Fibrillar Amyloid-β Peptides Kill Human Primary Neurons via NADPH Oxidase-mediated Activation of Neutral Sphingomyelinase
2004; Elsevier BV; Volume: 279; Issue: 49 Linguagem: Inglês
10.1074/jbc.m404635200
ISSN1083-351X
AutoresArundhati Jana, Kalipada Pahan,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoAlzheimer's disease is a major illness of dementia characterized by the presence of amyloid plaques, neurofibrillary tangles, and extensive neuronal apoptosis. However, the mechanism behind neuronal apoptosis in the Alzheimer's-diseased brain is poorly understood. This study underlines the importance of neutral sphingomyelinase in fibrillar Aβ peptide-induced apoptosis and cell death in human primary neurons. Aβ1–42 peptides induced the activation of sphingomyelinases and the production of ceramide in neurons. Interestingly, neutral (N-SMase), but not acidic (A-SMase), sphingomyelinase was involved in Aβ1–42-mediated neuronal apoptosis and cell death. Aβ1–42-induced production of ceramide was redox-sensitive, as reactive oxygen species were involved in the activation of N-SMase but not A-SMase. Aβ1–42 peptides induced the NADPH oxidase-mediated production of superoxide radicals in neurons that was involved in the activation of N-SMase, but not A-SMase, via hydrogen peroxide. Consistently, superoxide radicals generated by hypoxanthine and xanthine oxidase also induced the activation of N-SMase, but not A-SMase, through a catalase-sensitive pathway. Furthermore, antisense knockdown of p22phox, a subunit of NADPH oxidase, inhibited Aβ1–42-induced neuronal apoptosis and cell death. These studies suggest that fibrillar Aβ1–42 peptides induce neuronal apoptosis through the NADPH oxidase-superoxide-hydrogen peroxide-NS-Mase-ceramide pathway. Alzheimer's disease is a major illness of dementia characterized by the presence of amyloid plaques, neurofibrillary tangles, and extensive neuronal apoptosis. However, the mechanism behind neuronal apoptosis in the Alzheimer's-diseased brain is poorly understood. This study underlines the importance of neutral sphingomyelinase in fibrillar Aβ peptide-induced apoptosis and cell death in human primary neurons. Aβ1–42 peptides induced the activation of sphingomyelinases and the production of ceramide in neurons. Interestingly, neutral (N-SMase), but not acidic (A-SMase), sphingomyelinase was involved in Aβ1–42-mediated neuronal apoptosis and cell death. Aβ1–42-induced production of ceramide was redox-sensitive, as reactive oxygen species were involved in the activation of N-SMase but not A-SMase. Aβ1–42 peptides induced the NADPH oxidase-mediated production of superoxide radicals in neurons that was involved in the activation of N-SMase, but not A-SMase, via hydrogen peroxide. Consistently, superoxide radicals generated by hypoxanthine and xanthine oxidase also induced the activation of N-SMase, but not A-SMase, through a catalase-sensitive pathway. Furthermore, antisense knockdown of p22phox, a subunit of NADPH oxidase, inhibited Aβ1–42-induced neuronal apoptosis and cell death. These studies suggest that fibrillar Aβ1–42 peptides induce neuronal apoptosis through the NADPH oxidase-superoxide-hydrogen peroxide-NS-Mase-ceramide pathway. Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid-β; N-SMase, neutral sphingomyelinase; A-SMase, acidic sphingomyelinase; PBS, phosphate-buffered saline; DAG, diacylglycerol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; TNF-α, tumor necrosis factor α; NAC, N-acetylcysteine; DPI, diphenyliodonium; SAPK stress-activated protein kinase; TUNEL, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling.1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid-β; N-SMase, neutral sphingomyelinase; A-SMase, acidic sphingomyelinase; PBS, phosphate-buffered saline; DAG, diacylglycerol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; TNF-α, tumor necrosis factor α; NAC, N-acetylcysteine; DPI, diphenyliodonium; SAPK stress-activated protein kinase; TUNEL, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling. is a neurodegenerative disorder resulting in progressive neuronal death and memory loss. Neuropathologically, the disease is characterized by neurofibrillary tangles and neuritic plaques composed of aggregates of amyloid-β (Aβ) protein, a 40–43 amino acid proteolytic fragment derived from the amyloid precursor protein (1Martin J.B. N. Engl. J. Med. 1999; 340: 1970-1980Crossref PubMed Scopus (329) Google Scholar). The importance of Aβ in AD has been shown by means of several transgenic animal studies. The overexpression of mutant amyloid precursor protein results in neuritic plaque formation and synapse loss and correlative memory deficits, as well as behavioral and pathological abnormalities similar to those found in AD (2Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-102Crossref PubMed Scopus (3635) Google Scholar). Although deposition of Aβ peptides is one of the primary causes of neuronal loss in AD (1Martin J.B. N. Engl. J. Med. 1999; 340: 1970-1980Crossref PubMed Scopus (329) Google Scholar, 2Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-102Crossref PubMed Scopus (3635) Google Scholar), the mechanism by which Aβ causes neuronal loss is largely unknown. Increased TUNEL staining in postmortem AD brains suggests that neurons in the brains of AD patients die through apoptosis (3Gervais F.G. Xu D. Robertson G.S. Vaillancourt J.P. Zhu Y. Huang J. LeBlanc A. Smith D. Rigby M. Shearman M.S. Clarke E.E. Zheng H. Van Der Ploeg L.H.T. Ruffolo S.C. Thornberry N.A. Xanthoudakis S. Zamboni R.J. Roy S. Nicholson D.W. Cell. 1999; 97: 395-406Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 4Smale G. Nichols N.R. Brady D.R. Finch C.E. Horton Jr., W.E. Exp. Neurol. 1995; 133: 225-230Crossref PubMed Scopus (434) Google Scholar). Consistently, overexpression of the Aβ peptides intracellularly in transgenic mice causes chromatin segmentation, condensation, and increased TUNEL staining (5Games D. Adams D. Alessandrini R. Barbour R. Berthelette P. Blackwell C. Carr T. Clemens J. Donaldson T. Gillespie F. Nature. 1995; 373: 523-527Crossref PubMed Scopus (2217) Google Scholar, 6Masliah E. Sisk A. Mallory M. Mucke L. Schenk D. Games D. J. Neurosci. 1996; 16: 5795-5811Crossref PubMed Google Scholar, 7Holcomb L. Gordon M.N. McGowan E. Yu X. Benkovic S. Jantzen P. Wright K. Saad I. Mueller R. Morgan D. Sanders S. Zehr C. O'Campo K. Hardy J. Prada C.M. Eckman C. Younkin S. Hsiao K. Duff K. Nat. Med. 1998; 4: 97-100Crossref PubMed Scopus (1145) Google Scholar). Cell culture studies have also shown that Aβ peptides are apoptotic and cytotoxic to neuronal cells (8Lambert 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 (3056) Google Scholar, 9Bastianetto S. Ramassamy C. Dore S. Christen Y. Poirier J. Quirion R. Eur. J. Neurosci. 2000; 12: 1882-1890Crossref PubMed Scopus (333) Google Scholar). However, the mechanism by which Aβ peptides lead to neuronal loss is poorly understood. Although sphingomyelin was initially considered only a structural component of plasma membrane, several investigations established the involvement of sphingolipids and its metabolites in the key events of signal transduction associated with cell regulation, cell differentiation, and apoptosis (10Hannun Y.A. Science. 