- Email: [email protected]
Amyloid-beta-induced neurotoxicity is reduced by inhibition of glycogen synthase kinase-3
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –26 2
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Amyloid-beta-induced neurotoxicity is reduced by inhibition of glycogen synthase kinase-3 Seong-Ho Koh, Min Young Noh, Seung Hyun Kim⁎ Department of Neurology, College of Medicine, Hanyang University, Seoul, Republic of Korea
A R T I C LE I N FO
AB S T R A C T
Article history:
Deposition of amyloid-β protein (Aβ) is one of the most important pathologic features in
Accepted 18 October 2007
Alzheimer's disease. It is well known that Aβ induces neuronal cell death through several
Available online 1 November 2007
pathogenic mechanisms. Although the role of glycogen synthase kinase (GSK)-3β in the
Keywords:
direct GSK-3β inhibition on Aβ-induced neurotoxicity. Thus, in this study, the relationship
Amyloid beta
between GSK-3β activity and Aβ-induced neurotoxicity was explored. To investigate the
Glycogen synthase kinase-3
role of GSK-3β in Aβ-induced neurotoxicity, neurons were treated with amyloid beta-
Neurotoxicity
protein (1–42) (Aβ42) oligomers with or without the addition of a GSK-3β inhibitor for 72 h.
Alzheimer's dementia
An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, trypan blue
neurotoxicity of Aβ has been highlighted, there has been no report evaluating the effect of
staining, and DAPI staining all showed that Aβ42 treatment alone resulted in decreased neuronal cell viability in a concentration-dependent manner. Aβ42 treatment significantly increased the activity of GSK-3β and cell death signals such as phosphorylated Tau (pThr231), cytosolic cytochrome c, and activated caspase-3. Aβ42 treatment also resulted in decreased survival signals, including that of heat shock transcription factor-1. Treatment with a GSK-3β inhibitor prevented Aβ-induced cell death. These results suggest that the neurotoxic effect of Aβ42 is mediated by GSK-3β activation and that inhibition of GSK-3β can reduce Aβ42-induced neurotoxicity. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Alzheimer's disease (AD), in which patients show a progressive decline in multiple cognitive functions, is characterized by widespread neurodegeneration, deposition of amyloid beta-protein (Aβ), and formation of intraneuronal neurofibrillary tangles (NFTs) in the brain ( Jellinger and Bancher, 1998; Jellinger, 2002; Trojanowski and Lee, 2000). Although several pathogenic mechanisms are proposed to be related to AD, Aβ is thought to be a key molecule in the pathogenesis of AD (Verdile et al., 2004). According to the amyloid cascade hypothesis (Robinson and Bishop, 2002; Verdile et al., 2004),
an increase in amyloid beta-protein fragment (1–42) (Aβ42) levels induces accumulation, oligomerization, deposition, and aggregation of amyloid beta. Aggregated Aβ42 elicits an inflammatory response, causes oxidative stress, alters homeostasis of the central nervous system, and gives rise to neuronal cell death, which is linked to dementia. Glycogen synthase kinase (GSK)-3β has been suggested to play important roles in several proposed pathogenic mechanisms of Aβinduced neuronal cell death (Takashima et al., 1996). Glycogen synthase kinase (GSK)-3, originally identified as a regulator of glycogen synthesis (Embi et al., 1980), is now known to be a multifaceted enzyme, affecting a diverse range
⁎ Corresponding author. Department of Neurology, College of Medicine, Hanyang University, #17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, Republic of Korea. Fax: +82 2 2296 8370. E-mail address: [email protected] (S.H. Kim). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.10.064
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –2 62
of biological functions including gene expression, cellular architecture, and apoptosis ( Jope and Johnson, 2004). Of the two closely related isoforms, GSK-3α and GSK-3β, GSK-3β is known to play critical roles in oxidative stress-induced neuronal cell death mechanisms (Bijur and Jope, 2000, 2001; Grimes and Jope, 2001; Koh et al., 2003, 2004a,b; Takadera and Ohyashiki, 2004; Watcharasit et al., 2003). However, it has not been reported whether direct inhibition of GSK-3β can prevent Aβ-induced neuronal cell death. This study investigated the role of GSK-3β in Aβ42-induced primary cultured cortical neuron death. The effects of GSK-3β inhibition on Aβ42-induced neurotoxicity were also examined.
