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Address correspondence to Chung Y. Hsu, Center for the Study of Nervous System Injury, Dept. of Neurology, Washington University School of Medicine, 660 S. Euclid Ave., Box 8111, St. Louis, MO 63110. Tel.: (314) 362-3304. Fax: (314) 362-9462. email: hsuc{at}neuro.wustl.edu
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Abstract |
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Key Words: Alzheimer's disease; apoptosis; cell death; oxidative stress; white matter
Abbreviations used in this paper: 3-OMe-SM, 3-O-methyl-sphingomyelin; Aß, amyloid-ß peptide; AD, Alzheimer's disease; aSMase, acidic sphingomyelinase; bSMase, bacterial sphingomyelinase; BSO, buthionine sulfoximine; DEM, diethyl maleate; ESI/MS, electrospray ionization/mass spectrometry; GSH, glutathione; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC, N-acetylcysteine; nSMase, neutral sphingomyelinase; NOE, N-oleoyl-ethanolamine; OLG, oligodendrocyte; PLP, proteolipid protein.
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Introduction |
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Ceramide, a lipid second messenger that increases the cellular oxidative state, has been implicated in several apoptosis paradigms including trophic factor withdrawal and exposure to proinflammatory molecules (Coroneos et al., 1995; Kyriakis and Avruch, 1996; Kolesnick and Kronke, 1998). Cellular ceramide synthesis increases in response to stress or death signals (Haimovitz-Friedman et al., 1994; Tepper et al., 1995; Verheij et al., 1996). One pathway of ceramide formation involves sphingomyelin hydrolysis by either neutral sphingomyelinase (nSMase) or acidic sphingomyelinase (aSMase; Testi, 1996); both enzymes are involved in several cell death paradigms (Kolesnick and Kronke, 1998; Levade and Jaffrezou, 1999). Another pathway involves ceramide synthasecatalyzed de novo ceramide synthesis (Bose et al., 1995; Spiegel and Merrill, 1996; Xu et al., 1998).
Aß and ceramide share cell death signaling characteristics. Aß-induced apoptosis involves TNF-, p75 neurotrophin receptor, and Fas ligand (Blasko et al., 1997; de la Monte et al., 1997; Yaar et al., 1997), which are cell surface receptors that relay death signals through the sphingomyelinceramide pathway (Dobrowsky et al., 1995; Hannun, 1996). Moreover, both Aß (Kaneko et al., 1995; Askanas et al., 1996; Bruce-Keller et al., 1998; Xu et al., 2001) and ceramide (Garcia-Ruiz et al., 1997; Singh et al., 1998) cause mitochondrial dysfunction and induce oxidative stress. In vitro, OLG death induced by Aß (Xu et al., 2001) or ceramide (Larocca et al., 1997; Singh et al., 1998; Scurlock and Dawson, 1999) share similar apoptotic characteristics. Lower sphingomyelin levels and higher ceramide levels in AD brains have been reported (Soderberg et al., 1992), thereby implying that increased sphingomyelin degradation and ceramide accumulation contribute to AD pathogenesis. We have previously shown that Aß induced OLG death with characteristic features of apoptosis (Xu et al., 2001). In this paper, we demonstrate that ceramide mediates Aß-induced OLG death by activating the nSMaseceramide cascade.
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Results |
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Discussion |
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Results from this study also support the contention that Aß cytotoxicity is mediated via activation of nSMase leading to increased cellular ceramide generation. Aß 25-35 and Aß 1-40 activated nSMase, but not aSMase, in OLGs. Additionally, nSMase inhibitors such as 3-OMe-SM and NAC (also an antioxidant) prevented Aß 25-35induced nSMase activity, which resulted in decreased ceramide synthesis from sphingomyelin and protected OLGs from Aß 25-35 cytotoxicity. Antisense oligonucleotides specific for nSMase also attenuated Aß-induced OLG cell death, further implicating nSMase as a mediator. Chemical agents such as BSO and DEM that deplete cellular GSH content also activated nSMase in OLGs and caused cell death. The specific role of nSMase in Aß-induced OLG death is supported by the finding that pharmacological inhibition of nSMase, but not aSMase or ceramide synthase, prevented Aß 25-35induced OLG death.
