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Alcohol enhances Aβ42-induced neuronal cell death through mitochondrial dysfunction
FEBS Letters 582 (2008) 4185–4190
Alcohol enhances Ab42-induced neuronal cell death through mitochondrial dysfunction Do Yeon Leea,b,1, Kyu-Sun Leec,1, Hyun Jung Leea, Hee-Yeon Jungd, Jun Young Leed, Sang Hyung Leee, Young Chul Younf, Kyung Mook Seog, Jang Han Leeh, Won Bok Leea, Sung Su Kima,* a
c
Department of Anatomy and Cell biology, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea b Department of Herbal Resources Research, Korea Institute of Oriental Medicine, Daejeon 305-811, Republic of Korea Center for Regenerative Medicine, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea d Department of Psychiatry, College of Medicine, Seoul National University, Seoul 151-742, Republic of Korea e Department of Neurosurgery, College of Medicine, Seoul National University, Seoul 151-742, Republic of Korea f Department of Neurology, College of Liberal Arts, Chung-Ang University, Seoul 156-756, Republic of Korea g Department of Rehabilitation medicine, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea h Department of Psychology, College of Liberal Arts, Chung-Ang University, Seoul 156-756, Republic of Korea Received 5 October 2008; revised 2 November 2008; accepted 7 November 2008 Available online 19 November 2008 Edited by Jesus Avila
Abstract Mitochondrial dysfunction is a hallmark of beta-amyloid (Ab)-induced neuronal toxicity in AlzheimerÕs disease (AD). Epidemiological studies have indicated that alcohol consumption plays a role in the development of AD. Here we show that alcohol exposure has a synergistic effect on Ab-induced neuronal cell death. Ab-treated cultured neurons displayed spontaneous generation of reactive oxygen species (ROS), disruption of their mitochondrial membrane potential, induction of caspase-3 and p53 activities, and loss of cell viability. Alcohol exposure facilitated Ab-induced neuronal cell death. Our study shows that alcohol consumption enhances Ab-induced neuronal cell death by increasing ROS and mitochondrial dysfunction. Crown Copyright Ó 2008 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. All rights reserved. Keywords : : AlzheimerÕs disease; Ab; Ethanol; Mitochondria; ROS
1. Introduction AlzheimerÕs disease (AD), a neurodegenerative disorder clinically characterized by progressive dementia, is associated with neurofibrillary tangles and amyloid beta (Ab) plaques [1,2]. Since the discovery of the 4 kDa Ab peptide, much research has focused on understanding Ab toxicity and its relationship with AD progression and pathogenesis. Ab peptides are cleaved from amyloid precursor protein (APP) through the sequential proteolysis of aspartyl b-secretase and presenilindependent c-secretase in AD brains [3–5]. The progressive accumulation of Ab aggregates eventually triggers a cascade of cellular changes, including mitochondrial oxidative damage, hyperphosporylation of tau, synaptic failure, and inflammation [6–8].
*
Corresponding author. Fax: +82 2 826 1265. E-mail address: [email protected] (S.S. Kim). 1
These authors contributed equally to this work.
Mitochondrial dysfunction in AD patients has been well documented [6,7]. Several in vitro studies showed that Ab affects mitochondrial DNA and proteins, leading to impairments of the electronic transport chain (ETC) and, ultimately, to mitochondrial dysfunction [9,10]. Recently, Lustbader et al. [11] reported that Ab-binding alcohol dehydrogenase directly interacts with Ab in the mitochondria of AD patients and transgenic mice, and that this interaction promotes the leakage of reactive oxygen species (ROS), resulting in mitochondrial dysfunction. Investigations with animal models of fetal alcohol syndrome (FAS) suggest that neuronal vulnerability to ethanol-induced cell death may correlate with specific developmental events [12]. Ethanol exposure is a consistent and reliable producer of neuronal toxicity, particularly when the exposure occurs during periods of enhanced neuronal vulnerability. Neuronal dendritic shrinkage has been documented in alcoholics [13]. There does not appear to be a link between alcohol-related brain damage and AD [14], although some studies suggest a relationship between alcohol and aging [15]. To test the synergistic effect of ethanol in an Ab-rich environment in cultured neurons, we co-treated human neuroblastoma cells with Ab and ethanol. Here, we demonstrate that alcohol exposure is a risk factor facilitating Ab-induced neuronal cell death. Alcohol exposure had a synergistic effect on Abinduced cell stress via mitochondrial dysfunction. Inhibition of caspase-3 or p53 reduced the synergistic neurotoxicity of Ab and ethanol, whereas inhibition of Akt facilitated the susceptibility of cultured neurons to Ab and ethanol. Thus, our study shows that alcohol consumption enhances Ab-induced neuronal cell death by increasing ROS production and disruption of mitochondrial integrity.
