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assays. Morpholino-oligonucleotides (Gene Tools, LLC Philomath,. OR, USA) were .... (c) Cyto- chrome C release was performed by Western blots on cytoplasmic. (cytosol) and ..... Imaizumi K., Benito A., Kiryu-Seo S., Gonzalez. V., Inohara N.,.
Journal of Neurochemistry, 2006, 97, 631–640

doi:10.1111/j.1471-4159.2006.03774.x

Amyloid beta peptide increases DP5 expression via activation of neutral sphingomyelinase and JNK in oligodendrocytes Shawei Chen,*,1 Jin-Moo Lee,*,1 Chenbo Zeng,* Hong Chen,* Chung Y. Hsu  and Jan Xu* *Department of Neurology and the Hope Center for Neurological Disorders, Washington University, School of Medicine, St Louis, MO, USA  Taipei Medical University, Taipei, Taiwai

Abstract There is growing recognition that white matter pathology is a common feature in Alzheimer’s disease. We have previously reported that the amyloid beta peptide (Ab) induces apoptosis in oligodendrocytes (OLG), via activation of neutral sphingomyelinase (nSMase) and resultant generation of ceramide. In the current study, we report that both Ab and ceramide increased expression of the proapoptotic protein DP5/Hrk (DP5), and release of cytochrome C from mitochondria to cytoplasm in OLGs. We provide evidence that the Jun N-terminal kinase (JNK) signaling pathway mediates Ab- and ceramide-induced apoptosis: Both Ab and ceramide activated

JNK phosphorylation, and subsequent AP-1 DNA binding activity; JNK siRNA decreased AP-1 DNA binding, DP5 expression and reduced cell death. Furthermore, inhibition of nSMase attenuated Ab-induced JNK phosphorylation, AP-1 DNA binding activity, DP5 expression, and cytochrome C release. Collectively, these results suggest that Ab-induced apoptosis involves the sequential activation of nSMase with ceramide generation, JNK activation, AP-1 DNA binding, and DP5 expression. Keywords: Amyloid beta peptide, ceramide, DP5, Jun Nterminal kinase, neutral sphingomyelinase, oligodendrocyte. J. Neurochem. (2006) 97, 631–640.

The amyloid b (Ab) peptide, found in senile plaques in the brains of Alzheimer’s disease (AD) patients (Verbeek et al. 1997; Selkoe 1999; Tabira et al. 2002; Bateman and Chakrabartty 2004), has been hypothesized to be the primary neurotoxic factor leading to neurodegeneration in this disease (Verbeek et al. 1997; Colurso et al. 2003; Su et al. 2003; Li et al. 2004). In addition to neurons (Yankner et al. 1989; Behl et al. 1994; Yu et al. 1998), Ab is toxic to a variety of brain-derived cells, including endothelial cells (Thomas et al. 1996; Huang et al. 1998), vascular smooth muscle cells (Kawai et al. 1993; Davis-Salinas et al. 1995), and oligodendrocytes (Xu et al. 2001). Although the vast majority of studies have focused on grey matter pathology, accumulating evidence suggest that white matter degeneration may contribute to AD pathology as well. The observed white matter pathology, including loss of myelin and axons (Brun and Englund 1986; Lassmann et al. 1995), as well as oligodendrocyte (OLG) apoptosis, might be an indirect consequence of neuron damage in grey matter. Alternatively, such damage could be mediated directly by Ab deposition, which has been reported in oligodendrocytes (Yamada et al. 1997; Brown et al. 2000) and in white matter in animal

models (Kurt et al. 2001; Song et al. 2004). While Ab cytotoxicity in OLGs has been reported (Xu et al. 2001), the molecular mechanism underlying Ab cytotoxicity remains largely undefined. We have recently reported that Ab induced OLG death via activation of neutral sphingomyleinase (nSMase) with generation of ceramide (Lee et al. 2004). The lipid second messenger, ceramide, plays a pivotal role in cell cycle arrest and apoptosis (Dbaibo and Hannun 1998). Cellular ceramide synthesis increases in response to stress or death signals such as tumor necrosis factor-a (TNFa), Fas ligation, interleukin-1, serum deprivation, heat shock,

Received May 17, 2005; revised manuscript received September 7, 2005; accepted November 21, 2005. Address correspondence and reprint requests to Jan Xu, Department of Neurology, Washington University School of Medicine, 660 S Euclid Ave, Campus Box 8111, St Louis, MO 63110, USA. E-mail: [email protected] 1 Both authors contributed equally to this paper. Abbreviations used: Ab, amyloid-b peptide 25–35; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MT, mitochondria; nSMase, neutral sphingomyelinase; OLG, oligodendrocyte; siRNA, small interfering RNA.