The exact mechanism underlying Aß-mediated nSMase activation remains to be elucidated but may involve changes in the cellular redox state and/or GSH metabolism (Sawai and Hannun, 1999); GSH is the most abundant thiol-containing compound in living cells. nSMase enzymatic activity is directly regulated by cellular GSH content (Liu and Hannun, 1997; Liu et al., 1998a,b). Aß has been shown to deplete GSH in cultured cortical neurons (Muller et al., 1997), and depletion of cellular GSH stores by oxidative stress has been proposed as a prime mechanism underlying the Aß cytotoxic action (Muller et al., 1997; Pereira et al., 1999). Thus, it is plausible that a decrease in GSH level subsequent to Aß exposure may activate nSMase in OLGs. NAC, a GSH precursor, inhibited Aß 25-35 activation of nSMase and protected OLGs against Aß-induced death, whereas depletion of cellular GSH stores by BSO or DEM resulted in selective activation of nSMase and OLG death. Although the agents used to manipulate GSH levels may be relatively nonspecific, these findings raise the possibility that the activation of nSMase by Aß may involve the depletion of cellular GSH content.
Oxidative stress plays a prominent role in Aß-mediated neuronal and OLG death (Behl et al., 1994; Behl, 1999; Markesbery, 1999; Xu et al., 2001). Brain tissue is especially sensitive to oxidative injury because of its higher metabolic rate driven by glucose, lower concentrations of protective antioxidants, and higher levels of polyunsaturated fatty acids that are susceptible to lipid peroxidation (Behl and Sagara, 1997; Behl, 1999; Markesbery, 1999). Although Aß-mediated oxidative stress induces mtDNA damage (Bozner et al., 1997; Xu et al., 2001) and activates selected transcription factors including NF-B and AP-1 (Abate et al., 1990; Schreck et al., 1991; Pinkus et al., 1996; Xu et al., 2001), the mechanism by which Aß induces oxidative stress in the AD brain remains unknown. Ceramide has emerged as a potent second messenger in oxidative stress-induced apoptosis (Hannun and Luberto, 2000). Hydrogen peroxide (Goldkorn et al., 1998), 1-ß-D-arabinofuranosylcytosine (Bradshaw et al., 1996), daunorubicin (Jaffrezou et al., 1996), TNF-
(Bezombes et al., 1998),
-rays (Bruno et al., 1998), hypoxia (Yoshimura et al., 1998), CD40 activation (Segui et al., 1999), and sindbis virus infection (Jan et al., 2000) are among the agents that can mediate cell death via nSMase activation, thus emphasizing the central role of the nSMaseceramide cascade. Results shown here provide a unique signaling pathway from cell surface Aß engagement, induction of oxidative stress, and activation of the nSMaseceramide cascade culminating in OLG death.
In summary, this work demonstrates a novel mechanism for Aß-induced OLG death. These results reveal a causal relationship between Aß exposure and the activation of the nSMaseceramide pathway, which is likely to involve heightened oxidative stress after depletion of cellular GSH stores. In addition, we have evidence that activation of the nSMaseceramide cascade may also contribute to Aß-induced death of cortical neurons and cerebral endothelial cells (unpublished data), thereby suggesting that this cascade may be operating in many cell types other than OLGs. AßnSMase
ceramide cascade represents a novel signaling pathway that contributes at least in part to Aß cytotoxicity to various types of brain cells. Identification of this pathway may lead to the development of more effective therapeutic strategies aimed at preventing Aß-induced cell death. For instance, blockade of the Aß-activated death signaling process can be achieved by pharmacological modulation of nSMase activity as demonstrated in this work.
