Neurotoxic Mechanisms Caused by the Alzheimer's Disease-linked Swedish Amyloid Precursor Protein Mutation
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m212265200
ISSN1083-351X
AutoresCelio A. Marques, Uta Keil, Astrid Bonert, Barbara Steiner, Christian Haass, Wernér E.G. Müller, Anne Eckert,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoAutosomal dominant forms of familial Alzheimer's disease (FAD) are caused by mutations of the amyloid precursor protein (APP) gene and by mutations of the genes encoding for presenilin 1 or presenilin 2. Simultaneously, evidence is provided that increased oxidative stress might play a crucial role in the rapid progression of the Swedish FAD. Here we investigated the effect of the Swedish double mutation (K670M/N671L) in the β-amyloid precursor protein on oxidative stress-induced cell death mechanisms in PC12 cells. Western blot analysis and cleavage studies of caspase substrates revealed an elevated activity of the executor caspase 3 after treatment with hydrogen peroxide in cells containing the Swedish APP mutation. This elevated activity is the result of the enhanced activation of both intrinsic and extrinsic apoptosis pathways, including activation of caspase 2 and caspase 8. Furthermore, we observed an enhanced activation of JNK pathway and an attenuation of apoptosis by SP600125, a JNK inhibitor, through protection of mitochondrial dysfunction and reduction of caspase 9 activity. Our findings provide evidence that the massive neurodegeneration in early age of FAD patients could be a result of an increased vulnerability of neurons through activation of different apoptotic pathways as a consequence of elevated levels of oxidative stress. Autosomal dominant forms of familial Alzheimer's disease (FAD) are caused by mutations of the amyloid precursor protein (APP) gene and by mutations of the genes encoding for presenilin 1 or presenilin 2. Simultaneously, evidence is provided that increased oxidative stress might play a crucial role in the rapid progression of the Swedish FAD. Here we investigated the effect of the Swedish double mutation (K670M/N671L) in the β-amyloid precursor protein on oxidative stress-induced cell death mechanisms in PC12 cells. Western blot analysis and cleavage studies of caspase substrates revealed an elevated activity of the executor caspase 3 after treatment with hydrogen peroxide in cells containing the Swedish APP mutation. This elevated activity is the result of the enhanced activation of both intrinsic and extrinsic apoptosis pathways, including activation of caspase 2 and caspase 8. Furthermore, we observed an enhanced activation of JNK pathway and an attenuation of apoptosis by SP600125, a JNK inhibitor, through protection of mitochondrial dysfunction and reduction of caspase 9 activity. Our findings provide evidence that the massive neurodegeneration in early age of FAD patients could be a result of an increased vulnerability of neurons through activation of different apoptotic pathways as a consequence of elevated levels of oxidative stress. Alzheimer's disease is the most frequent neurodegenerative disorder in the aged population and is characterized by the presence of senile plaques and neurofibrillary tangles in the brain. Senile plaques are deposits of amyloid-β-protein (Aβ), 1The abbreviations used are: Aβ, amyloid-β-protein; APP, amyloid precursor protein; AD, Alzheimer's disease; FAD, familial Alzheimer's disease; APPsw, Swedish double mutation form of APP; APPwt, wild-type APP; JNK, c-Jun N-terminal kinase; pNA, para-nitroaniline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PI, propidium iodide; ANOVA, analysis of variance; CHO, aldehyde. which arises by proteolysis from the amyloid precursor protein (APP) through an initial β-secretase cleavage by the aspartyl protease beta-site APP-cleaving enzyme (1Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. 