1996; 274: 1855-1858Crossref PubMed Scopus (1483) Google Scholar, 11Pettus B.J. Chalfant C.E. Hannun Y.A. Biochim. Biophys. Acta. 2002; 1585: 114-125Crossref PubMed Scopus (662) Google Scholar). Because ceramide, the lipid second messenger molecule, produced from the degradation of sphingomyelin by sphingomyelinases (neutral and acidic) induces apoptosis and cell death in various cell types, including glial and neuronal cells (10Hannun Y.A. Science. 1996; 274: 1855-1858Crossref PubMed Scopus (1483) Google Scholar, 11Pettus B.J. Chalfant C.E. Hannun Y.A. Biochim. Biophys. Acta. 2002; 1585: 114-125Crossref PubMed Scopus (662) Google Scholar, 12Singh I. Pahan K. Khan M. Singh A.K. J. Biol. Chem. 1998; 273: 20354-20362Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 13Brugg B. Mitchel P.P. Agid Y. Ruberg M. J. Neurochem. 1996; 66: 733-739Crossref PubMed Scopus (182) Google Scholar, 14Wiesner D.A. Dawson G. J. Neurochem. 1996; 66: 1418-1425Crossref PubMed Scopus (173) Google Scholar, 15Keane R.W. Srinivasan A. Foster L.M. Testa M.P. Ord T. Nonner D. Wang H.G. Reed J.C. Bredesen D.E. Kayalar C. J. Neurosci. Res. 1997; 48: 168-180Crossref PubMed Scopus (146) Google Scholar), we decided to investigate the effect of Aβ peptides on the induction of ceramide production in neuronal cells. Here we report that fibrillar Aβ1–42 peptides induce the activation of sphingomyelinases and the production of ceramide in human primary neurons. We also show that the activation of neutral, but not acidic, sphingomyelinase plays the vital role in neuronal apoptosis and that NADPH oxidase-mediated superoxide production in neurons is responsible for Aβ-induced activation of neutral sphingomyelinase. Reagents—Neurobasal medium and B27 supplement were purchased from Invitrogen. Human Aβ peptides 1–42 and 42–1 were obtained from Bachem Bioscience. Antibodies against p22phox were purchased from Santa Cruz Biotechnology. Phosphorothioate-labeled antisense and scrambled oligodeoxynucleotides were synthesized in the DNA-synthesizing facility of Invitrogen. Isolation of Human Primary Neurons—Human primary neurons were prepared as described by Zhang et al. (16Zhang Y. McLaughlin R. Goodyer C. LeBlanc A. J. Cell Biol. 2002; 156: 519-529Crossref PubMed Scopus (368) Google Scholar) with some modifications. All of the experimental protocols were reviewed and approved by the Institutional Review Board (IRB number 224–01-FB) of the University of Nebraska Medical Center. Briefly, 11–17-week-old fetal brains obtained from the Human Embryology Laboratory (University of Washington, Seattle, WA) were dissociated by trituration and trypsinization (0.25% trypsin in PBS at 37 °C for 15 min). The trypsin was inactivated with 10% heat-inactivated fetal bovine serum (Mediatech). The dissociated cells were filtered through 380- and 140-μm meshes (Sigma) and pelleted by centrifugation. The cell pellet was washed once with PBS and once with Neurobasal medium containing 2% B27 and 1% antibiotic-antimycotic mixture (Sigma). In the first step, neurons were enriched by allowing the cells (3 × 106/ml) to adhere to poly-d-lysine-coated plates or coverslips for 5 min. Nonadherent cells were removed, and adherent cells (mostly neurons) were further treated with 10 μm Ara-C to prevent the proliferation of dividing cells. After 10 days of Ara-C treatment, the cells were used for this study. More than 98% of this preparation was positive for microtubule-associated protein-2 (MAP-2), a marker for neurons. Preparation of Fibrillar Aβ—Fibrillar Aβ1–42 and control reverse peptide Aβ42–1 (Bachem Bioscience) were prepared by incubating freshly solubilized peptides at 50 μm in sterile distilled water at 37 °C for 5 days (17Pike C.J. Burdick D. Walencewicz A.J. Glabe C.G. Cotman C.W. J. Neurosci. 