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Materials and methods |
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OLG culture
Neurospheres were cultured using the methods of Zhang et al. (1999) with modifications. In brief, embryonic rat brains (E1416 d) were dissected, homogenized gently in DME/Ham's F12, and centrifuged at 350 g for 5 min. The pellet was digested with 0.05% trypsin in 1.5 ml of 0.53 M EDTA for 30 min at 37°C, followed by the addition of 1.5 ml DME/Ham's F12 with 20% FBS, and filtered through 10-µm nylon mesh. The filtrate was centrifuged at 350 g for 5 min, and the pellet was washed twice with DME/Ham's F12. Dissociated cells were layered on a preequilibrated Percoll gradient (formed by centrifuging 50% Percoll and 50% DME/Ham's F12 at 23,500 g for 1 h at 4°C) and centrifuged at 3,500 g for 15 min. The fraction containing glial progenitors banding between myelin and blood cell layers was recovered and washed twice with DME/Ham's F12 followed by another wash with neurosphere culture medium (DME/Ham's F12/Hepes, N1 supplement, 25 µg/ml insulin, 130 ng/ml progesterone, 20 ng/ml of basic FGF, and 20 ng/ml EGF). The cell pellet was resuspended in 20 ml of neurosphere culture medium and seeded in 75-mm culture flasks. After 24 h, when neurospheres had formed, 5 ml of fresh medium was added to each culture every other day for 7 d, and then the neurosphere cultures were split (1:2). The neurospheres were dissociated gently 10 times with a syringe and a 25-gauge needle and centrifuged at 350 g. The resulting cell pellets were treated with 0.05% trypsin/0.53 mM EDTA and centrifuged at 350 g for 10 min. The cells were resuspended in progenitor medium (69% DME/Ham's F12/Hepes containing N1 supplement, 10 µg/ml insulin, 20 nM progesterone, 30% conditioned medium from B104 cells, and 1% FBS) and plated on 100-mm culture dishes precoated with poly-L-ornithine. For differentiated OLG cultures, progenitor cells were detached with trypsin/EDTA and cultured on poly-L-ornithinecoated plates or coverslips in mature OLG medium (DME/Ham's F12; N1 supplement, 20 µg/ml biotin; 20 µg/ml triiodo-L-thyronine, T3, and 1% FBS).
Immunocytochemistry
Differentiated OLG cultures were fixed in 4% PFA, washed in PBS, and blocked with 5% goat serum. Fixed cells were incubated with the primary antibody overnight at 4°C. The following primary antibodies were used: antimouse CNP, antimouse Rip, antimouse PLP (Chemicon), and antimouse GalC (Cedarlane Laboratories) at a concentration of 1:100. The secondary antibody, antimouse IgG conjugated to FITC (Vector Laboratories), was added for 1 h at RT. The cells were washed in PBS and visualized using a confocal microscope (model LSM 5 Pascal; Carl Zeiss MicroImaging, Inc.) equipped with a CCD camera (model Axiocam HR; Carl Zeiss MicroImaging, Inc.). Images were collected and processed using Adobe Photoshop software.
Sphingomyelinase assay
The cells were washed twice with PBS, pH 7.4, and lysed in 0.2% Triton X-100 for 10 min at 4°C. The lysates were sonicated for 30 s in ice-cold bath, and protein concentrations were determined by Lowry assay (Lowry et al., 1951). A sphingomyelinase substrate, [methyl-14C]sphingomyelin (55 mCi/mmol; Amersham Biosciences), was evaporated to dryness and resuspended in either 25 µl of nSMase assay buffer (40 mM Hepes, 5 mM MgCl2, and 0.2% Triton X-100, pH 7.4) or aSMase assay buffer (250 mM sodium acetate and 0.2% Triton X-100, pH 5.2) and sonicated to form micelles on ice until use. Each reaction containing 25 µl of cell lysate protein (1 mg/ml) and 25 µl [methyl-14C]sphingomyelin (0.23 nmol) in nSMase or aSMase assay buffer was incubated for 2 h at 37°C. The reaction was terminated with 200 µl CHCl3/methanol (1:1) and 90 µl H2O followed by vigorous agitation. The samples were centrifuged at 6,000 g for 5 min. [14C]Phosphocholine in the aqueous phase (120 µl) was collected for liquid scintillation counting. Phosphocholine is the degraded moiety of sphingomyelin after ceramide is released by nSMase or aSMase. The aSMase or nSMase activity was calculated as picomoles of sphingomyelin hydrolyzed by 1 mg of total proteins per hour and expressed as a percentage of control values.
TLC
OLGs with or without Aß 25-35 treatment were cultured with 10 µCi [3H]palmitate (1 mCi/ml; Amersham Biosciences; Kaneko et al., 1995). The labeled cells were collected and washed twice with PBS, pH 7.4, to remove free isotope before lipid extraction (Xu et al., 1998). The cell pellet was resuspended in 400 µl methanol/1 N HCl (100:6, vol/vol) followed by 800 µl chloroform and 240 µl H2O. The sample was mixed and centrifuged at 6,000 g for 5 min. The lipid fraction was reextracted with 1 ml chloroform/methanol (2:1, vol/vol) and applied to a TLC plate. The solvent was chloroform/methanol/acetic acid/H2O (85:4.5:5:0.5, vol/vol) for ceramide and chloroform/methanol/acetic acid/water (65:25:8.8:4.5, vol/vol) for sphingomyelin. Plates were air dried and sprayed with 1 M sodium salicylate for autoradiography. Standard lipids were stained by rhodamine 6G (Sigma-Aldrich) and visualized by UV light.