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Furthermore, we investigated the protective effects of caspase inhibitors as well as JNK inhibitor in preventing oxidative stress-mediated cell death. Materials—Phospho-JNK (Thr183/Tyr185) and phospho-c-Jun (Ser63) antibodies were purchased from Cell Signaling Technology, and cytochrome c antibody was from BD Biosciences. Rat-specific caspase 3 and caspase 9 antibodies were also purchased from Cell Signaling Technology. Caspase 2 antibody was purchased from Alexis. Caspase 8 antibody was from Biocat. The caspase substrates were purchased from Calbiochem (Ac-DEVD-para-nitroaniline (pNA), Ac-VEID-pNA, Ac-VDVAD-pNA, and Ac-IETD-pNA for caspase 3, caspase 6, caspase 2, and caspase 8, respectively). JNK inhibitor II (SP600125; anthrax(1,9-cd)pyrazol-6(2H)-one) was from Calbiochem. Cell Culture and Transfection—The generation and characterization of PC12 cell lines expressing human APPwt-, mutant APP (APPsw)-, and vector-transfected control clones have been described previously (12Eckert 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). In brief, PC12 cells were transfected with DNA constructs harboring human mutant (APPsw; K670M/N671L) or wild-type APP (APPwt) gene, inserted downstream of a cytomegalovirus promotor, using the FUGENE technique (Roche Applied Science). The transfected cell lines APPwt PC12, APPsw PC12, and vector-transfected PC12 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 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 stably transfected clones (APPwt M5, N10, and U7 and APPsw Q8 and Q9) were selected for the present study based on their similar expression of APP695 and the 5-fold increased secretion of Aβ-(1–40) in the APPsw clones (Fig. 1). To rule out potential influences on the APP expression levels, APPsw and APPwt clones were routinely screened in parallel to the experiments presented in this work. No alterations in the expression of APP were found. Detection of Aβ—For the detection of secreted Aβ-(1–40), we used a specific sandwich enzyme-linked immunosorbent assay employing monoclonal antibodies as described previously (50Steiner H. Capell A. Pesold B. Citron M. Kloetzel P.M. Selkoe D.J. Romig H. Mendla K. Haass C. J. Biol. Chem. 1998; 273: 32322-32331Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Oxidative Stress-induced Cell Death—Cell death was induced with hydrogen peroxide (freshly prepared solution; 250 μm; Sigma). In respective experiments, caspase inhibitors were added 3 h before exposure to H2O2 (250 μm, 24 h) at a final concentration of 10 μm: caspase 3 inhibitor (AC-DEVD-CMK), caspase 6 inhibitor (VEID-CHO), caspase 8 inhibitor (IETD-CHO), and caspase 9 inhibitor (LEHD-CHO). Only cell-permeable caspase inhibitors were used. Measurement of Caspase Activity—PC12 cells were plated the day before at a density of 5 × 106 cells/culture dish (10 cm diameter). After treatment with H2O2, the cells were harvested, and after a centrifugation step, the culture medium was aspirated. The cell-containing pellet was washed with PBS and lysed in 100 μl of lysis buffer (10 mm HEPES, 0.1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 0.1 mm EDTA, 1 mm dithiothreitol, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 0.1% CHAPS, pH 7.4). Lysates were centrifuged at 20,000 × g for 10 min at 4 °C, and the supernatant was used for caspase assay. The approximate protein amount of the probes was confirmed by a Lowry protein test. Caspase activity was measured by cleavage of colorimetric substrates (Ac-DEVD-pNA, Ac-VEID-pNA, Ac-VDVAD-pNA, Ac-IETD-pNA, and Ac-LEHD-pNA for caspase 3, caspase 6, caspase 2, caspase 8, and caspase 9, respectively). The production of pNA was monitored over 30 min in a photometer (λ = 405 nm). The caspase activity is expressed as change in absorption units. One unit was defined as the amount of enzyme required to cleave 1 pmol of NA per min of incubation per 5 × 106 cells. Quantification of Apoptosis by Flow Cytometry—Apoptosis was determined by propidium iodide (PI) staining and fluorescence-activated cell sorting analysis as described previously (12Eckert 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). Briefly, H2O2-treated PC12 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 (Becton Dickinson). Cells with a lower DNA content showing less propidium iodide staining than G1 have been defined as apoptotic cells (sub- G1 peak). Phospho-JNK Pathway Western Blot Analysis—PC12 cells were plated at a density of 5 × 106 cells/culture dish (10 cm diameter) and treated with H2O2. Cells were harvested and lysed in SDS sample buffer: Tris-HCl (62.5 mm, pH 6.8), 2% SDS, 10% glycerol, 50 mm dithiothreitol, 0.01% bromphenol blue. After sonicating and boiling, proteins were separated by electrophoresis on a 16% polyacrylamide gel. The separated proteins were transferred to a polyvinylidene difluoride membrane (ImmobilonTM-P; Millipore). These membranes were stained with Ponceau S red (reversible stain) to visualize the proteins. Nonspecific sites on the membranes were blocked with 5% fat-free milk in Tris-buffered saline, 0.1% Tween 20 (TBST). The phosphorylated JNK was visualized with the appropriate primary phosphoantibody, phospho-stress-activated protein kinase/JNK (Thr183/Tyr185), which detects stress-activated protein kinase/JNK only dually phosphorylated at Thr183/Tyr185. The phosphorylated c-Jun was detected by a phospho-c-Jun (Ser63) antibody. After thorough washing with TBST, membranes were covered with ECL™ detection reagents and quickly exposed to an autoradiography film. Membranes were routinely stripped for actin control. Caspase Immunoblot Analysis—PC12 cells were plated at a density of 5 × 106 cells/culture dish (10 cm diameter) and treated with H2O2. After the indicated periods of time, the medium was aspirated, and cells were processed for Western blotting with rat-specific cleaved caspase 3, caspase 8, and caspase 9 antibodies. Blots were scanned (Umax Astra 4000 U), and the band intensity was determined after background subtraction using densitometric analysis. Determination of Cytochrome c Release—The amount of cytochrome c released from the mitochondrial intermembrane space into the cytosol was determined by digitonin permeabilization (51Gottlieb R.A. Granville D.J. Methods. 2002; 26: 341-347Crossref PubMed Scopus (69) Google Scholar). Briefly, 5 × 106 cells were exposed to oxidative stress for different periods of time. After washing with ice-cold phosphate-buffered saline, cells were resuspended in permeabilization buffer containing 75 mm NaCl, 1 mm NaH2PO4, 8 mm Na2PO4, 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 mitochondria and cellular debris. The supernatant containing cytoplasmic proteins was purified by centrifugation at 13,000 × g at 4 °C for 10 min. Equal amounts of protein (10 μg) were loaded on an 18% acrylamide gel and separated by SDS-PAGE. After immunoblotting with a monoclonal cytochrome c antibody, polyvinylidene difluoride membranes were stripped and reprobed either for COX4 or actin, ensuring equal protein loading and the absence of mitochondrial contamination. Determination of Mitochondrial Membrane Potential—PC12 cells were plated the day before at a density of 2 × 105 cells/well in a 24-well plate. The cells were pretreated for 1 h with the JNK inhibitor SP600125, and H2O2 was added for 6 h. The membrane potential of the inner mitochondrial membrane was measured using the dye rhodamine 123. The dye was added to the cell culture medium at 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; PerkinElmer Life Sciences). Transmembrane distribution of the dye depends on the mitochondrial membrane potential (Δψm). Statistical Analysis—Data are given as mean ± S.E. For statistical comparison, paired t test, Student's t test, or one-way ANOVA followed by Tukey's post hoc test or two-way ANOVA were used. p values less than 0.05 were considered statistically significant. Generation and Characterization of PC12 Cell Lines Overexpressing APPwt and APPsw—To investigate the effects of APP overexpression in oxidative stress-induced apoptosis, we used several clones of PC12 cells stably transfected with APP cDNAs. Similar expression of human APP in the investigated cell lines was confirmed by Western blotting. As expected, the human-specific antibody does not recognize any APP species in vector-transfected PC12 cells (Fig. 1A), whereas equal expression levels of human APP were detected in clones of APPwt cells and in clones of cells containing the Swedish mutation. Culture supernatants of the APP-transfected cells were also analyzed for Aβ-(1–40) production revealing 4–5-fold elevated Aβ-(1–40) levels in clones of APPsw cells compared with clones of APPwt cells (Fig. 1B). APPsw Mutation Leads to an Enhanced Vulnerability to Oxidative Stress-induced Apoptosis—We induced apoptosis in transfected PC12 cells using the oxidative stressor hydrogen peroxide. Nuclear DNA fragmentation was quantitatively detected by propidium iodide staining and flow cytometry. Very importantly, clones bearing the APPsw mutation are sensitized to an exposure to oxidative stress for 24 h in a concentration-dependent manner with a maximum increase of apoptotic cells at a concentration of 500 μm H2O2 (12Eckert 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). Treatment with H2O2 led to a significantly enhanced maximum increase of apoptotic cells in APPsw-bearing PC12 cells (51.4 ± 5.3%) compared with APPwt-bearing cells (28.4 ± 4.7%) and vector control cells (21.3% ± 4.3%). Different clones with the same transfectant behave similarly in their sensitivity to H2O2-induced cell death (Fig. 1C). Oxidative Stress Induces Activation of Caspase 2 in PC12 Cells—Oxidative stress in the human brain has been implicated as one major cause of neuronal cell loss in AD patients. However, the exact mechanism still remains unknown. Aβ appears to bind redox-active metals like zinc, copper, or iron with high affinity, resulting in production of hydrogen peroxide and autoxidation of the metallopeptide complex (52Rottkamp C.A. Raina A.K. Zhu X. Gaier E. Bush A.I. Atwood C.S. Chevion M. Perry G. Smith M.A. Free Radic. Biol. Med. 2001; 30: 447-450Crossref PubMed Scopus (362) Google Scholar, 53Tabner B.J. Turnbull S. El Agnaf O.M. Allsop D. Free Radic. Biol. Med. 2002; 32: 1076-1083Crossref PubMed Scopus (227) Google Scholar). The signaling cascades activated following the oxidative stress have not been widely studied. Therefore, we studied the implication of caspase 2 by measuring the activity and by Western blotting. Cleavage of the photometric substrate Ac-VDVAD-p-nitroanilide by cytosolic protein extracts indicates the presence of caspase 2 protease activity in the cultures after exposure to hydrogen peroxide. The activity was already induced early in APPsw cells within 2 h of induction (Fig. 2A). Caspase 2 activity was continuously elevated over time compared with APPwt and vector-transfected cells. Maximum caspase 2 activity was measured after 2 h, with a 4-fold higher activity in APPsw cells than in APPwt and vector PC12 cells, respectively (Fig. 2B). The mechanism of activation of caspases occurs by a sequential cleavage of the zymogen to release the large and the small cleavage products. In accordance with our findings from the determination of activity, Western blotting confirmed cleavage of caspase 2 within 2 h of hydrogen peroxide induction (Fig. 2C). Again, the activation of caspase 2 was noticeably higher in APPsw cells compared with vector-transfected controls. Involvement of Caspase 8 —Apoptosis associated with the Fas/tumor necrosis factor receptor family of death receptors requires caspase 8 activity and adaptor proteins such as FADD. The involvement of caspase 8 in processing of APP during apoptosis by caspase 8 has been reported recently (46Pellegrini L. Passer B.J. Tabaton M. Ganjei J.K. D'Adamio L. J. Biol. Chem. 1999; 274: 21011-21016Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The blocking of neuronal death by caspase 8 inhibitor IETD-fmk in Aβ-induced cell death was also demonstrated (54Ivins K.J. Thornton P.L. Rohn T.T. Cotman C.W. Neurobiol. Dis. 1999; 6: 440-449Crossref PubMed Scopus (182) Google Scholar). Hence, we examined the activation of caspase 8 in our transfected cell lines overexpressing intracellular high levels of Aβ in order to ascertain the involvement of this initiator caspase in oxidative stress. We utilized Ac-IETD-pNA to measure caspase 8 activity in lysates of treated cells. In parallel to the early activation of caspase 2, caspase 8 activity increased about 3-fold after 2 h of stress induction in PC12 cells expressing the Swedish double mutation and was significantly enhanced in a time-dependent manner compared with vector transfected controls (Fig. 3, A and B). Activation of caspase 8 involves a two-step proteolysis: the cleavage of the procaspase 8 to g
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