1993; 13: 1676-1687Crossref PubMed Google Scholar). Treatment of Primary Neurons—During treatment with fibrillar Aβ peptides, the cells were incubated in Neurobasal medium containing 2% B27 supplement without antioxidant (B27-AO) (Invitrogen). Assay of Neutral and Acidic Sphingomyelinases (N-SMase and A-SMase)—Activities of SMase(s) were assayed as described by Liu et al. (18Liu B. Andrieu-Abadie N. Levade T. Zhang P. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1998; 273: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Briefly, after stimulation with fibrillar Aβ peptides, the cells were washed with PBS, harvested in PBS, divided into two halves, and centrifuged. The fraction for N-SMase was resuspended in buffer A (100 mm Tris-HCl, pH 7.4, 0.1% Triton X-100, 1 mm EDTA, and protease inhibitors), and the cell suspension was sonicated and centrifuged at 500 × g at 4 °C for 5 min. The supernatant was used as the enzyme source for N-SMase. The reaction mixture contained enzyme preparation in buffer A containing 5 nmol of [14C]sphingomyelin, 5 nmol of phosphatidylserine, 5 mm dithiothreitol, and 5 mm MgCl2 in a final volume of 100 μl. Similarly, the fraction for A-SMase was resuspended in buffer B (100 mm sodium acetate, pH 5.0, 0.1% Triton X-100, and protease inhibitors). The cell suspension was sonicated and centrifuged. The supernatant was used as the source of A-SMase. The activity of A-SMase was measured in a 100-μl reaction mixture consisting of the enzyme preparation in buffer B and 5 nmol of [14C]sphingomyelin. The enzyme reaction was initiated by the addition of 50 μl of substrate and stopped by the addition of 1.5 ml of chloroform:methanol (2:1, v/v) and 0.2 ml of water. After vortexing and phase separation, the aqueous phase was removed for counting. Lipid Extraction—Approximately 5 × 105 cells were exposed to fibrillar Aβ peptides for different periods of time, and the lipids were extracted as described previously (19Pahan K. Sheikh F.G. Khan M. Namboodiri A.M. Singh I. J. Biol. Chem. 1998; 273: 2591-2600Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 20Pahan K. Khan M. Singh I. J. Neurochem. 2000; 75: 576-582Crossref PubMed Scopus (66) Google Scholar). Quantification of Ceramide Levels by Diacylglycerol Kinase Assay— Ceramide content was quantified using diacylglycerol (DAG) kinase and [γ-32P]ATP as described earlier (19Pahan K. Sheikh F.G. Khan M. Namboodiri A.M. Singh I. J. Biol. Chem. 1998; 273: 2591-2600Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 20Pahan K. Khan M. Singh I. J. Neurochem. 2000; 75: 576-582Crossref PubMed Scopus (66) Google Scholar). Briefly, dried lipids were solubilized in 20 μl of an octyl β-d-glucoside/cardiolipin solution (7.5% octyl β-d-glucoside, 5 mm cardiolipin in 1 mm diethylenetriaminepentaacetic acid) by sonication in a sonicator bath. The reaction was then carried out in a final volume of 100 μl containing the 20-μl sample solution, 50 mm imidazole HCl, pH 6.6, 50 mm NaCl, 12.5 mm MgCl2, 1 mm EGTA, 2 mm dithiothreitol, 6.6 μg of DAG kinase, and 1 mm [γ-32P]ATP (specific activity of 1–5 × 105 cpm/nmol) for 30 min at room temperature. The labeled ceramide-1-phosphate was resolved with a solvent system consisting of methyl acetate:n-propyl alcohol:chloroform:methanol:0.25% KCl in water:acetic acid (100:100:100:40:36:2). A standard sample of ceramide was phosphorylated under identical conditions and developed in parallel. Both standard and experimental samples had an identical RF value (0.46). Quantification of ceramide-1-phosphate was carried out by autoradiography and densitometric scanning using a Fluor Chem 8800 imaging system (Alpha Innotech