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ultraviolet light, chemotherapeutic agents, and others (Andrieu-Abadie and Levade 2002). Two general pathways lead to ceramide formation: one involves sphingomyelin hydrolysis by either nSMase or acidic sphingomyelinase (aSMase) (Testi 1996); where both enzymes have been implicated in a variety of cell death paradigms (Kolesnick and Kronke 1998; Levade and Jaffrezou 1999); another pathway involves de novo synthesis via ceramide synthase (Bose et al. 1995; Spiegel and Merrill 1996; Xu et al. 1998). Ceramide, in turn, has been shown to activate several stress-related kinases, including stress-activated protein kinases (SAPKs) (Ruvolo 2001), c-jun NH2 terminal kinase (JNK) (Basu and Kolesnick 1998), kinase suppressor of Ras (KSR) (Mathias et al. 1991; Joseph et al. 1994; Yao et al. 1995; Basu et al. 1998); as well as proteins phosphatases, protein phosphatase 1 (PP1) (Chalfant et al. 2001; Chalfant et al. 2004) and protein phosphatase 2 A (PP2A) (Dobrowsky et al. 1993; Wolff et al. 1994). Several of these regulatory pathways have been implicated in programmed cell death (Pettus et al. 2002). In AD, where neurodegeneration is an integral process in pathogenesis, elevated levels of ceramide have been reported in AD brains at the very earliest clinical stage of the disease (Gottfries et al. 1996; Han et al. 2002). While increased cellular ceramide generation has been demonstrated in a variety of cell death models, the exact apoptotic regulatory mechanisms downstream of ceramide have not been defined. Several studies have demonstrated that ceramide alters key apoptotic regulatory proteins. For example, ceramide has been shown to activate caspase-3 in a Bcl-2-dependent manner (Mizushima et al. 1996; Smyth et al. 1996). Furthermore, ceramide induced the dephosphorylation of Bcl-2 (via PP2A), resulting in subsequent cell death (Ruvolo et al. 1999). More recent data suggests that ceramide may regulate alternative splicing of the bcl-x gene, favoring the pro-apoptotic bcl-xs transcript (Chalfant et al. 2002; Massiello et al. 2004). This process appears to involve activation of PP1 and dephosphorylation of SR proteins, resulting in the formation of the bcl-xs splice product (Chalfant et al. 2002; Massiello et al. 2004). We have previously shown that Ab induces OLG death with characteristic features of apoptosis (Xu et al. 2001). Two recent reports have suggested that Ab neurotoxicity may involve the induction of DP5 (a.k.a. HRK), a BH3-only proapoptotic protein belonging to the Bcl-2 family of apoptotic regulators (Imaizumi et al. 1999; Bozyczko-Coyne et al. 2001). In this report, we investigated the possibility that Abinduced OLG death involved DP5 induction via nSMase activation, ceramide generation, and activation of stress kinase pathways. Materials and methods All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and cell culture supplies from Invitrogen