Quantitative ceramide analysis by ESI/MS
After three washes with PBS, pH 7.4, OLGs harvested from 100-mm culture plates with or without Aß 25-35 treatment were homogenized in 0.5 ml PBS, pH 7.4, with a glass tissue grinder. A bicinchomic protein assay kit was used to determine the protein concentration before lipid extraction (Pierce Chemical Co.). Lipids from the homogenates were extracted as described previously with modifications (Bligh and Dyer, 1959) using 50 mM LiOH in the aqueous layer and C17:0 ceramide (2 nmol/mg protein) as an internal standard for quantitation of ceramide content. These molecular species represent <1% of the endogenous cellular lipid mass. The lipid extracts were dried under a nitrogen stream, dissolved in chloroform, desalted with Sep-Pak columns, and filtered with 0.2 µm PTFE syringe filters (Fisher Scientific). Lipids were reextracted with 20 mM LiOH in the aqueous layer, dried under a nitrogen stream, and resuspended in 0.5 ml of chloroform/methanol (1:1, vol/vol) for ESI/MS analysis.
ESI/MS analysis was performed using a spectrometer (model TSQ-7000; Finnigan) equipped with an electrospray ion source as described previously (Han et al., 1996, 2001). A 5-min period of signal averaging in the profile mode was used for each spectrum of a lipid extract. All extracts were directly infused into the ESI chamber using a syringe pump at 1 µl/min flow rate. Ceramide in the lipid extracts was quantitated directly as deprotenated ions ([M-H]-) in comparison with an internal standard (C17:0 ceramide) after correction for 13C isotope effects in the negative-ion mode. Ion peaks were identified using tandem mass spectroscopic analyses as described previously (Han and Gross, 1995).
Cell death assays
OLG viability was quantitated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue exclusion method. Cell death was also assessed by the amount of lactate dehydrogenase (LDH) release into the culture medium after Aß or C2-ceramide treatment (Koh and Choi, 1987; Shaikh et al., 1997; Xu et al., 1998). The amount of LDH released by cells killed with Triton X-100 was considered maximal cell death or "full kill" (Xu et al., 1998).
Assay for cellular GSH content
Cellular GSH levels were determined using a GSH-400 colorimetric assay kit (Calbiochem-Novabiochem). Triplicate samples (3 x 106 cells) were collected by centrifugation and washed twice with PBS, pH 7.4. The cell pellets were treated with 5% metaphosphoric acid (Sigma-Aldrich). A Teflon pestle was used to homogenize the cells. Protein concentrations were determined by Lowry assay (Lowry et al., 1951). The homogenates were centrifuged at 3,000 g for 10 min at 4°C. Supernatants were assayed for GSH according to the instructions provided with the kit. A standard curve was generated with graded concentrations of GSH (540 µM). GSH concentration was measured by absorbance at 400 nm with a spectrophotometer.
nSMase antisense oligonucleotides
Morpholino sense (GCCGCAGAGAAAAGTTGTGCTTCAT) and antisense (CCTCTTACCTCAGTTACAATTTATA) oligonucleotides were generated for nSMase (Gene Tools, LLC). OLGs in serum-free medium were treated with 1.4 µM oligonucleotides in EPEI delivery solution (per manufacturer's instructions; Gene Tools) for 4 h. The medium was exchanged and Aß was added for 24 h, after which cells were harvested for nSMase activity or cell death determination.
Statistical analysis
Results are expressed as mean ± SD. Differences among groups were analyzed by one-way ANOVA followed by Bonferroni's post-hoc t test to determine statistical significance. Comparison between two experimental groups was based on two-tailed t test. P < 0.05 was considered statistically significant.
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Acknowledgments |
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This work was supported by National Institutes of Heath grants NS37230 and NS40525.
Submitted: 2 July 2003
Accepted: 19 November 2003
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References |
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