2. Materials and methods 2.1. Cell culture and pharmacological treatments SK-N-SH human neuroblastoma cells were cultured at 37 °C in DulbeccoÕs Modified EagleÕs Medium (DMEM) (Gibco-BRL) supplemented with 10% heat-inactivated FBS in a humidified 95% air, 5% CO2 incubator. For aging, Ab1-42 (Biosource Inc.) was incubated
0014-5793/$34.00 Crown Copyright Ó 2008 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. All rights reserved. doi:10.1016/j.febslet.2008.11.007
4186 for 1 week at 37 °C before use. In experiments requiring a 48 h incubation with ethanol (Sigma), the culture plates were sealed with plastic tape and wrapped in plastic wrap to prevent ethanol evaporation. 2.2. Cell viability assay Ab- and ethanol-treated cells were stained with 10 ll of alamarBlue (Serotec) [16]. For assessment of apoptosis, cells were fixed with 4% paraformaldehyde and then stained with 8 mg/ml of Hoechst dye 33258 (Sigma) for 5 min. Nuclear morphology was visualized using a fluorescence microscope (IX70 microscope, Olympus). 2.3. Determination of ROS generation Ab- or ethanol-induced hydrogen peroxide generation was measured by incubating the cells with the fluorescent probe 2 0 ,7 0 -dichlorofluorescein diacetate (DCF-DA) (Sigma). DCF fluorescein intensity was measured with a fluorometer (TECAN, GENios). 2.4. Effect of p53 antisense oligonucleotides and Akt inhibitor on neuronal viability To access the role of p53 and Akt inhibitor on cell viability in Ab- and ethanol-mediated neuronal cell death, antisense 20-mer phosphorothioate oligonucleotides were synthesized and added to the culture medium at a final concentration of 10 lM 6 h prior to
D.Y. Lee et al. / FEBS Letters 582 (2008) 4185–4190 Ab and/or ethanol addition. The p53 sequence of the p53 antisense olignucleotides (5 0 -CGCTAGGATCTGACTGC-3 0 ) was complementary to the translation initiation site of the human p53 mRNA. Akt inhibitor, LY294002 (CalBiochem), also pre-treated for 2 h before Ab and/or ethanol addition in culture medium. 2.5. RT-PCR analysis and Western blotting Total RNA was isolated from the cells by using Trizol Reagent (Invitrogen) according to the manufacturerÕs instructions. cDNA was synthesized by using Superscript II Reverse Transcription system (Invitrogen). For RT-PCR, AccuPower PCR premix (Bioneer) was mixed with each primer. Catalase primers 5 0 -TTAATCCATTCGATCTCACC-3 0 (forward), 5 0 -GGCGGTGAGTGTCAGGATAG-3 0 (reverse); Cu/ZnSOD primers 5 0 -CAGTGCAGGTCCTCACTTTA-3 0 (forward), 5 0 -CCTGTCTTTGTACTTTCTTC-3 0 (reverse); MnSOD primers 5 0 -GTTTTGGGGTATCTGGGCTC-3 0 (forward), 5 0 -TCCCTGATTTGGACAAGCAGC-3 0 (reverse); GAPDH-specific primers 5 0 -GGGGCTCTCCAGAACATCAT-3 0 (for(reverse). To ward), 5 0 -AAGTGGTCGTTGAGGGCAAT-3 0 measure the expression levels of p53 and phospho-Akt, immunoblotting was performed with anti-p53 and anti-phospho-Akt (Santa Cruz Biotech, CA, USA). An anti-b-actin antibody (SantaCruz Biotechology; 1:1000 dilution) was used as a loading control. Protein preparation and immunoblotting were performed as described [16].
Fig. 1. Ab and EtOH synergistically enhance neuronal apoptosis in SK-N-SH cells. (A) The relative cell viability was decreased in all cases compared with the untreated control. (B) Morphological changes in cultured SK-N-SH cells treated with Ab and/or EtOH. (C) Effect of Ab and EtOH on induction of caspase-3 activity. (D) Cells were stained with Hoechst 33258 staining. Dead cells were identified by morphological changes, such as nuclei condensation and fragmentation (arrow), compared with normal cell nuclei (arrowhead). Data were presented as means ± S.D. from at least three independent experiments. Scale bar is 20 lm. *P < 0.05, **P < 0.001.
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2.6. Analysis of mitochondrial membrane potential (DWm) and activity of caspase-3 Changes in DWm were estimated using tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes). Carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP) was used as a positive control for complete mitochondrial depolarization. Caspase-3 activities were measured as described [16]. 2.7. Statistical analysis All data are expressed as the means ± S.D. Statistical significance was determined via ANOVA followed by ScheffeÕs post hoc tests. A P-value of <0.05 was considered to be significant.
3. Results 3.1. Characterization of the synergistic neurotoxicity effects of combined treatment with Ab and ethanol We first performed a series of experiments to evaluate the neurotoxicity effects of single agent treatment with Ab or ethanol on cell survival in human neuroblastoma cell line, SK-N-SH. To confirm the synergistic cytotoxic effect of Ab and ethanol, we also co-treated with Ab and ethanol under the same conditions. At a concentration of 1 lM, Ab showed no cell-killing effects, even after 48 h of treatment, compared with un-treated control; however, this concentration did selectively inhibit neurite growth. In contrast, cell viability decreased with time when cells were treated with 15 mM ethanol, which is a similar concentration of the drink/drive limit in the USA and UK (0.08% w/v, 17.4 mM) (Fig. 1A). Interestingly, co-treatment with Ab and ethanol showed a significant synergistic effect on cell death compared to treatment with Ab or ethanol alone for 48 h (Fig. 1A). This synergistic effect on neuronal cell death was also detected in the morphological changes (Fig. 1B and D) and caspase-3 activation (Fig. 1C).