2.
Results
2.1.
Role of GSK-3β in Aβ42-induced neurotoxicity
To evaluate the viability of cells following Aβ42 treatment, cortical cells were incubated with Aβ42 [0 (control), 5, 10, 20, or
255
50 μM] for 72 h. After incubation, cell viability was measured with an MTT assay, and surviving cells were counted using trypan blue stain. Following Aβ42 treatment, cell viability was significantly decreased in a concentration-dependent pattern (Fig. 1A). Based on these results, 20 μM was selected as an optimal Aβ42 concentration for subsequent experiments as cell viability was approximately 60% at that concentration (Fig. 1A). Additionally, cell death at 20 μM Aβ42 appeared to be due mainly to apoptosis (Fig. 2). To investigate the effects of GSK-3β inhibitor VIII itself, cortical cells were treated with several concentrations of GSK3β inhibitor VIII (0, 10, 25, 50, 100, 200, 500, and 1000 nM) for 24 h. Compared with the control, cell viability was not decreased at the 100 nM treatment level (97.9 ± 6.2% in TBS and 97.8 ± 3.9% in MTT, p N 0.10), but was significantly decreased at treatment levels of 200 nM or greater (89.8 ± 3.2% in TBS and 90.1 ± 4.5% in MTT, p < 0.01) (Fig. 1B). These results suggest that excessive inhibition of GSK-3β induces neuronal cell death. To investigate the importance of GSK-3β in Aβ42-induced neurotoxicity, cortical cells were incubated with both 20 μM
Fig. 1 – Measurement of primary cultured cortical neuronal cell viability by MTT assay and trypan blue stain. All data are presented as means (% of control) ± SEM from five or more independent experiments. Each treatment group was compared with the other groups using Duncan's Multiple Range Test after One-Way ANOVA. *p < 0.05 when compared with the control group. # p < 0.05 when compared with the group treated only with 20 μM Aβ42.
256
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –26 2
Fig. 2 – Detection of apoptosis in cortical cells by DAPI staining. The percent of apoptosis was increased to 34.0 ± 2.9% in cortical cells treated only with 20 μM Aβ42, but decreased to 13.3 ± 2.6% in cells treated with both 20 μM Aβ42 and 50 nM GSK-3β inhibitor VIII (p < 0.01). The data are represented as means (% of control) ± SEM. Each treatment group was compared with the other groups using Duncan's Multiple Range Test after One-Way ANOVA. *p < 0.01 when compared with the control group. #p < 0.01 when compared with the group treated only with 20 μM Aβ42.
Aβ42 and several concentrations of either GSK-3β inhibitor VIII (0, 25, 50, or 100 nM) or lithium chloride (0, 1, 5, 10, or 100 mM) for 72 h. Compared with the control, cell viability was significantly decreased following 20 μM Aβ42 treatment (59.0 ± 9.0% in MTT and 59.6 ± 5.8% in TBS, p < 0.01). However, cotreatment with 50 nM GSK-3β inhibitor VIII increased cell viability (81.5 ± 5.3% in MTT and 83.4 ± 5.6% in PBS, p < 0.01) (Fig. 1C). Co-treatment with 5 mM lithium chloride increased cell viability approximately 23% (p < 0.05, Fig. 1D). These results suggest that GSK-3β might play very important roles in Aβ42induced neuronal cell death. DAPI staining demonstrated that the percent of apoptotic cells was significantly increased with 20 μM Aβ42 treatment (34.0 ± 2.9%) compared with control (4.5 ± 1.3%). The percent of apoptotic cells was markedly reduced by treatment with 50 nM GSK-3β inhibitor VIII (13.3 ± 2.6%) ( p < 0.01) (Fig. 2).
2.2.