(Carlsbad, CA, USA), unless otherwise specified. B104 cells were obtained from the ATCC (Manassas, VA, USA). OLG culture Neurospheres were cultured as previously described (Zhang et al. 1999; Lee et al. 2004) with modifications. Briefly, embryonic rat brains (E14-16) were dissected, homogenized gently in Dulbeccos Modified Eagles Medium (DMEM)/F-12 medium and centrifuged at 350 · g for 5 min. The pellet was digested with 1.5 mL of 0.025% trypsin/0.53 mM ethylene diamine tetraacetic acid (EDTA) at 37C for 30 min followed by the addition of 1.5 mL DMEM/F-12 with 20% fetal bovine serum (FBS) and then filtered through 10 lm nylon mesh. The filtrate was centrifuged at 350 · g for 5 min and the pellet was washed twice with DMEM/F-12. Dissociated cells were layered on a pre-equilibrated Percoll gradient [formed by centrifuging 50% Percoll (Sigma-Aldrich, St Louis, MO, USA) and 50% DMEM/F-12 at 23 500 · g for 1 h at 4C] and centrifuged at 23 500 · g for 15 min. The fraction containing glial progenitors banding between myelin and red blood cell layers was recovered and washed twice with DMEM/F-12 followed by a wash with neurosphere culture medium [DMEM/F-12; N1 supplement; 25 lg/ mL insulin; 130 ng/mL progesterone; 20 ng/mL basic fibroblast growth factor (bFGF) and 20 ng/mL epidermal growth factor (EGF)]. The cell pellet was re-suspended in 20 mL of neurosphere culture medium and seeded in 75 mm culture flasks. After 24 h when neurospheres were formed, 5 mL of fresh medium was added to each culture every other day for 7 days and then the neurosphere cultures were split (1 : 2). For derivation of neurospheres into progenitors, the neurospheres were dissociated gently 10 times with a syringe with needle (25 gauge) and centrifuged at 350 · g. The resulting cell pellets were then 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% DMEM/F-12/HEPES containing N1 supplement, 10 lg/mL insulin, 20 nM progesterone, 100 unit penicillin/streptomycin, 30% conditioned medium from B104 cells and 1% FBS) and plated on 100-mm culture dishes pre-coated with poly L-ornithine. Disaggregated oligosphere cells displayed bipolar or tripolar morphology. For differentiated OLG cultures, progenitor cells were detached with trypsin and cultured on poly L-ornithine-coated dishes in mature OLG medium (DMEM/F-12; N1 supplement, 20 lg/mL biotin; 20 lg/mL triiodo-L-thyronine, T3 and 1% FBS). Immunoreactivity to OLG surface markers including galacto cerebroside (GalC), Rip and cyclic nucleotide 3¢-phophodiesterase (CNP) was observed in the vast majority of these differentiated cells within 48 h (Lee et al. 2004). RT-PCR DP5 gene mRNA expression was detected by RT-PCR. Total RNA was isolated with TriReagent (Molecular Research Center Inc., Cincinnati, OH). RNA was quantified by spectrophotometry. Equal amounts of total RNA (600 ng) were reverse-transcribed with 1.0 lM oligodT and 0.5 mM each dATP, dCTP, dGTP, dUTP (Qiagen, CA), 10 unit RNasin (Promega), and 4 unit Omniscript reverse transcriptase (Qiagen, CA, USA) for 1 h at 37C. Complementary DNA (cDNA) (transcribed from 600 ng RNA) was amplified in 0.2 mM each dATP, dCTP, dGTP, dUTP, 1 lM of each primer, 1.5 mmol/L MgCl2, and 2.5 unit Taq polymerase

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Ab induces DP5 by activation of nSMase and JNK in OLGs 633