Corporation). Values are expressed either as arbitrary units (absorbance) or as percent of control, considering control as 100%. Statistical comparisons were made using one-way analysis of variance followed by Student's t test. Assay of Superoxide Production—Superoxide production by human primary neurons was detected by LumiMax™ superoxide anion-detection kit (Stratagene) following the manufacturer's protocol. Briefly, 5 × 105 cells suspended in 100 μl of superoxide anion assay medium were added to 100 μl of reagent mixture containing 0.2 mm luminol, 0.25 mm enhancer, and either fibrillar Aβ1–42 or Aβ42–1 at 1 μm in superoxide anion assay medium. Light emission was recorded at regular intervals in a TD-20/20 Luminometer (Turner Designs). Assay of Hydrogen Peroxide (H2O2) Production—The production of H2O2 by human primary neurons was quantified by a colorimetric H2O2 assay kit (Assay Designs, Inc.) following the manufacturer's protocol. Briefly, different amounts of supernatants were allowed to react with 100 μl of a color reagent containing xylenol orange in an acidic solution with sorbitol and ammonium iron sulfate followed by reading of the optical density at 550 nm. Immunostaining—Coverslips containing 200–300 cells/mm2 were fixed with 4% paraformaldehyde for 20 min followed by treatment with cold ethanol (-20 °C) for 5 min and 2 rinses in PBS. The samples were blocked with 3% bovine serum albumin in PBS containing Tween 20 (PBST) for 30 min and incubated in PBST containing 1% bovine serum albumin and goat anti-p22phox (1:50), rabbit anti-GFAP (1:50), or goat anti-MAP-2 (1:50), as described previously (21Dasgupta S. Zhou Y. Jana M. Banik N.L. Pahan K. J. Immunol. 2003; 170: 3874-3882Crossref PubMed Scopus (62) Google Scholar, 22Dasgupta S. Jana M. Zhou Y. Fung Y.K. Ghosh S. Pahan K. J. Immunol. 2004; 173: 1344-1354Crossref PubMed Scopus (103) Google Scholar). After three washes in PBST (15 min each), the slides were further incubated with Cy5 (Jackson ImmunoResearch Laboratories, Inc.). For negative controls, a set of culture slides was incubated under similar conditions without the primary antibodies. The samples were mounted and observed under a Bio-Rad MRC1024ES confocal laser scanning microscope. Fragment End Labeling of DNA—Fragmented DNA was detected in situ by the terminal deoxynucleotidyltransferase-mediated binding of 3′-OH ends of DNA fragments generated in response to fibrillar Aβ1–42, using a commercially available kit (TdT FragEL™) from Calbiochem. Briefly, cover slips were treated with 20 μg/ml proteinase K for 15 min at room temperature and washed prior to terminal deoxynucleotidyltransferase staining. Cell Viability Measurement—Mitochondrial activity was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma). The cells were grown on 24-well culture plates with 500 μl of medium and treated with various reagents according to the experimental design. At the end of the treatment period, 300 μl of culture medium were removed from each well, and 20 μl of MTT solution (5 mg/ml) were added and incubated for 1 h. Lactate Dehydrogenase Measurement—The activity of lactate dehydrogenase (LDH) was measured using the direct spectrophotometric assay using an assay kit from Sigma. Fibrillar Aβ1–42 Peptides Induce the Production of Ceramide in Human Primary Neurons—Because the fibrillar form of Aβ is commonly found in the senile plaques in AD brains (1Martin J.B. N. Engl. J. Med. 1999; 340: 1970-1980Crossref PubMed Scopus (329) Google Scholar), we examined whether fibrillar Aβ1–42 is capable of inducing apoptosis in human primary neurons. As demonstrated in Fig. 1, fibrillar Aβ1–42, but not reverse (Aβ42–1), peptides markedly induced the formation of apoptotic bodies. Because ceramide is an important inducer of apoptosis in different cells (10Hannun Y.A. Science. 