, Christian Czech, Laurent Pradier, Wernér E.G. Müller, Anne Eckert,
Tópico(s)Dementia and Cognitive Impairment Research
ResumoIncreasing evidence suggests an important role of mitochondrial dysfunction in the pathogenesis of Alzheimer's disease. Thus, we investigated the effects of acute and chronic exposure to increasing concentrations of amyloid β (Aβ) on mitochondrial function and nitric oxide (NO) production in vitro and in vivo. Our data demonstrate that PC12 cells and human embryonic kidney cells bearing the Swedish double mutation in the amyloid precursor protein gene (APPsw), exhibiting substantial Aβ levels, have increased NO levels and reduced ATP levels. The inhibition of intracellular Aβ production by a functional γ-secretase inhibitor normalizes NO and ATP levels, indicating a direct involvement of Aβ in these processes. Extracellular treatment of PC12 cells with comparable Aβ concentrations only leads to weak changes, demonstrating the important role of intracellular Aβ. In 3-month-old APP transgenic (tg) mice, which exhibit no plaques but already detectable Aβ levels in the brain, reduced ATP levels can also be observed showing the in vivo relevance of our findings. Moreover, we could demonstrate that APP is present in the mitochondria of APPsw PC12 cells. This presence might be directly involved in the impairment of cytochrome c oxidase activity and depletion of ATP levels in APPsw PC12 cells. In addition, APPsw human embryonic kidney cells, which produce 20-fold increased Aβ levels compared with APPsw PC12 cells, and APP tg mice already show a significantly decreased mitochondrial membrane potential under basal conditions. We suggest a hypothetical sequence of pathogenic steps linking mutant APP expression and amyloid production with enhanced NO production and mitochondrial dysfunction finally leading to cell death. Increasing evidence suggests an important role of mitochondrial dysfunction in the pathogenesis of Alzheimer's disease. Thus, we investigated the effects of acute and chronic exposure to increasing concentrations of amyloid β (Aβ) on mitochondrial function and nitric oxide (NO) production in vitro and in vivo. Our data demonstrate that PC12 cells and human embryonic kidney cells bearing the Swedish double mutation in the amyloid precursor protein gene (APPsw), exhibiting substantial Aβ levels, have increased NO levels and reduced ATP levels. The inhibition of intracellular Aβ production by a functional γ-secretase inhibitor normalizes NO and ATP levels, indicating a direct involvement of Aβ in these processes. Extracellular treatment of PC12 cells with comparable Aβ concentrations only leads to weak changes, demonstrating the important role of intracellular Aβ. In 3-month-old APP transgenic (tg) mice, which exhibit no plaques but already detectable Aβ levels in the brain, reduced ATP levels can also be observed showing the in vivo relevance of our findings. Moreover, we could demonstrate that APP is present in the mitochondria of APPsw PC12 cells. This presence might be directly involved in the impairment of cytochrome c oxidase activity and depletion of ATP levels in APPsw PC12 cells. In addition, APPsw human embryonic kidney cells, which produce 20-fold increased Aβ levels compared with APPsw PC12 cells, and APP tg mice already show a significantly decreased mitochondrial membrane potential under basal conditions. We suggest a hypothetical sequence of pathogenic steps linking mutant APP expression and amyloid production with enhanced NO production and mitochondrial dysfunction finally leading to cell death. Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid β peptide; APP, amyloid precursor protein; FAD, familial Alzheimer's disease; APPsw, Swedish double mutation form of APP; APPwt, wild-type APP; NO, nitric oxide; NOS, nitric-oxide synthase; ELISA, enzyme-linked immunosorbent assay; HEK, human embryonic kidney; tg, transgenic; COX, cytochrome c oxidase; ROS, reactive oxygen species; Ψm, mitochondrial membrane potential; Smac, second mitochondria-derived activator of caspase; AIF, apoptosis-inducing factor; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TMRE, tetramethylrhodamineethyl ester; ANOVA, analysis of variance; DAPT, N-[N-(3,5-trifluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. is the most common neurodegenerative disorder (1Yankner B.A. Neuron. 