Fig. 3 – GSK-3β enzyme activity assay. Kinase activity was measured in cortical cell lysates 72 h after the initial treatment under several different conditions. Treatment with 20 μM Aβ42 increased kinase activity, but 50 nM GSK-3β inhibitor VIII treatment effectively inhibited the increase in kinase activity from 20 μM Aβ42 treatment. The data are represented as means (% of control) ± SEM. Each treatment group was compared with the other groups using Duncan's Multiple Range Test after One-Way ANOVA. *p < 0.05 when compared with the control group. #p < 0.05 when compared with the group treated with only 20 μM Aβ42.
DA (Fig. 4). The level of free radicals was significantly increased in cells treated with 20 μM Aβ42 (197.35 ± 22.66%), compared with the control (100.00 ± 30.93%, p < 0.01). However, co-treatment with GSK-3β inhibitor VIII did not decrease the amounts of free radicals (189.47 ± 37.55%). These findings suggest that Aβ42 induces the production of free radicals and that GSK-3β inhibitor VIII does not reduce
Effects of Aβ42 on GSK-3 enzyme activity
Based on the viability results, GSK-3β enzyme activity was evaluated in the cultured cells. Twenty micromolar Aβ42 treatment for 72 h increased GSK-3β enzyme activity to 210.7 ± 30.0% (p < 0.001) compared with control cortical cells without any treatment (100.0 ± 10.2%), while the combined treatment of 20 μM Aβ42 and 50 nM GSK-3β inhibitor VIII for 72 h successfully inhibited the activity increase (127.3 ± 15.8%, p < 0.001) (Fig. 3). These findings suggest that Aβ42 treatment increased GSK-3β enzyme activity and GSK-3β inhibitor VIII effectively inhibited the activity.
2.3. Effects of Aβ42 and GSK-3β inhibitor VIII on free radical formation To appreciate the oxidant effects of Aβ42, the amounts of free radicals in the cortical cells treated only with 0 or 20 μM Aβ42 for 30 min were measured using the fluorescent probe DCFH-
Fig. 4 – Measurement of free radical production. Data are represented as means ± SEM. Each treatment group was compared with the other groups using Duncan's Multiple Range Test. Cortical cells were treated with 20 μM Aβ42 for 30 min to measure free radicals generated in the presence of Aβ42 using the fluorescent probe DCFH-DA (see Materials and methods). The amount of free radicals was significantly different in the cells treated with 20 μM Aβ42. However, this change was not blocked by combined treatment with 50 nM GSK-3β inhibitor VIII. * = p < 0.05 (level of free radicals compared with that of the control group).
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –2 62
the production of free radicals induced by Aβ42. Thus, the blocking of Aβ42-induced neurotoxic effects by GSK-3β inhibitor VIII is not related to the inhibition of free radical production by Aβ42.
2.4.
257
Effect of Aβ42 on intracellular signal levels
To ascertain the effects of Aβ42 treatment on intracellular signal levels, cortical cell homogenates from 72-hour post-
Fig. 5 – Immunoreactivity (IR) of heat shock transcription factor-1, phosphorylated Tau (Thr231), Tau, cytosolic cytochrome c, and activated caspase-3 in cortical cells 72 h after the initial treatment with 20 μM Aβ42. Quantitative data from cortical cells are expressed in arbitrary units normalized to the simultaneously assayed control group value. Each treatment group was compared with the other groups using Duncan's Multiple Range Test. Representative ECL radiographs of immunoblots, showing the IRs of heat shock transcription factor-1 (HSTF-1) (A), phosphorylated Tau (Thr231) and Tau (B), cytosolic cytochrome c (C), and activated caspase-3 (D). Quantitative data expressing the IR ratios of HSTF-1 (A), phosphorylated Tau (Thr231)/Tau (B), cytosolic cytochrome c (C), and activated caspase-3 (D) normalized to actin immunostaining in the control group, the 20 μM Aβ42-treated group, and groups simultaneously treated with 20 μM Aβ42 and 50 nM GSK-3β inhibitor VIII. The data are represented as means (% of control) ± SEM. Each treatment group was compared with the other groups using Duncan's Multiple Range Test after One-Way ANOVA. *p < 0.05 when compared with the control group. #p < 0.05 when compared with the group treated with only 20 μM Aβ42.