(Roche Applied Science, Indianapolis, IN, USA). PCR was performed for 26 cycles alternating between 95C for 20 s, 53C for 30 s, followed by extension at 72C for 10 min. Primers were designed based on the rat DP5 sequences (forward primer: 5¢AGACCCAGCCCGGACCGAGCAA-3¢, reverse primer: 5¢-ATAGCACTGAGGTGGCTATC-3¢). The PCR reaction conditions (RT input and PCR cycles) were determined to be within a linear range for quantification. The amplified products were analyzed on 1% agarose gel containing 0.1 lg/mL ethidium bromide, visualized with UV light. The relative mRNA levels of DP5 were normalized to endogenous cyclophilin mRNA for each sample. Subfractionization of mitochondria and nuclear proteins from OLGs After treatment, OLGs in 100 mm dishes were harvested by centrifugation at 350 · g for 10 min at 4C. The cell pellets were washed twice with ice-cold phosphate buffered saline (PBS), followed by centrifugation at 350 · g for 5 min at 4C, and resuspended with 5 volume of Buffer A (20 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 0.1 mM PMSF, 1 mM dithiothreitol (DTT), 4 lg/mL pepstatin, 4 lg/mL leupeptin, 5 lg/mL aprotinin, pH 7.9). After 10 min incubation on ice, cells were homogenized with a mini-pestle. The lysates were centrifuged at 750 · g for 15 min at 4C. The supernatant contained mitochondrial and cytosolic proteins while the pellet contained nuclei. The pellets were resuspended and mixed in 45 lL of buffer B (20 mM HEPES, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, and 10 lg/mL leupeptin and aprotinin, pH 7.9), and 15 lL of buffer C (20 mM HEPES, 1.2 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 1 mg/mL leupeptin and aprotinin, pH 7.9). The samples were placed on ice for 30 min and centrifuged at 12 000 · g. Supernatants containing nuclear protein were transferred and stored at ) 80C for electrophoretic mobility shift assay (EMSA). The cytosolic/mitochondrial fractions were centrifuged at 10 000 · g for 15 min at 4C, and the resulting cytosolic supernatant and mitochondrial pellets were analyzed by Western blot analysis. Protein concentrations were measured using the Lowry method (Lowry et al. 1951). Western blot analysis Samples (20–40 lg of protein) were electrophoresed onto a 12% SDS/polyacrylamide gel (SDS/PAGE), and transferred to polyvinylidine difluoride membranes. The membranes were blocked with 5% of non-fat milk in tris-buffered saline tween (TBST) containing 20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween-20 (pH 7.5) for 1 h at 22C. Thereafter the blot was incubated with either rabbit anti-DP5 antibody (1 : 100, custom made, Bethyl, MA, USA), rabbit anti-phospho-JNK antibody (1 : 500; BD Biosciences), anticytochrome C (1 : 1,000, Pharmigen, CA, USA) or anti-actin antibodies (1 : 1000; Santa Cruz, CA, USA) overnight at 4C. The membrane was washed with TBST, incubated with secondary antibody (1 : 5000; anti-rabbit, antimouse goat IgG conjugated with alkaline phosphatase, Promega; Madison, WI, USA) at 22C for 1 h, then washed with TBST and TBS. The color reaction was developed by the Blot AP System according to the technical manual provided by Promega. The ratio of density of each band from RT-PCR or Western blot/internal

control (cyclophilin or actin or COX IV) was captured and quantified on a UVP Bioimaging and Analysis system (UVP. Inc., Upland, CA, USA). The mean values of the ratios from at least three experiments were graphed. JNK small interfering RNA (siRNA) and nSMase antisense oligonucleotide treatment OLGs grown 70% confluence were transfected with JNK2/SAPK1a siRNA (Si,100 nM) or non-specific siRNA according to manufacture protocol (Upstate, NY, USA) for 24 h followed by treatment of Ab (20 lM) or C2-ceramide (25 lM) for various times for different assays. Morpholino-oligonucleotides (Gene Tools, LLC Philomath, OR, USA) were generated as follows: 5¢-GCCGCAGAGAAAAGTTGTGCTTCAT-3¢ for nSMase antisense; 5¢-CCTCTTACCTCAGTTACAATTTATA-3¢ for a scrambled control oligonucleotide provided by the manufacture. OLGs in serum-free medium were treated with 1.4 lM oligonucleotides in delivery solution, ethoxylated polyethylenimine (EPEI), for 4 h (according to the manufacture’s instructions, Gene Tools). The medium was changed and Ab or C2-ceramide was added for 24 h, after which cells were harvested for assay of AP1 binding activities. Cell death assays OLG viability was quantitated by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium b romide (MTT) assay (Xu et al. 2001). The MTT assay is determining the degree of survival cells and based on the cleavage of the yellow tetrazolium salt MTT into purple formazan by metabolically active cells. The solubilized formazan product can be photometrically quantitated. The stronger purple color of formazan represents more number of living cells. AP-1 DNA binding Nuclear protein extraction and DNA binding have been described previously (Xu et al. 2001). Briefly, OLGs were treated with a JNK siRNA, and non-specific siRNA for 24 h and nSMase antisense oligonucleotide and a scrambled control oligonucleotide for 4 h, followed by the addition of Ab or C2-ceramide for 8 h or 24 h. Nuclear proteins were extracted and incubated with 32P-labeled AP1 consensus oligonucleotides (5¢-CGCTTGATGAGTCAGCCGGAA-3¢; Promega). The AP-1 oligonucleotide was labeled with c-32P[ATP] (3000 Ci/mMole, PerkinElmer Life Science, Boston, MA) according to Promega technical bulletin (No. 106). The binding reaction was performed in 20 lL of binding buffer (10 mM Tris-HCl, 20 mM NaCl, 1 mM DTT, 1 mM EDTA, 5% glycerol, pH 7.6) containing 0.0175 pmol of the labeled probe (> 10 000 cpm), 20 lg of nuclear protein and 1 lg of poly dIdC. After incubation for 20 min at room temperature, the reaction mixture was subjected to electrophoresis on a non-denaturing 6% polyacrylamide gel at 180 V for 2 h under low ionic strength conditions. The gel was dried and subjected to autoradiography as described previously (An et al. 1993; Xu et al. 2001). Statistical analysis Results are expressed as mean ± SD. Differences among groups were analyzed by one-way ANOVA followed by Bonferroni’s posthoc t-test to determine statistical significance. Comparison between two experimental groups was based on a 2-tailed t-test. A p-value < 0.05 was considered statistically significant.