1996; 16: 921-932Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar) marked by progressive loss of memory and cognitive ability. 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On the basis of these findings, we set out to investigate the precise mechanisms underlying the action of Aβ and/or expression of mutant APP on mitochondrial function in multiple experimental designs mimicking different in vivo situations that are discussed to occur in AD patients: 1) in cell lines overexpressing different levels of Aβ (APPsw PC12 cells exhibiting low physiological concentrations of Aβ within picomolar range and APPsw HEK cells expressing Aβ levels within low nanomolar range) to study dose-dependent effects of Aβ in an in vitro setting characterized by chronic Aβ production due to increased APP processing; 2) after extracellular Aβ treatment to distinguish chronic from acute and/or extracellular actions from effects of intracellular Aβ or APP-processing products on mitochondria; and 3) in a secondary insult model to examine the additional impact of oxidative stress. Finally, we checked the in vivo relevance of our in vitro findings by studying mitochondrial function in brain cells from APP transgenic (tg) mice. Materials—Rhodamine 123 and tetramethylrhodamineethylester (TMRE) were purchased from Molecular Probes. 4,5-Diaminofluorescein diacetate was obtained from Calbiochem. ViaLight HT kit was purchased from Cambrex. Cytochrome c oxidase assay kit, hydrogen peroxide, rotenone, thenoyltrifluoroacetone, antimycin, sodium azide (NaN3), and oligomycin were obtained from Sigma. l-NAME was purchased from Cayman. Nω-propyl-l-arginine and 1400W were purchased from Biotrend. Aβ1–42 was supplied by Bachem. DAPT was obtained from Merck Biosciences. Aβ1–42 was dissolved in Tris-buffered saline (pH 7.4) at a concentration of 1 mm and stored at -20 °C. The stock solution was diluted in Tris-buffered saline to the desired concentrations and incubated at 37 °C for 24 h to have aged preparations of Aβ1–42. Cell Culture and Transfection—PC12 cells and HEK cells were transfected with DNA constructs harboring human mutant APP (APPsw, K670M/N671L) gene, the APPwt gene, inserted downstream of a cytomegalovirus promoter using the FUGENE 6 technique (Roche Diagnostics) (37Eckert A. Steiner B. Marques C. Leutz S. Romig H. Haass C. Muller W.E. J. Neurosci. Res. 2001; 64: 183-192Crossref PubMed Scopus (89) Google Scholar). The transfected cells APPwt PC12, APPsw PC12, and control PC12 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum and 5% heat-inactivated horse serum, 50 units/ml penicillin, 50 μg/ml streptomycin, and 400 μg/ml G418 at 37 °C in a humidified incubator containing 5% CO2. The transfected cell lines APPwt HEK, APPsw HEK, and control HEK were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 50 units/ml penicillin, 50 μg/ml streptomycin, and 400 μg/ml G418 at 37 °C in a humidified incubator containing 5% CO2. The Aβ levels of APP-transfected PC12 cells and HEK cells are shown in Table I.Table IAβ secretion of PC12 cells and HEK cells in pg/ml medium unter basal conditions and after incubation with DAPT One-way ANOVA, post hoc Tukeys multiple comparison test. Values are means ± S.E. from 3–4 experiments.CoAPPwtAPPswAPPsw + DAPT (24 h 1 μm)APPsw + DAPT (48 h 1 μm)PC12 cells3.67 ± 1.5217.33 ± 3.1897.33 ± 9.56ap < 0.001 versus control PC12 and HEK cells19.81 ± 0.40bp < 0.001 versus untreated APPsw cells19.44 ± 0.41bp < 0.001 versus untreated APPsw cellsHEK cells35.47 ± 1.16259.1 ± 7.91ap < 0.001 versus control PC12 and HEK cells2598 ± 26.29ap < 0.001 versus control PC12 and HEK cells73.69 ± 0.65bp < 0.001 versus untreated APPsw cells15.77 ± 0.15bp < 0.001 versus untreated APPsw cellsa p < 0.001 versus control PC12 and HEK cellsb p < 0.001 versus untreated APPsw cells Open table in a new tab Detection of Aβ levels—For the detection of secreted Aβ1–40, a specific sandwich enzyme-linked immunosorbent assay (ELISA) employing monoclonal antibodies was used. The ELISA was performed according to the instructions given in the Abeta-ELISA kit by BIOSOURCE. The assay principle is that of a standard sandwich ELISA, which utilizes a monoclonal mouse anti-human Abeta1–16 capture antibody, a cleavage site-specific rabbit anti-human Abeta1–40 C-terminal detection antibody, and anti-rabbit IgG peroxidase-conjugated secondary antibody. Transgenic Animal Brain Tissue—Female C57BL/6 mice bearing the human Swedish and London mutations in the 751 amino acid form of human amyloid precursor protein (tg APP) under the control of a murine Thy-1 promoter at an age of 3 months and non-transgenic littermate animals were used for the experiments (38Wirths O. Multhaup G. Czech C. Feldmann N. Blanchard V. Tremp G. Beyreuther K. Pradier L. Bayer T.A. Brain Pathol. 