1996; 274: 1855-1858Crossref PubMed Scopus (1483) Google Scholar, 11Pettus B.J. Chalfant C.E. Hannun Y.A. Biochim. Biophys. Acta. 2002; 1585: 114-125Crossref PubMed Scopus (662) Google Scholar, 12Singh I. Pahan K. Khan M. Singh A.K. J. Biol. Chem. 1998; 273: 20354-20362Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 13Brugg B. Mitchel P.P. Agid Y. Ruberg M. J. Neurochem. 1996; 66: 733-739Crossref PubMed Scopus (182) Google Scholar, 14Wiesner D.A. Dawson G. J. Neurochem. 1996; 66: 1418-1425Crossref PubMed Scopus (173) Google Scholar, 15Keane R.W. Srinivasan A. Foster L.M. Testa M.P. Ord T. Nonner D. Wang H.G. Reed J.C. Bredesen D.E. Kayalar C. J. Neurosci. Res. 1997; 48: 168-180Crossref PubMed Scopus (146) Google Scholar), and cell-permeable C2-ceramide induced apoptosis in human fetal neurons (Fig. 1), we examined the effect of fibrillar Aβ1–42 and Aβ42–1 on the induction of ceramide production. We have found that fibrillar Aβ1–42, but not Aβ42–1, induced the production of ceramide in human primary neurons at different times of incubation. Within 15 min of treatment, fibrillar Aβ1–42 peptides were able to induce the level of ceramide by more than 2-fold, and with further increase in duration of treatment, the level of ceramide increased markedly (Fig. 2A). After 10 h of treatment of Aβ1–42, a ∼12-fold increase in ceramide production was observed (Fig. 2A). In contrast to the fibrillar form, soluble Aβ1–42 peptides were weakly efficient in inducing the level of ceramide (Fig. 2A). For example, soluble Aβ1–42 peptides were unable to induce the production of ceramide at 15 or 30 min of stimulation. However, after 6 h of stimulation, soluble Aβ1–42 peptides induced a 4-fold increase in ceramide production compared with an 8-fold increase by fibrillar Aβ1–42 (Fig. 2A). Although Aβ1–42-induced production of ceramide was dose-dependent, Aβ1–42 effectively induced the production of ceramide even at 0.25 μm (Fig. 2B). Because Aβ1–42 induced neuronal apoptosis effectively at 1 μm, we have chosen the particular dose for further studies. On the other hand, under similar treatment conditions, fibrillar reverse (Aβ42–1) peptides were unable to induce the production of ceramide (Fig. 2A), suggesting the specificity of the effect. Because DAG kinase phosphorylates both DAG and ceramide using [γ-32P]ATP as substrate, both lipids can be quantified in the same assay. In contrast to a time-dependent increase in ceramide production, the level of DAG was unchanged at different time points of stimulation (Fig. 2A).Fig. 2Fibrillar Aβ1–42, but not Aβ42–1, peptides induce the production of ceramide and the activation of neutral (N-SMase) and acidic (A-SMase) sphingomyelinases in human primary neurons. Cells were incubated with 1 μm of fibrillar Aβ1–42, fibrillar Aβ42–1, or soluble Aβ1–42 in Neurobasal medium containing 2% B27-AO for different time intervals. Lipids were extracted, and ceramide and DAG contents (A) were determined. a, p < 0.001 versus control. B, after 6 h of incubation with different concentrations of Aβ1–42, ceramide contents were determined. b, p < 0.05 versus control. C, after incubation with either fibrillar Aβ1–42 or Aβ42–1 at 1 μm for different time intervals, activities of N-SMase and A-SMase were assayed in total cell extract.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fibrillar Aβ1–42 Peptides Induce the Activation of Sphingomyelinases in Human Primary Neurons—Five distinct sphingomyelinases have been identified based upon their pH optima, cellular localization, and cation dependence (10Hannun Y.A. Science. 