Fig. 2. Effects of Ab and EtOH on ROS production by and antioxidant enzyme mRNA expression in SK-N-SH cells. (A) Aband EtOH-induced ROS generation was measured by incubation with DCF-DA. Data were presented as means ± S.D. from three independent experiments. *P < 0.05. (B) The expression of antioxidant genes was measured by RT-PCR analysis. GAPDH is used for a loading control.
3.2. Synergistic effects of Ab and ethanol in triggering oxidative stress To determine whether Ab and/or ethanol treatment induces oxidative stress in cultured neurons, we measured the level of ROS production using a DCF-DA-dependent fluorescence assay. When SK-N-SH neurons were exposed to Ab or ethanol alone, intracellular ROS levels did not significantly change. However, ROS production was significantly increased in cotreated with both Ab and ethanol (Fig. 2A). To determine whether a combined treatment with Ab and ethanol disturbs the internal defense mechanism for ROS, we examined the expression of antioxidant genes. ROS are regulated by endogenous antioxidant defense systems, which include catalase, superoxide dismutase (SOD), and glutathione peroxidase [17]. Of these antioxidant genes, catalase and mitochondrial manganese-containing MnSOD, but not cytosolic Cu/ZnSOD, decreased following combination treatment with Ab and ethanol (Fig. 2B). Expression of catalase and MnSOD were decreased by 3-fold and 5-fold, respectively. These results indicate that Ab and ethanol have a synergistic effect on ROS generation in SK-N-SH cells.
cause a change in p53 expression. In contrast, p53 was significantly increased after 3 h following co-treatment of cells with Ab and ethanol (Fig. 3A). To further demonstrate that p53 induction correlates with cell death, we blocked the expression of the p53 protein by pre-incubating with p53-specific antisense oligonucleotides. Inhibition of p53 (p53 AS) significantly rescued the synergistic cell death effect of Ab and ethanol in SK-N-SH cells (Fig. 3B). We also investigated whether a combined treatment with Ab and ethanol influences neuronal cell survival signaling by measuring the activity of Akt with a phospho-specific Akt antibody. The level of pAkt in neurons was significantly decreased after 3 h with a combined treatment with Ab and ethanol (Fig. 3C). In addition, pre-treatment of Akt specific inhibitor, LY294002, significantly enhanced the synergistic Ab- and ethanol-induced neuronal cell death (Fig. 3D). Together, these findings suggest that Ab and ethanol show synergistic effects on neuronal cell death through activating the p53dependent apoptotic pathway and inhibiting Akt-dependent survival signaling.
3.3. Synergistic effects of Ab and ethanol on apoptotic or survival pathways To investigate whether Ab and ethanol have a synergistic effect on apoptosis, we measured the expression of pro-apoptotic marker protein p53. Treatment of Ab or ethanol alone did not
3.4. Effects of p53 antisense oligonucleotide and pAkt inhibitor on ROS generation To demonstrate that p53 and Akt signaling directly influence the ROS production, we pre-treated cultured neurons with a p53 antisense oligonucleotide or an Akt inhibitor before mea-
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Fig. 3. p53 and pAkt are involved in Ab- and EtOH-induced neuronal cell death in SK-N-SH cells. Western blot analysis was performed using antip53 (A) and anti-pAkt (C) antibodies. Beta-actin is used for a protein loading control. Involvement of p53 and pAkt in Ab- and EtOH-induced cell viability. Cells were pre-treated with p53 antisense oligonucleotides (B, open squares) or with LY294002 (D, open squares). Data were represented as means ± S.D. from three independent experiments. *P < 0.05 versus vehicle alone, Ab alone, or EtOH alone; **P < 0.05 versus Ab/EtOH together.
suring the ROS level. As above, we confirmed that combined treatment with Ab and ethanol significantly increased ROS production in SK-N-SH cells (Fig. 2A). Inhibition of p53 significantly reduced ROS production in Ab plus ethanol-treated cells, similar to the positive control group where cells were pretreated for 2 h with a well-known glutathione precursor, NAC (Fig. 4A). Glutathione plays an important role in the detoxification of ROS in brain [18]. In contrast, Akt inhibition did not significantly change ROS production.
duce depolarization of DWm. Pre-treatment with p53 antisense oligonucleotides attenuated and an Akt inhibitor enhanced this synergistic effect on DWm reduction (Fig. 4B), indicating that p53 and/or Akt plays a role in Ab- and ethanol-induced depolarization of DWm.