2002; 12: 275-286Crossref PubMed Scopus (114) Google Scholar). APP tg mice exhibited the onset of Aβ plaques at an age of 6 months, but intracellular Aβ load was already detectable at the age of 3 months (39Blanchard V. Moussaoui S. Czech C. Touchet N. Bonici B. Planche M. Canton T. Jedidi I. Gohin M. Wirths O. Bayer T.A. Langui D. Duyckaerts C. Tremp G. Pradier L. Exp. Neurol. 2003; 184: 247-263Crossref PubMed Scopus (249) Google Scholar). Mice were sacrificed by decapitation, and brains were quickly dissected on ice (method modified after Stoll et al. (40Stoll L. Schubert T. Muller W.E. Neurobiol. Aging. 1992; 13: 39-44Crossref PubMed Scopus (47) Google Scholar)). After removing the cerebellum, the tissue was minced into 2 ml of medium I (138 NaCl, 5.4 KCl, 0.17 Na2HPO4, 0.22 K2PO4, 5.5 glucose, and 58.4 sucrose, all in mmol/liter, pH 7.35) with a scalpel and further dissociated by trituration through a nylon mesh (pore diameter 1 mm) with a Pasteur pipette. The resulting suspension was filtered by gravity through a fresh nylon mesh with a pore diameter of 102 μm, and the dissociated cell aggregates were washed twice with medium II (110 NaCl, 5.3 KCl, 1.8 CaCl2·H2O, 1 MgCl2·6 H2O, 25 glucose, 70 sucrose, and 20 HEPES, all in mmol/l, pH 7.4) by centrifugation (400 × g for 3 min at 4 °C). 100 μl of the suspension were used for protein determination. After centrifugation, cells were resuspended in 6 ml of Dulbecco's modified Eagle's medium and 500 μl/well were distributed on a 24-well plate for the measurement of mitochondrial membrane potential. For the measurement of ATP levels, 100 μl/well were distributed on a white 96-well plate. The preparations of APP tg mice and non-transgenic littermate mice (overcross design) were made under the same conditions and in parallel. Quantification of Apoptosis by Flow Cytometry—Apoptosis was determined by propidium iodide staining and fluorescence-activated cell sorter analysis as described previously (37Eckert A. Steiner B. Marques C. Leutz S. Romig H. Haass C. Muller W.E. J. Neurosci. Res. 2001; 64: 183-192Crossref PubMed Scopus (89) Google Scholar). PC12 cells and HEK cells were lysed in buffer (0.1% sodium citrate and 0.1% Triton X-100) containing 50 μg/ml propidium iodide. Samples were analyzed by flow cytometry (FACSCalibur) using Cell Quest software (BD Biosciences). Cells with a lower DNA content showing less propidium iodide staining than G1 have been defined as apoptotic cells (sub-G1 peak). Determination of Intracellular Nitric Oxide Levels—PC12 cells and HEK cells were plated the day before at a density of 2 × 105 cells/well in a 24-well plate. To measure the NO levels, the fluorescence dye 4,5-diaminofluorescein diacetate was used in a concentration of 10 μm for 30 min (41Kojima H. Hirata M. Kudo Y. Kikuchi K. Nagano T. J Neurochem. 2001; 76: 1404-1410Crossref PubMed Scopus (44) Google Scholar). The cells were washed twice with Hanks' balanced salt solution, and the fluorescence was determined with a fluorescence reader (Victor® multilabel counter) at 490/535 nm. NO levels were also determined after a 48-h incubation in the absence or in the presence of the NO synthase inhibitors 20 mm l-NAME, 1 mmNω-propyl-l-arginine, and 1 mm 1400W. Determination of the Mitochondrial Membrane Potential (Ψm)—PC12 cells and HEK cells were plated the day before at a density of 2 × 105 cells/well in a 24-well plate. PC12 cells were incubated with H2O2 (0.5 mm) for different periods of time. The mitochondrial membrane potential was measured using the fluorescence dye Rhodamine 123 (42Baracca A. Sgarbi G. Solaini G. Lenaz G. Biochim. Biophys. Acta. 2003; 1606: 137-146Crossref PubMed Scopus (410) Google Scholar). Transmembrane distribution of the dye depends on the mitochondrial membrane potential. The dye was added