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Fig. 2 Ab and C2-ceramide induce JNK phosphorylation. OLGs were grown in the presence or absence of Ab (20 lM) or C2-ceramide (25 lM) for varying periods of time (0.5–24 h), as indicated. Cells were fractionated and Western blots were performed to detect phosphoJNK (JNK-p) in the nuclear fraction (Nuc) and total JNK in the cytoplasmic fraction (Cyto). A rapid increase in JNK-p was induced by both Ab and C2-ceramide. Dihydro-ceramide (DH, inactive form of ceramide) did not alter JNK-p at 4 h after treatment. Actin expression remained unchanged and served as a control demonstrating equal protein loading. Data shown in bar graphs are mean values of ratios of densities (JNK-p/actin) from at least three independent experiments from Western blot by densitometric analyses. *compared to no treatment (p < 0.05).

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Fig. 1 Ab and C2-ceramide induce mRNA and protein expression of proapoptotic genes. OLGs were grown in the presence or absence of Ab (20 lM) or C2-ceramide (25 lM) for varying periods of time (1–24 or 48 h), as indicated. (a) Total RNA was extracted and subjected to RT-PCR using DP5 and cyclophilin primers. (b) Western blot was performed using anti-DP5 antibody. (c) Cytochrome C release was performed by Western blots on cytoplasmic (cytosol) and mitochondrial (MT) fractions. COX IV is served as MT control and actin as cytosol control for equal protein loading. Data shown in bar graphs are mean values of ratios of densities (DP5/ cyclophilin, Fig. 1a or DP5/actin, Fig. 1b and cytochrome C/COX IV or actin, Fig. 1c) from at least three independent experiments from Western blot by densitometric analyses. *compared to no treatment (p < 0.05).

DP5 is induced by Ab and C2-ceramide treatment in OLGs We have previously reported that Ab and the cell-permeable ceramide analog, C2-ceramide, induced apoptosis in neurosphere-derived differentiated OLGs (Lee et al. 2004). To determine if the BH3-only protein, DP5, was involved in Aband C2-ceramide-induced OLG apoptosis, DP5 gene and protein expression was examined using RT-PCR and Western blot. OLGs treated with Ab (20 lM) or C2-ceramide (25 lM) demonstrated a rapid induction of DP5 mRNA and protein within hours after treatment (Figs 1a and b). In parallel with these changes in DP5 expression, cytosolic cytochrome C increased, while mitochondrial cytochrome C decreased, suggesting release from mitochondria to cytosol in a time dependent manner (Fig. 1c).

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Ab induces DP5 by activation of nSMase and JNK in OLGs 635

JNK signaling pathway is involved in Ab- and C2-ceramide-induced apoptosis It has been reported that the JNK signaling pathway mediates apoptosis by regulating expression of proapoptotic Bcl-2 family proteins (Lei et al. 2002; Lei and Davis 2003). Because of our results suggesting that DP5 was induced by Ab and C2-ceramide, we tested the hypothesis that DP5 expression was regulated by JNK activation. Phosphorylation of JNK was determined by Western blot, using antiphospho-JNK antibodies. Ab (20 lM) and C2-ceramide (25 lM) rapidly increased JNK phosphorylation within 1 h (Fig. 2). Dihydro-ceramide which is an inactive ceramide as a negative control, did not affect JNK phosphorylation at 4 h