3.5. Effects of p53 antisense oligonucleotide and pAkt inhibitor on mitochondrial membrane potential To investigate the synergistic effect of Ab and ethanol on mitochondrial integrity, we examined the mitochondrial membrane potential (DWm) with TMRE staining. A slight disruption of DWm was observed in neurons treated with Ab or ethanol alone. Co-treatment with Ab and ethanol triggered the collapse of DWm, whereas pre-treatment with a p53 antisense oligonucleotide significantly blocked DWm depolarization (Fig. 4B). In contrast, Akt inhibition slightly enhanced the depolarization of DWm in cultured neurons co-treated with Ab and ethanol. Treatment with NAC, the p53 antisense oligonucleotide, or the Akt inhibitor alone did not affect DWm. These results indicate that Ab and ethanol synergistically in-
The results of this study are the first to show the synergistic effects of ethanol exposure and an Ab-rich environment, relative to ROS production, disruption of mitochondrial integrity, and cytotoxicity. In late-onset, sporadic AD, an age-dependent increase of ROS has been identified as a key factor in the development and progression of AD [1,8]. In this study, we suggest that alcohol poisoning increases the risk of sporadic AD, with damage to the neuronal tissues or cells through enhanced ROS production. It is well recognized that one of the main effects of ROS exposure is damage to DNA molecules. Among the proteins that play an important role in the cellular response to DNA damage is the tumor suppressor p53 [19]. Neuronal cells in which p53 was inhibited were less vulnerable to ROS-induced
4. Discussion
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Fig. 4. Effect of p53 and pAkt on Ab- and EtOH-induced synergistic induction of ROS production and loss of mitochondrial membrane potential (DWm) in SK-N-SH cells. ROS (A) and DWm (B) were analyzed using DCF-DA and TMRE. Data were presented as means ± S.D. from three independent experiments. FCCP was used as a positive control for complete mitochondrial depolarization. *P < 0.05 versus vehicle alone, Ab alone, or EtOH alone; **P < 0.05 versus Ab/EtOH together.
oxidative injury. This protective mechanism involved the impairment of ROS-activated, p53-dependent apoptosis. Over the past decade, the Akt pathway has been shown to be sufficient, and in some cases necessary, for the growth factorinduced cell survival of several neuronal cell types [20]. Akt may induce the expression of survival genes, such as Bcl-XL and A1, by activating CREB or NF-jB, and directly inhibits members of the apoptotic machinery [20]. In this study, we show that neuronal cell death induced by Ab and ethanol through decreasing Akt activity. Recently, Smith et al. [21] proposed that Ab toxicity is elicited through the generation of ROS, leading to activation of the JNK pathway, which in turn activates p66Shc by phosphorylation at Ser36. Activated p66Shc then triggers the phosphorylation of FKH, thereby down-regulating target genes, such as MnSOD, and leading to an even greater accumulation of cellular ROS. This biochemical cycle causes cell death. Here, we found that MnSOD expression was significantly decreased by combined treatment with Ab and ethanol.
Mitochondria are unique organelles in the consumption of oxygen, production of ATP, generation of oxygen radicals, and mobilization of calcium [22]. Mitochondrial dysfunction is evaluated by multiple parameters of mitochondrial integrity. Regulation of changes in mitochondrial membrane potential (DWm), the release of cytochrome c from the mitochondria into the cytoplasm and the activation of caspase-9 are important processes during mitochondria-dependent apoptotic process [23]. In this study, depolarization of DWm was increased by over half in combined treatment with Ab and ethanol. This study demonstrates that a low-dose of Ab or ethanol alone does not affect cell viability, whereas the combined exposure to both two agents activates apoptotic pathways through ROS production and the disruption of mitochondrial integrity. This study also indicates that the p53 and Akt pathways contribute to pro- or anti-apoptosis in cultured neurons. Acknowledgement: This work was supported by Grant (20080401034034) from BioGreen 21 Program, Rural Development Administration, Republic of Korea.
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References [1] Mattson, M.P. (2004) Pathways towards and away from AlzheimerÕs disease. Nature 430, 631–639. [2] Tanzi, R.E. and Bertram, L. (2005) Twenty years of the AlzheimerÕs disease amyloid hypothesis: a genetic perspective. Cell 120, 545–555. [3] Hardy, J. and Selkoe, D.J. (2002) The amyloid hypothesis of AlzheimerÕs disease: progress and problems on the road to therapeutics. Science 297, 353–356. [4] Sisodia, S.S., Annaert, W., Kim, S.H. and De Strooper, B. (2001) Gamma-secretase: never more enigmatic. Trends Neurosci. 24, S2–S6. [5] Walsh, D.M., Klyubin, I., Fadeeva, J.V., Cullen, W.K., Anwyl, R., Wolfe, M.S., Rowan, M.J. and Selkoe, D.J. (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535–539. [6] Hirai, K. et al. (2001) Mitochondrial abnormalities in AlzheimerÕs disease. J. Neurosci. 21, 3017–3023. [7] Reddy, P.H. (2006) Amyloid precursor protein-mediated free radicals and oxidative damage: implications for the development and progression of AlzheimerÕs disease. J. Neurochem. 96, 1–13. [8] Tanzi, R.E. (2005) The synaptic Abeta hypothesis of Alzheimer disease. Nat. Neurosci. 8, 977–979. [9] Cardoso, S.M., Santos, S., Swerdlow, R.H. and Oliveira, C.R. (2001) Functional mitochondria are required for amyloid betamediated neurotoxicity. FASEB J. 15, 1439–1441. [10] Casley, C.S., Canevari, L., Land, J.M., Clark, J.B. and Sharpe, M.A. (2002) Beta-amyloid inhibits integrated mitochondrial respiration and key enzyme activities. J. Neurochem. 80, 91–100. [11] Lustbader, J.W. et al. (2004) ABAD directly links Abeta to mitochondrial toxicity in AlzheimerÕs disease. Science 304, 448–452. [12] Berman, R.F. and Hannigan, J.H. (2000) Effects of prenatal alcohol exposure on the hippocampus: spatial behavior, electrophysiology, and neuroanatomy. Hippocampus 10, 94–110.