1996; 274: 1855-1858Crossref PubMed Scopus (1483) Google Scholar, 11Pettus B.J. Chalfant C.E. Hannun Y.A. Biochim. Biophys. Acta. 2002; 1585: 114-125Crossref PubMed Scopus (662) Google Scholar). However, the neutral membrane-bound Mg2+-independent sphingomyelinase (N-SMase) and the lysosomal acid pH optima sphingomyelinase (A-SMase) have been the best studied for their roles in ceramide generation (10Hannun Y.A. Science. 1996; 274: 1855-1858Crossref PubMed Scopus (1483) Google Scholar, 11Pettus B.J. Chalfant C.E. Hannun Y.A. Biochim. Biophys. Acta. 2002; 1585: 114-125Crossref PubMed Scopus (662) Google Scholar). Therefore, we investigated whether fibrillar Aβ1–42 peptides are capable of inducing the activation of N-SMase and A-SMase. Marked induction of N-SMase activity was observed even at 15 min of treatment with Aβ1–42, with the maximum induction (∼5-fold) observed at 1 h (Fig. 2C). However, the induction of N-SMase activation decreased afterward. On the other hand, Aβ42–1 peptides were unable to induce the activation of N-SMase at different time points of stimulation. Although Aβ1–42, but not Aβ42–1, peptides also induced the activation A-SMase showing the peak at 30 min, the level of its induction was much lower than that of N-SMase (Fig. 2C). Because TNF-α is a prototype inducer of N-SMase (10Hannun Y.A. Science. 1996; 274: 1855-1858Crossref PubMed Scopus (1483) Google Scholar, 18Liu B. Andrieu-Abadie N. Levade T. Zhang P. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1998; 273: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar), we were prompted to investigate whether TNF-α had any role in fibrillar Aβ1–42-induced neuronal activation of N-SMase. However, neutralizing antibodies against human TNF-α capable of blocking TNF-α-induced activation N-SMase had no effect on Aβ1–42-induced activation of N-SMase in human primary neurons (data not shown). Role of N-SMase and A-SMase in Aβ1–42-induced Apoptosis and Cell Death in Human Primary Neurons—Antisense oligonucleotides provide the most effective tool with which to investigate the in vivo functions of different proteins in human primary neurons. Therefore, we adopted the antisense-knockdown technique to investigate the role of N-SMase and A-SMase in Aβ1–42-induced neuronal apoptosis and cell death. Using the method of Bloomfield and Giles (23Bloomfeild M.R. Giles I.G. Biochem. Soc. Trans. 1992; 20: 293SCrossref PubMed Scopus (4) Google Scholar) and based on the translational initiation site, we selected different antisense (ASO) and scrambled (ScO) oligonucleotide sequences against human N-SMase and A-SMase and screened their efficacy to inhibit Aβ1–42-induced activation of respective SMase in primary neurons. After screening of several antisenses, we found the following specific ASO and ScO against N-SMase and A-SMase: N-SMase, ASO, 5′-CAGCGAGCCCGTCCACCAGCC-3′; ScO, 5′-CACGCGTCCGACGCCGCACGA-3′ and A-SMase, ASO, 5′-GACATCTCGGAGCCGGGGCA-3′; ScO, 5′-GGAAACCCGGTTAGGCCCGG-3′. As observed in Fig. 3, ASO against N-SMase dose-dependently inhibited Aβ1–42-induced activation of N-SMase but not A-SMase. Similarly, ASO against A-SMase inhibited Aβ1–42-induced activation of A-SMase but not N-SMase. On the other hand, respective ScO did not influence the activation of either N-SMase or A-SMase (Fig. 3). Next, we investigated the effect of antisense knockdown of N-SMase and/or A-SMase on Aβ1–42-induced neuronal apoptosis and cell death. Under the experimental condition of ASO/ScO treatment, the control neurons showed a few apoptotic bodies, but Aβ1–42 treatment resulted in marked increase in apoptosis (Fig. 4, A and B). However, ASO, but not ScO, against