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Fig. 3 JNK siRNA attenuates Ab-induced DP5 expression and AP-1 activation. OLGs were grown in the presence or absence (control, C) of JNK siRNA (Si) or non-specific siRNA (NS) for 24 h, followed by treatment with Ab (20 lM) or C2-ceramide (25 lM) for another 12 h for the protein levels of JNK-p, JNK and 8 h for AP1 binding activity. Nuclear proteins (for detection of phospho-JNK) and cytoplasmic proteins (for detection of DP5 and actin) were subjected to Western blot analysis using antiphospho-JNK, anti-DP5 or antiactin antibodies (a). JNK Si RNA attenuated basal JNK level, Ab-induced JNK and JNK-p (a), as well as DP5 expression (b). In a parallel set of cultures treated as above except that treatment time with Ab and c2-ceramide

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was 8 h, nuclear proteins were extracted for AP-1 binding by EMSA (c). JNK siRNA decreased AP-1 DNA binding induced by both Ab and C2-ceramide. Lane 1: free probe; Lane 2: 50 fold of cold AP-1 oligonucleitide consensus sequence for competition. Data in bar graphs shown are mean values of ratios of densities (JNK-p or JNK/actin, DP5/actin) from at least three independent experiments by Western blot (Fig. 3a and b) with densitometric analyses. Data in bar graphs shown are mean values of relative intensities from the bands of AP1 DNA binding detected by EMSA (Fig. 3c) with densitometric analyses. *compared to no siRNA treatment (p < 0.05).

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level), while the non-specific siRNA did not reduce JNK level (Fig. 3a). Ab- and C2-ceramide-induced phospho-JNK (JNK-p) was inhibited by JNK siRNA in nuclei (Fig. 3a). Ab- and C2-ceramide-induced DP5 expression was significantly reduced by treatment with JNK siRNA (Fig. 3b); the non-specific siRNA did not reduce DP5 expression compared to control. The protein c-Jun, a known substrate of JNK, is a component of the AP-1 transcription factor, and AP-1 DNA binding consensus sequences are present in the promoter region of DP5 (predicted using Transcription Element Search System – TESS, http://www.cbil.upenn.edu/tess). Phosphorylation of c-Jun results in heterodimer formation of AP-1 components, increased DNA-binding activities, and up-regulation of target genes (Lee et al. 1998; Marinissen et al. 2004; Nateri et al. 2004). Therefore, we investigated the possibility that Ab and C2-ceramide induced DP5 gene expression by increasing AP-1 DNA binding activity, using an electrophoresis mobility shift assay (EMSA). AP-1 DNA binding activities in nuclear extracts from OLGs were increased with Ab and C2-ceramide treatment for 8 h. This increased DNA binding activities were significantly reduced by pre-treatment with JNK siRNA, but were not reduced by the non-specific siRNA (Fig. 3c). To examine if inhibition of JNK activity with JNK siRNA altered Ab- or C2-ceramideinduced OLG death, we performed an MTT assay. OLG cell death induced by both agents was attenuated by JNK siRNA, but unaltered by non-specific siRNA (Fig. 4a). In addition, Ab- and C2-ceramide induced cell death, AP-1 binding activity and DP5 expression was decreased by the specific JNK inhibitor, SP600125 (Figs 4b,c and d). Collectively, these data suggest that Ab and C2-ceramide induce OLG apoptosis via activation of JNK, increased AP-1 DNA