D.Y. Lee et al. / FEBS Letters 582 (2008) 4185–4190 [13] Harper, C. and Corbett, D. (1990) Changes in the basal dendrites of cortical pyramidal cells from alcoholic patients—a quantitative Golgi study. J. Neurol. Neurosurg. Psychiatr. 53, 856– 861. [14] Morikawa, Y. et al. (1999) Cerebrospinal fluid tau protein levels in demented and nondemented alcoholics. Alcohol Clin. Exp. Res. 23, 575–577. [15] Caine, D., Halliday, G.M., Kril, J.J. and Harper, C.G. (1997) Operational criteria for the classification of chronic alcoholics: identification of WernickeÕs encephalopathy. J. Neurol. Neurosurg. Psychiatr. 62, 51–60. [16] Lee do, Y. et al. (2008) Kynurenic acid attenuates MPP(+)induced dopaminergic neuronal cell death via a Bax-mediated mitochondrial pathway. Eur. J. Cell Biol. 87, 389–397. [17] Finkel, T. and Holbrook, N.J. (2000) Oxidants, oxidative stress and the biology of ageing. Nature 408, 239–247. [18] Dringen, R. (2000) Metabolism and functions of glutathione in brain. Prog. Neurobiol. 62, 649–671. [19] Grilli, M. and Memo, M. (1999) Possible role of NF-kappaB and p53 in the glutamate-induced pro-apoptotic neuronal pathway. Cell Death Differ. 6, 22–27. [20] Brunet, A., Datta, S.R. and Greenberg, M.E. (2001) Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr. Opin. Neurobiol. 11, 297– 305. [21] Smith, W.W., Norton, D.D., Gorospe, M., Jiang, H., Nemoto, S., Holbrook, N.J., Finkel, T. and Kusiak, J.W. (2005) Phosphorylation of p66Shc and forkhead proteins mediates Abeta toxicity. J. Cell Biol. 169, 331–339. [22] Melov, S. (2000) Mitochondrial oxidative stress. Physiologic consequences and potential for a role in aging. Ann. NY Acad. Sci. 908, 219–225. [23] Kroemer, G. and Reed, J.C. (2000) Mitochondrial control of cell death. Nat. Med. 6, 513–519.
Alcohol enhances Ab42-induced neuronal cell death through mitochondrial dysfunction Do Yeon Leea,b,1, Kyu-Sun Leec,1, Hyun Jung Leea, Hee-Yeon Jungd, Jun Young Leed, Sang Hyung Leee, Young Chul Younf, Kyung Mook Seog, Jang Han Leeh, Won Bok Leea, Sung Su Kima,* a
c
Department of Anatomy and Cell biology, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea b Department of Herbal Resources Research, Korea Institute of Oriental Medicine, Daejeon 305-811, Republic of Korea Center for Regenerative Medicine, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea d Department of Psychiatry, College of Medicine, Seoul National University, Seoul 151-742, Republic of Korea e Department of Neurosurgery, College of Medicine, Seoul National University, Seoul 151-742, Republic of Korea f Department of Neurology, College of Liberal Arts, Chung-Ang University, Seoul 156-756, Republic of Korea g Department of Rehabilitation medicine, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea h Department of Psychology, College of Liberal Arts, Chung-Ang University, Seoul 156-756, Republic of Korea Received 5 October 2008; revised 2 November 2008; accepted 7 November 2008 Available online 19 November 2008 Edited by Jesus Avila
Abstract Mitochondrial dysfunction is a hallmark of beta-amyloid (Ab)-induced neuronal toxicity in AlzheimerÕs disease (AD). Epidemiological studies have indicated that alcohol consumption plays a role in the development of AD. Here we show that alcohol exposure has a synergistic effect on Ab-induced neuronal cell death. Ab-treated cultured neurons displayed spontaneous generation of reactive oxygen species (ROS), disruption of their mitochondrial membrane potential, induction of caspase-3 and p53 activities, and loss of cell viability. Alcohol exposure facilitated Ab-induced neuronal cell death. Our study shows that alcohol consumption enhances Ab-induced neuronal cell death by increasing ROS and mitochondrial dysfunction. Crown Copyright Ó 2008 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. All rights reserved. Keywords : : AlzheimerÕs disease; Ab; Ethanol; Mitochondria; ROS
1. Introduction AlzheimerÕs disease (AD), a neurodegenerative disorder clinically characterized by progressive dementia, is associated with neurofibrillary tangles and amyloid beta (Ab) plaques [1,2]. Since the discovery of the 4 kDa Ab peptide, much research has focused on understanding Ab toxicity and its relationship with AD progression and pathogenesis. Ab peptides are cleaved from amyloid precursor protein (APP) through the sequential proteolysis of aspartyl b-secretase and presenilindependent c-secretase in AD brains [3–5]. The progressive accumulation of Ab aggregates eventually triggers a cascade of cellular changes, including mitochondrial oxidative damage, hyperphosporylation of tau, synaptic failure, and inflammation [6–8].
*
Corresponding author. Fax: +82 2 826 1265. E-mail address: [email protected] (S.S. Kim). 1
These authors contributed equally to this work.