N-SMase markedly blocked Aβ1–42-induced neuronal apoptosis (Fig. 4, A and B). On the other hand, antisense knockdown of A-SMase had no effect on Aβ1–42-induced apoptosis (Fig. 4B). After 18 h of treatment, Aβ1–42 reduced cell viability, as evidenced by a decrease in MTT metabolism (Fig. 4C). Interestingly, ASO against N-SMase, but not A-SMase, effectively prevented Aβ1–42-induced loss of MTT metabolism. Similarly, Aβ1–42 also induced an increase in LDH release, and treatment of cells with ASO against N-SMase, but not A-SMase, resulted in significant reduction in LDH release (Fig. 4C). On the other hand, ScO against N-SMase had no effect on Aβ1–42-induced loss of MTT metabolism and increase in LDH release (Fig. 4C). These studies suggest that activation of N-SMase, but not A-SMase, plays the key role in fibrillar Aβ1–42-mediated neuronal apoptosis and cell death. Involvement of Reactive Oxygen Species in Aβ1–42-induced Production of Ceramide and Activation of N-SMase in Human Primary Neurons—Next we investigated the mechanisms by which Aβ1–42 induced the activation of N-SMase in neurons. Earlier observations (12Singh I. Pahan K. Khan M. Singh A.K. J. Biol. Chem. 1998; 273: 20354-20362Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 18Liu B. Andrieu-Abadie N. Levade T. Zhang P. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1998; 273: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar) that reactive oxygen species are involved in cytokine-induced production of ceramide and activation of N-SMase prompted us to investigate whether reactive oxygen species play a role in Aβ1–42-induced production of ceramide and activation of N-SMase in neurons. We examined the effect of antioxidants, N-acetylcysteine (NAC) and diphenyliodonium (DPI), on Aβ1–42-induced production of ceramide and activation of SMases. Interestingly, both NAC and DPI dose-dependently prevented Aβ1–42-induced generation of ceramide (Fig. 5A), suggesting that Aβ1–42-induced ceramide generation in neurons is redox-sensitive. Next, we investigated whether Aβ1–42-induced activation of N-SMase and/or A-SMase is/are redox-sensitive. Although both NAC and DPI dose-dependently inhibited Aβ1–42-induced activation of N-SMase (Fig. 5B), these two antioxidants had no effect on the activation of A-SMase (Fig. 5C), suggesting that Aβ1–42-induced activation of N-SMase, but not A-SMase, is redox-sensitive. Involvement of NADPH Oxidase in Aβ1–42-induced Activation of N-SMase in Human Primary Neurons—In an attempt to identify the reactive oxygen species-producing molecule that couples fibrillar Aβ1–42 to N-SMase in neurons, we examined the production of superoxide in response to fibrillar Aβ peptides using the LumiMax™ superoxide anion-detection kit (Stratagene). Interestingly, treatment of primary neurons with fibrillar Aβ1–42, but not Aβ42–1, peptides resulted in time-dependent release of superoxide (Fig. 6). The production of superoxide was observed as early as 30 s of stimulation, which peaked at 50 s of stimulation and decreased afterward. On the other hand, the production of superoxide by Aβ1–42 was close to the control value, even after 120 s of stimulation (Fig. 6). This Aβ1–42-induced production of superoxide was completely inhibited when the cells were pretreated with 25 units/ml superoxide dismutase for 15 min (data not shown). On the other hand, catalase (25 units/ml) had no effect on fibrillar Aβ1–42-induced superoxide production (data not shown), suggesting that the LumiMax™ superoxide kit detects superoxide but not H2O2. Because DPI inhibited Aβ1–42-induced activation of N-SMase (Fig. 5B), we examine
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