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nSMase activation occurs upstream of JNK activation We have recently reported that Ab-induced OLG death was mediated, at least in part, through the nSMase-ceramide pathway (Lee et al. 2004). In addition, numerous reports indicate that ceramide activates JNK in a variety of cell types (Pascual et al. 2003; Willaime-Morawek et al. 2003). Therefore, we sought to determine if nSMase was an upstream activator of JNK in Ab- and C2-ceramide-induced apoptosis in OLGs. It has been known that both nSMase inhibitor, 3-O-methyl-sphingomyelin and nSMase antisense oligonucleotides inhibited nSMase activities (data not shown; also see Lee et al. 2004; Zeng et al. 2005). OLGs, pre-treated with the nSMase inhibitor, 3-O-methyl-sphingomyelin (I, 1 lM) for 1 h followed by Ab (20 lM) treatment for 24 h, were subjected to Western blot using antiphosphoJNK, anti-DP5, anticytochrome C, or antiactin antibodies. Inhibition of nSMase with 3-O-methyl-sphingomyelin resulted in a significant attenuation of Ab-induced phosphorylation of JNK (Fig. 5a), and DP5 expression, as well as cytosolic cytochrome C release (Fig. 5b). Furthermore, inhibition of nSMase with antisense oligonucleotides or with 3-O-methyl-sphingomyelin, resulted in a decrease in Ab-induced AP-1 DNA binding activity (Fig. 5c). These data suggest that nSMase is activated upstream of JNK, resulting in AP-1 DNA binding, up-regulation of DP5 expression, and subsequent cytochrome C release from mitochondria. Neutral SMase antisense oligonucleotides had no effect on C2-ceramide-induced AP-1 DNA binding (Fig. 5C), suggesting that ceramide acted downstream of nSMase.

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24 h for DP5 expression by Western blotting (d). Inhibition of JNK activity by JNK si RNA or by SP600125 attenuated Ab-induced OLG cell death (*compared to the control; **compared to Ab or C2 alone). Shown are representative data from three independent experiments with triplicates.

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Ab induces DP5 by activation of nSMase and JNK in OLGs 637

(a)

C

I

Aβ I+Aβ

JNK-P Actin (b)

C

Aβ I+Aβ

DP5 Cyt C (c)

Cont



C S AS S AS I

C2 S AS

AP-1 DNA complex

Labelled probe Fig. 5 Inhibition of nSMase decreases Ab-induced JNK phosphorylation, DP5 expression, cytochrome C and AP-1 binding activity. OLGs were grown in the presence or absence of the nSMase inhibitor (I), 3O-methyl-sphingomyelin (1 lM) for 1 h, followed by Ab (20 lM) for 24 h. Nuclear proteins (for detection of phospho-JNK, panel a) and cytoplasmic proteins (for detection of DP5 and cytochrome c, panel b) were analyzed by Western blot. Inhibition of nSMase resulted in an attenuation of Ab-induced JNK phophorylation (a), DP5 expression, and cytochrome C release (b). In another experiment OLGs were grown in the presence of 1.4 lM nSMase antisense oligonucleotides (AS), nSMase scrambled oligonucleotides (S) or nSMase inhibitor (I) for 4 h, followed by treatment with 20 lM Ab or 25 lM C2-ceramide for 24 h. Nuclear proteins were extracted for EMSA (C). Neutral SMase inhibition resulted in a marked attenuation of Ab-induced AP-1 DNA binding activity. Shown are representative blots from three independent experiments.

Discussion

We have found that Ab rapidly induced DP5 expression in OLGs in a time-dependent manner. DP5 induction, which preceded cytochrome C release from mitochondria to cytosol, occurred in parallel with JNK phosphorylation (total JNK levels remained unchanged during this period of time). Furthermore, inhibition of JNK activation with JNK siRNA reduced JNK induction in basal level, prevented Ab-induced DP5 induction, AP-1 DNA binding, and OLG cell death. The nSMase inhibitor, 3-O-methyl-sphingomyelin, also prevented Ab-induced JNK activation, AP-1 binding, DP5 expression and cytochrome C release. We have previously reported that nSMase inhibition attenuated Ab-induced OLG death (Lee et al. 2004). Therefore, our data suggest that Ab