Mitochondrial dysfunction in AD patients has been well documented [6,7]. Several in vitro studies showed that Ab affects mitochondrial DNA and proteins, leading to impairments of the electronic transport chain (ETC) and, ultimately, to mitochondrial dysfunction [9,10]. Recently, Lustbader et al. [11] reported that Ab-binding alcohol dehydrogenase directly interacts with Ab in the mitochondria of AD patients and transgenic mice, and that this interaction promotes the leakage of reactive oxygen species (ROS), resulting in mitochondrial dysfunction. Investigations with animal models of fetal alcohol syndrome (FAS) suggest that neuronal vulnerability to ethanol-induced cell death may correlate with specific developmental events [12]. Ethanol exposure is a consistent and reliable producer of neuronal toxicity, particularly when the exposure occurs during periods of enhanced neuronal vulnerability. Neuronal dendritic shrinkage has been documented in alcoholics [13]. There does not appear to be a link between alcohol-related brain damage and AD [14], although some studies suggest a relationship between alcohol and aging [15]. To test the synergistic effect of ethanol in an Ab-rich environment in cultured neurons, we co-treated human neuroblastoma cells with Ab and ethanol. Here, we demonstrate that alcohol exposure is a risk factor facilitating Ab-induced neuronal cell death. Alcohol exposure had a synergistic effect on Abinduced cell stress via mitochondrial dysfunction. Inhibition of caspase-3 or p53 reduced the synergistic neurotoxicity of Ab and ethanol, whereas inhibition of Akt facilitated the susceptibility of cultured neurons to Ab and ethanol. Thus, our study shows that alcohol consumption enhances Ab-induced neuronal cell death by increasing ROS production and disruption of mitochondrial integrity.
2. Materials and methods 2.1. Cell culture and pharmacological treatments SK-N-SH human neuroblastoma cells were cultured at 37 °C in DulbeccoÕs Modified EagleÕs Medium (DMEM) (Gibco-BRL) supplemented with 10% heat-inactivated FBS in a humidified 95% air, 5% CO2 incubator. For aging, Ab1-42 (Biosource Inc.) was incubated
0014-5793/$34.00 Crown Copyright Ó 2008 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. All rights reserved. doi:10.1016/j.febslet.2008.11.007
4186 for 1 week at 37 °C before use. In experiments requiring a 48 h incubation with ethanol (Sigma), the culture plates were sealed with plastic tape and wrapped in plastic wrap to prevent ethanol evaporation. 2.2. Cell viability assay Ab- and ethanol-treated cells were stained with 10 ll of alamarBlue (Serotec) [16]. For assessment of apoptosis, cells were fixed with 4% paraformaldehyde and then stained with 8 mg/ml of Hoechst dye 33258 (Sigma) for 5 min. Nuclear morphology was visualized using a fluorescence microscope (IX70 microscope, Olympus). 2.3. Determination of ROS generation Ab- or ethanol-induced hydrogen peroxide generation was measured by incubating the cells with the fluorescent probe 2 0 ,7 0 -dichlorofluorescein diacetate (DCF-DA) (Sigma). DCF fluorescein intensity was measured with a fluorometer (TECAN, GENios). 2.4. Effect of p53 antisense oligonucleotides and Akt inhibitor on neuronal viability To access the role of p53 and Akt inhibitor on cell viability in Ab- and ethanol-mediated neuronal cell death, antisense 20-mer phosphorothioate oligonucleotides were synthesized and added to the culture medium at a final concentration of 10 lM 6 h prior to
D.Y. Lee et al. / FEBS Letters 582 (2008) 4185–4190 Ab and/or ethanol addition. The p53 sequence of the p53 antisense olignucleotides (5 0 -CGCTAGGATCTGACTGC-3 0 ) was complementary to the translation initiation site of the human p53 mRNA. Akt inhibitor, LY294002 (CalBiochem), also pre-treated for 2 h before Ab and/or ethanol addition in culture medium. 2.5. RT-PCR analysis and Western blotting Total RNA was isolated from the cells by using Trizol Reagent (Invitrogen) according to the manufacturerÕs instructions. cDNA was synthesized by using Superscript II Reverse Transcription system (Invitrogen). For RT-PCR, AccuPower PCR premix (Bioneer) was mixed with each primer. Catalase primers 5 0 -TTAATCCATTCGATCTCACC-3 0 (forward), 5 0 -GGCGGTGAGTGTCAGGATAG-3 0 (reverse); Cu/ZnSOD primers 5 0 -CAGTGCAGGTCCTCACTTTA-3 0 (forward), 5 0 -CCTGTCTTTGTACTTTCTTC-3 0 (reverse); MnSOD primers 5 0 -GTTTTGGGGTATCTGGGCTC-3 0 (forward), 5 0 -TCCCTGATTTGGACAAGCAGC-3 0 (reverse); GAPDH-specific primers 5 0 -GGGGCTCTCCAGAACATCAT-3 0 (for(reverse). To ward), 5 0 -AAGTGGTCGTTGAGGGCAAT-3 0 measure the expression levels of p53 and phospho-Akt, immunoblotting was performed with anti-p53 and anti-phospho-Akt (Santa Cruz Biotech, CA, USA). An anti-b-actin antibody (SantaCruz Biotechology; 1:1000 dilution) was used as a loading control. Protein preparation and immunoblotting were performed as described [16].