to the cell culture medium in a concentration of 0.4 μm for 15 min. The cells were washed twice with Hanks' balanced salt solution, and the fluorescence was determined with a fluorescence reader (Victor multilabel counter) at 490/535 nm. The mitochondrial membrane potential of dissociated neurons was also measured with Rhodamine 123 in a concentration of 0.4 μm for 15 min. For detailed information regarding the preparation, see “Transgenic Animal Brain Tissue.” To test acute and fast changes in Ψm, the fluorescence dye TMRE (43Collins T.J. Berridge M.J. Lipp P. Bootman M.D. EMBO J. 2002; 21: 1616-1627Crossref PubMed Scopus (464) Google Scholar) was used in a concentration of 0.4 μm for 15 min. TMRE exhibits a characteristic increase in fluorescence at 490/590 nm after challenging mitochondria with membrane potential-decreasing drugs (44Krohn A.J. Wahlbrink T. Prehn J.H. J. Neurosci. 1999; 19: 7394-7404Crossref PubMed Google Scholar). The mitochondrial membrane potential was recorded, and then the complex inhibitors (2 μm rotenone, 10 μm thenoyltrifluoroacetone, 2 μm antimycin, 10 μm oligomycin, and 10 mm sodium azide) were added. Determination of ATP Levels with a Bioluminescence Assay (ViaLight HT)—PC12 cells and HEK cells were plated the day before at a density of 2 × 104 cells/well in a white 96-well plate. PC12 cells were incubated for different periods of time with H2O2 (0.1 mm). The ATP levels of dissociated neurons were also measured with the bioluminescence assay. For detailed information regarding this preparation, see “Transgenic Animal Brain Tissue.” The kit is based upon the bioluminescent measurement of ATP (45Crouch S.P. Kozlowski R. Slater K.J. Fletcher J. J. Immunol. Methods. 1993; 160: 81-88Crossref PubMed Scopus (739) Google Scholar). The bioluminescent method utilizes an enzyme, luciferase, which catalyzes the formation of light from ATP and luciferin. The emitted light is linearly related to the ATP concentration and is measured using a luminometer. Confocal Microscopy—The amount of mitochondria was measured using the cell-permanent mitochondrion-selective dye Mitotracker Red. This probe can accumulate in active mitochondria and then react with accessible thiol groups of proteins and peptides to form fluorescent aldehydic-fixable conjugates. PC12 cells were plated the day before at a density of 2 × 105 cells/chamber slide. Cells were incubated for 30 min at 37 °C with Mitotracker Red or for 15 min with Rhodamine 123. Cells were washed twice with phosphate-buffered saline, fixed with 2% paraformaldehyde for 15 min, and then washed twice with phosphate-buffered saline. The samples were embedded in Mowiol and analyzed using a laser-scanning confocal microscope. Isolation of Cytosolic and Mitochondrial Fractions—Cytosolic and mitochondrial fractions were isolated by digitonin permeabilization (46Gottlieb R.A. Granville D.J. Methods. 2002; 26: 341-347Crossref PubMed Scopus (69) Google Scholar). 5 × 106 cells were washed with ice-cold phosphate-buffered saline, and cells were resuspended for 15 min on ice in permeabilization buffer containing 75 mm NaCl, 1 mm NaH2PO4, 250 mm sucrose, 1 mm phenylmethylsulfonyl fluoride, additional protease inhibitors, and 0.05% digitonin. Following a centrifugation step at 800 × g at 4 °C for 10 min, the supernatant was separated from the pellet consisting of cellular debris. The crude mitochondrial pellet was collected by centrifugation at 13,000 × g at 4 °C for 10 min. The supernatant containing cytoplasmic proteins was stored at -20 °C for further investigation. The pellet consisting of mitochondria was dissolved in phosphate-buffered saline for the cytochrome c oxidase assay and resuspended in 0.1% Triton X-100 and mechanically lysed for the Western blot. The total protein content was determined by the method of Lowry (Bio-Rad). Determination of Cytochrome c Oxidase Activity in Isolated Mitochondria with Cytochrome c Oxidase Assay Kit (47Rasmussen U.F. Rasmussen H.N. Mol. Cell Biochem. 