induces apoptosis in OLGs by initiating a series of events: nSMase activation and ceramide generation, followed by JNK activation and an increase in AP-1 DNA binding, induction of DP5 expression, cytochrome C release and the activation of downstream apoptotic pathway. In this study, we report the novel finding that nSMase activity and ceramide generation may regulate DP5 gene induction by Ab, via JNK activation. This contention is supported by several lines of evidence. Both Ab and C2ceramide rapidly induced DP5 gene expression and both agents increased JNK phosphorylation and AP-1 DNA binding. C-Jun, which is a component of the AP-1 transcription factor, is an exclusive substrate of JNK (Marinissen et al. 2004; Nateri et al. 2004); therefore, JNK activation (via phosphorylation) would be expected to activate c-Jun and AP-1 DNA binding. Select target genes with AP-1 consensus sequences in their promoter, such as DP5, are subsequently up-regulated. Further support is derived from the experiments of blocking nSMase activity (Fig. 5). 3-O-methyl-sphingomyelin, a specific inhibitor of nSMase, attenuated Ab-induced JNK activation, AP-1 DNA binding, and DP5 gene expression. Our findings that Ab induced DP5 induction via JNK activation are in agreement with several recent studies. Imaizumi et al. (Imaizumi et al. 1999) reported that Ab induced DP5 expression in rat cortical neurons undergoing apoptosis. In another study the JNK inhibitor, CEP-1347, attenuated DP5 expression and neuronal cell death induced by Ab (Bozyczko-Coyne et al. 2001). The neurons cultured from DP5 knockout mice display more resistance to NGF deprivation compared to the cultures from wild-type mice indicating that DP5 causes cell death (Imaizumi et al. 2004). The regulation of DP5 induction during apoptosis by JNK has also been described in cerebellar granule cells deprived of serum and potassium, a Bax-dependent process (Harris and Johnson 2001; Harris et al. 2002). Like other members of the BH3-only family, DP5 interacts with other Bcl-2 family regulators on the outer mitochondrial membrane (Harris and Johnson 2001; Harris et al. 2002). Through a poorly understood process, this interaction results in the release of cytochrome C from mitochondria to cytosol, and subsequent activation of enzymes in the effector arm of apoptosis (Bozyczko-Coyne et al. 2001). The involvement of ceramide as a mediator in the pathogenesis of AD is supported by several studies which report lower sphingomyelin and higher ceramide levels in AD brains compared to controls (Soderberg et al. 1992). More recent findings suggest that this profile of sphingomyelin/ceramide changes is more pronounced in the white matter compared to grey matter in AD brains (Han et al. 2002). These data suggest an increase in sphingomyelin degradation and accumulation of ceramide, and are consistent with our finding that nSMase is activated by Ab in OLGs (Xu et al. 2001; Lee et al. 2004).

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Increasing evidence demonstrates that there is marked damage and dysfunction not only in the grey matter but also in the white matter in AD. It was reported that Ab deposition and levels increase over time in transgenic mice overexpressing b-amyloid precursor protein under control of the platelet-derived growth factor promoter mice. These changes lead to primary or secondary white matter injury (Song et al. 2004). Brain imaging and morphometric technologies such as magnetic resonance imaging (MRI) and computed stereology have demonstrated that in normal aging there is a proportionately greater loss of white matter relative to grey matter (Tang et al. 1997; Guttmann et al. 1998), and these differences are even more pronounced in AD patients. Biochemical analyses of AD white matter suggest that extensive white matter axonal demyelination underlies Alzheimer’s pathology, resulting in loss of capacitance and serious disturbances in nerve conduction, severely damaging brain function. These white matter alterations undoubtedly contribute to AD pathogenesis (Roher et al. 2002). Indeed, pathology in the white matter of AD brains is increasingly recognized as a consequence of the disease, evidenced by the frequency of MRI abnormalities found in the white matter of AD patients and transgenic mouse model (Bozzali et al. 2002; Song et al. 2004). In addition, a high density of apoptotic OLGs has been reported in white matter lesions of AD brains (Kobayashi et al. 2002). In summary, we have found that Ab-activated nSMase/ ceramide pathway upstream of JNK and DP5 which may play a role in Ab-induced OLG apoptosis, and suggest a mechanism for white matter pathology in AD. Based on our current and previous studies we propose the following model: Ab induces OLG apoptosis through a sequence of molecular events including activation of nSMase-ceramide pathway, activation of JNK, increased AP-1 DNA binding activity, up-regulation of DP5 expression, subsequent cytochrome C release from mitochondria, and activation of apoptotic cascades. Acknowledgements This work was supported by National Institute of Health (NIH) grants RO1 NS40525 (JX), NS40162 (JX), NS51625 (JML), P01 NS51644 (JML), American Heart Association (AHA) 0050597 N (JX) and 0460066Z (JML). We thank Dr QL Xiao for technical assistance.

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