Fig. 1. Ab and EtOH synergistically enhance neuronal apoptosis in SK-N-SH cells. (A) The relative cell viability was decreased in all cases compared with the untreated control. (B) Morphological changes in cultured SK-N-SH cells treated with Ab and/or EtOH. (C) Effect of Ab and EtOH on induction of caspase-3 activity. (D) Cells were stained with Hoechst 33258 staining. Dead cells were identified by morphological changes, such as nuclei condensation and fragmentation (arrow), compared with normal cell nuclei (arrowhead). Data were presented as means ± S.D. from at least three independent experiments. Scale bar is 20 lm. *P < 0.05, **P < 0.001.
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2.6. Analysis of mitochondrial membrane potential (DWm) and activity of caspase-3 Changes in DWm were estimated using tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes). Carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP) was used as a positive control for complete mitochondrial depolarization. Caspase-3 activities were measured as described [16]. 2.7. Statistical analysis All data are expressed as the means ± S.D. Statistical significance was determined via ANOVA followed by ScheffeÕs post hoc tests. A P-value of <0.05 was considered to be significant.
3. Results 3.1. Characterization of the synergistic neurotoxicity effects of combined treatment with Ab and ethanol We first performed a series of experiments to evaluate the neurotoxicity effects of single agent treatment with Ab or ethanol on cell survival in human neuroblastoma cell line, SK-N-SH. To confirm the synergistic cytotoxic effect of Ab and ethanol, we also co-treated with Ab and ethanol under the same conditions. At a concentration of 1 lM, Ab showed no cell-killing effects, even after 48 h of treatment, compared with un-treated control; however, this concentration did selectively inhibit neurite growth. In contrast, cell viability decreased with time when cells were treated with 15 mM ethanol, which is a similar concentration of the drink/drive limit in the USA and UK (0.08% w/v, 17.4 mM) (Fig. 1A). Interestingly, co-treatment with Ab and ethanol showed a significant synergistic effect on cell death compared to treatment with Ab or ethanol alone for 48 h (Fig. 1A). This synergistic effect on neuronal cell death was also detected in the morphological changes (Fig. 1B and D) and caspase-3 activation (Fig. 1C).
Fig. 2. Effects of Ab and EtOH on ROS production by and antioxidant enzyme mRNA expression in SK-N-SH cells. (A) Aband EtOH-induced ROS generation was measured by incubation with DCF-DA. Data were presented as means ± S.D. from three independent experiments. *P < 0.05. (B) The expression of antioxidant genes was measured by RT-PCR analysis. GAPDH is used for a loading control.
3.2. Synergistic effects of Ab and ethanol in triggering oxidative stress To determine whether Ab and/or ethanol treatment induces oxidative stress in cultured neurons, we measured the level of ROS production using a DCF-DA-dependent fluorescence assay. When SK-N-SH neurons were exposed to Ab or ethanol alone, intracellular ROS levels did not significantly change. However, ROS production was significantly increased in cotreated with both Ab and ethanol (Fig. 2A). To determine whether a combined treatment with Ab and ethanol disturbs the internal defense mechanism for ROS, we examined the expression of antioxidant genes. ROS are regulated by endogenous antioxidant defense systems, which include catalase, superoxide dismutase (SOD), and glutathione peroxidase [17]. Of these antioxidant genes, catalase and mitochondrial manganese-containing MnSOD, but not cytosolic Cu/ZnSOD, decreased following combination treatment with Ab and ethanol (Fig. 2B). Expression of catalase and MnSOD were decreased by 3-fold and 5-fold, respectively. These results indicate that Ab and ethanol have a synergistic effect on ROS generation in SK-N-SH cells.
cause a change in p53 expression. In contrast, p53 was significantly increased after 3 h following co-treatment of cells with Ab and ethanol (Fig. 3A). To further demonstrate that p53 induction correlates with cell death, we blocked the expression of the p53 protein by pre-incubating with p53-specific antisense oligonucleotides. Inhibition of p53 (p53 AS) significantly rescued the synergistic cell death effect of Ab and ethanol in SK-N-SH cells (Fig. 3B). We also investigated whether a combined treatment with Ab and ethanol influences neuronal cell survival signaling by measuring the activity of Akt with a phospho-specific Akt antibody. The level of pAkt in neurons was significantly decreased after 3 h with a combined treatment with Ab and ethanol (Fig. 3C). In addition, pre-treatment of Akt specific inhibitor, LY294002, significantly enhanced the synergistic Ab- and ethanol-induced neuronal cell death (Fig. 3D). Together, these findings suggest that Ab and ethanol show synergistic effects on neuronal cell death through activating the p53dependent apoptotic pathway and inhibiting Akt-dependent survival signaling.