2000; 208: 37-44Crossref PubMed Google Scholar)—The colorimetric assay is based on the observation of the decrease in absorbance at 550 nm of ferrocytochrome c caused by its oxidation to ferricytochrome c by cytochrome c oxidase. The cytochrome c oxidase assay was performed according to the instructions given in the kit. Western Blot—After determination of the total protein content by the method of Lowry, the cytosolic and mitochondrial fraction was mixed with 4× Laemmli sample buffer and denatured for 10 min at 95 °C. An equal amount (10–20 μg) of protein was loaded per lane on acrylamide gels and examined by SDS-PAGE. Dependent on the protein to be detected, we performed glycine or Tricine Western blots with acrylamide amounts of 10–18% at 90 V for 2–3 h. For proteins with a molecular mass <30 kDa, Tricine Western blots were more convenient. The proteins were transferred onto polyvinylidene difluoride membranes (Amersham Biosciences) at 25 V for 90 min. After this procedure, membranes were saturated with 5% nonfat dry milk in Tris-buffered saline with Tween 20 for 1 h after antibody exposure. The polyvinylidene difluoride membranes then were exposed to the following antibodies overnight: rabbit anti-Bcl-xL (Cell Signaling); rabbit anti-Bax (Cell Signaling); rabbit anti-apoptosis-inducing factor (AIF) (Chemicon); rabbit anti-Smac (Chemicon); rabbit anti-cytochrome c (BD Biosciences), mouse anti-COX4 (BD Biosciences); goat anti-actin (Santa Cruz Biotechnology); rabbit anti-calreticulin (Chemicon); mouse anti-Na+/K+-ATPase (Chemicon); rabbit anti-nitrotyrosine (Merck Biosciences); and mouse anti-human APP W02 (Abeta; W02 antibody is directed against amino acids 4–10 of the human Aβ sequence, thereby detecting the mature and immature APP695). After treatment for 1 h with the corresponding horseradish peroxidase-coupled secondary antibodies (Merck Biosciences), the protein bands were detected by ECL reagent (Amersham Biosciences). After detection, the membranes were treated with stripping buffer (100 mm glycine, pH 2.5) for 2 h after reprobing with different antibodies. Statistical Analysis—The data are given as the mean ± S.E. For statistical comparison, Student's t test and one-way ANOVA followed by Tukey's post hoc test, repeated measures ANOVA, or two-way ANOVA were used. p values < 0.05 were considered statistically significant. APP Expression and Aβ Levels of APP-transfected PC12 Cells and HEK Cells—Increasing knowledge was obtained that Aβ is produced intracellularly (48Bayer T.A. Wirths O. Majtenyi K. Hartmann T. Multhaup G. Beyreuther K. Czech C. Brain Pathol. 2001; 11: 1-11Crossref PubMed Scopus (163) Google Scholar) and that intraneuronal accumulation of Aβ precedes plaque formation in APP tg mice bearing the Swedish double mutation (49Wirths O. Multhaup G. Czech C. Blanchard V. Moussaoui S. Tremp G. Pradier L. Beyreuther K. Bayer T.A. Neurosci. Lett. 2001; 306: 116-120Crossref PubMed Scopus (289) Google Scholar, 50Hartmann T. Eur. Arch. Psychiatry Clin. Neurosci. 1999; 249: 291-298Crossref PubMed Scopus (64) Google Scholar). Thus, intracellular accumulation of Aβ might be a primary step in the neurotoxicity cascade of Aβ in vivo and in vitro. APP containing the Swedish mutation resulted in an overall increase of Aβ including both Aβ1–40 and Aβ1–42. In our cell model, the APPsw mutation resulted in a markedly increased Aβ secretion of PC12 cells (0.20 nmol/liter) as well as in HEK cells (5.42 nmol/liter) compared with APPwt PC12 cells and APPwt HEK cells. APPsw PC12 cells secreted low Aβ levels, reflecting the physiological situation in vivo during cellular metabolism (7Citron M. Oltersdorf T. Haass C. McConlogue L. Hung A.Y. Seubert P. Vigo-Pelfrey C. Lieberburg I. Selkoe D.J. Nature. 1992; 360: 672-674Crossref PubMed Scopus (1535) Google Scholar), whereas human APP was equally expressed in APPwt and APPsw PC12 cells and was increased 2-fold compared with the endogenous APP expression of control PC12 cells (37Eckert A. Steiner B. Marques C. Leutz S. Romig H. Haass C. Muller W.E. J. Neurosci. Res. 2001; 64: 183-192Crossref PubMed Scopus (89) Google Scholar). Of note, HEK cells bearing the APPsw mutation exhibited an ∼20-fold increased Aβ secretion compared with APPsw-bearing PC12 cells (Table I), a scenario that might occur in familial AD and that could explain the more drastic effects in some assay designs. Furthermore, this might indicate that, in APPsw HEK cells, the secretion pathway is more pronounced than in PC12 cells. The endogenous human APP expression of control HEK cells was increased 2-fold compared with APPs
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