3.3. Synergistic effects of Ab and ethanol on apoptotic or survival pathways To investigate whether Ab and ethanol have a synergistic effect on apoptosis, we measured the expression of pro-apoptotic marker protein p53. Treatment of Ab or ethanol alone did not
3.4. Effects of p53 antisense oligonucleotide and pAkt inhibitor on ROS generation To demonstrate that p53 and Akt signaling directly influence the ROS production, we pre-treated cultured neurons with a p53 antisense oligonucleotide or an Akt inhibitor before mea-
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Fig. 3. p53 and pAkt are involved in Ab- and EtOH-induced neuronal cell death in SK-N-SH cells. Western blot analysis was performed using antip53 (A) and anti-pAkt (C) antibodies. Beta-actin is used for a protein loading control. Involvement of p53 and pAkt in Ab- and EtOH-induced cell viability. Cells were pre-treated with p53 antisense oligonucleotides (B, open squares) or with LY294002 (D, open squares). Data were represented as means ± S.D. from three independent experiments. *P < 0.05 versus vehicle alone, Ab alone, or EtOH alone; **P < 0.05 versus Ab/EtOH together.
suring the ROS level. As above, we confirmed that combined treatment with Ab and ethanol significantly increased ROS production in SK-N-SH cells (Fig. 2A). Inhibition of p53 significantly reduced ROS production in Ab plus ethanol-treated cells, similar to the positive control group where cells were pretreated for 2 h with a well-known glutathione precursor, NAC (Fig. 4A). Glutathione plays an important role in the detoxification of ROS in brain [18]. In contrast, Akt inhibition did not significantly change ROS production.
duce depolarization of DWm. Pre-treatment with p53 antisense oligonucleotides attenuated and an Akt inhibitor enhanced this synergistic effect on DWm reduction (Fig. 4B), indicating that p53 and/or Akt plays a role in Ab- and ethanol-induced depolarization of DWm.
3.5. Effects of p53 antisense oligonucleotide and pAkt inhibitor on mitochondrial membrane potential To investigate the synergistic effect of Ab and ethanol on mitochondrial integrity, we examined the mitochondrial membrane potential (DWm) with TMRE staining. A slight disruption of DWm was observed in neurons treated with Ab or ethanol alone. Co-treatment with Ab and ethanol triggered the collapse of DWm, whereas pre-treatment with a p53 antisense oligonucleotide significantly blocked DWm depolarization (Fig. 4B). In contrast, Akt inhibition slightly enhanced the depolarization of DWm in cultured neurons co-treated with Ab and ethanol. Treatment with NAC, the p53 antisense oligonucleotide, or the Akt inhibitor alone did not affect DWm. These results indicate that Ab and ethanol synergistically in-
The results of this study are the first to show the synergistic effects of ethanol exposure and an Ab-rich environment, relative to ROS production, disruption of mitochondrial integrity, and cytotoxicity. In late-onset, sporadic AD, an age-dependent increase of ROS has been identified as a key factor in the development and progression of AD [1,8]. In this study, we suggest that alcohol poisoning increases the risk of sporadic AD, with damage to the neuronal tissues or cells through enhanced ROS production. It is well recognized that one of the main effects of ROS exposure is damage to DNA molecules. Among the proteins that play an important role in the cellular response to DNA damage is the tumor suppressor p53 [19]. Neuronal cells in which p53 was inhibited were less vulnerable to ROS-induced
4. Discussion
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Fig. 4. Effect of p53 and pAkt on Ab- and EtOH-induced synergistic induction of ROS production and loss of mitochondrial membrane potential (DWm) in SK-N-SH cells. ROS (A) and DWm (B) were analyzed using DCF-DA and TMRE. Data were presented as means ± S.D. from three independent experiments. FCCP was used as a positive control for complete mitochondrial depolarization. *P < 0.05 versus vehicle alone, Ab alone, or EtOH alone; **P < 0.05 versus Ab/EtOH together.
oxidative injury. This protective mechanism involved the impairment of ROS-activated, p53-dependent apoptosis. Over the past decade, the Akt pathway has been shown to be sufficient, and in some cases necessary, for the growth factorinduced cell survival of several neuronal cell types [20]. Akt may induce the expression of survival genes, such as Bcl-XL and A1, by activating CREB or NF-jB, and directly inhibits members of the apoptotic machinery [20]. In this study, we show that neuronal cell death induced by Ab and ethanol through decreasing Akt activity. Recently, Smith et al. [21] proposed that Ab toxicity is elicited through the generation of ROS, leading to activation of the JNK pathway, which in turn activates p66Shc by phosphorylation at Ser36. Activated p66Shc then triggers the phosphorylation of FKH, thereby down-regulating target genes, such as MnSOD, and leading to an even greater accumulation of cellular ROS. This biochemical cycle causes cell death. Here, we found that MnSOD expression was significantly decreased by combined treatment with Ab and ethanol.
Mitochondria are unique organelles in the consumption of oxygen, production of ATP, generation of oxygen radicals, and mobilization of calcium [22]. Mitochondrial dysfunction is evaluated by multiple parameters of mitochondrial integrity. Regulation of changes in mitochondrial membrane potential (DWm), the release of cytochrome c from the mitochondria into the cytoplasm and the activation of caspase-9 are important processes during mitochondria-dependent apoptotic process [23]. In this study, depolarization of DWm was increased by over half in combined treatment with Ab and ethanol. This study demonstrates that a low-dose of Ab or ethanol alone does not affect cell viability, whereas the combined exposure to both two agents activates apoptotic pathways through ROS production and the disruption of mitochondrial integrity. This study also indicates that the p53 and Akt pathways contribute to pro- or anti-apoptosis in cultured neurons. Acknowledgement: This work was supported by Grant (20080401034034) from BioGreen 21 Program, Rural Development Administration, Republic of Korea.
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