258
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –26 2
Fig. 6 – Schematic diagram of a model for the prevention of Aβ42-induced neurotoxicity by GSK-3β inhibition. treatment samples were immunoblotted to examine levels of heat shock transcription factor-1 (HSTF-1), phosphorylated Tau (Thr231), Tau, cytosolic cytochrome c, and activated caspase-3. Compared with non-treated cells (1.00 ± 0.17), the immunoreactivity (IR) of HSTF-1 was significantly decreased in 20 μM Aβ42-treated cells (0.10 ± 0.08, p < 0.01) at 72 h (Fig. 5). However, this decrease was recovered with a combined treatment of Aβ42 and 50 nM GSK-3β inhibitor VIII (0.71 ±0.12, p < 0.01). In contrast, treatment with 20 μM Aβ42 increased the IRs of phosphorylated Tau (Thr231)/Tau, cytosolic cytochrome c, and activated caspase-3 (4.69 ± 0.82, 7.32 ±0.78, and 3.77 ± 0.54, respectively), compared with non-treated cells (1.00 ± 0.31, 1.00 ± 0.26, and 1.00 ± 0.15, respectively) (p < 0.01) (Fig. 5). The combined treatment with 50 nM GSK-3β inhibitor VIII resulted in decreased expression levels of these proteins (1.71 ± 0.57, 1.79 ± 0.43, and 1.50 ± 0.49, respectively) (p < 0.01). These findings suggest that Aβ42 decreases survival signals, including HSTF-1, and increases cell death signals including phosphorylated Tau (Thr231), cytochrome c, and activated caspase-3 and that these cytotoxic effects of Aβ42 can be blocked by GSK-3β inhibition.
3.
Discussion
In recent years, numerous studies have suggested that neurotoxicity of amyloid beta (Aβ) is one of the most important pathogenic factors in Alzheimer's disease (Robinson and Bishop, 2002; Verdile et al., 2004). With the recent emphasis on the role of GSK-3β in neurodegenerative diseases, it has been reported that treatment of neuronal cells with Aβ increased GSK-3β activity (Takashima et al., 1996). Lithium, a GSK-3β inhibitor, reduced production of Aβ in an animal model of AD (Rockenstein et al., 2007) and inhibited Aβ-induced stress in the endoplasmic reticulum of rabbit hippocampus (Ghribi et al., 2003). However, there has been no report of the effects of the direct inhibition of GSK-3β, GSK-3β activity, or the GSK-3β signaling pathway on Aβ-induced neuronal cell death.
In the present study, we aimed to investigate what mechanisms, in addition to the reduction of phosphorylation of Tau (Bhat et al., 2003), may contribute to the protective effects of GSK-3β inhibition against Aβ-induced neurotoxicity. Therefore, we studied whether GSK-3β inhibition could have an antioxidant effect or could alter other cellular signals such as HSTF-1 and caspase-3. This study is an extension of similar studies that have used highly selective GSK-3β inhibitors to define the role of GSK-3β in cellular signaling in the pathogenesis of Alzheimer's disease (Bhat et al., 2003). Our results showed that Aβ42 treatment induced neuronal cell death, and this neurotoxic effect was blocked by treatment with either GSK-3β inhibitor VIII, a direct and highly selective GSK-3β inhibitor (Bhat et al., 2003; Gould et al., 2004; Vasdev et al., 2005) which effectively inhibited GSK-3β activity in a concentration-dependent manner in a previous study (Lee et al., 2007), or lithium chloride, another GSK-3β inhibitor. Aβ42 treatment induced neuronal cell death, and this neurotoxicity was associated with GSK-3β activation and decreased survival signals, including HSTF-1, which is a direct substrate of GSK-3β and an important survival signal (Bijur and Jope, 2000). Aβ42 treatment also resulted in increased cell death signals, including phosphorylated Tau (Thr231), cytosolic cytochrome c, and activated caspase-3. These neurotoxic effects of Aβ42 were ameliorated by combined treatment with the specific GSK-3β inhibitor. The GSK-3β inhibitor inhibited the activation of GSK-3β, the reduction of HSTF-1, the increase of phosphorylated Tau (Thr231) and cytosolic cytochrome c, the activation of caspase-3, and the neuronal cell death induced by Aβ42. These effects of the GSK-3β inhibitor on Aβ42-induced neurotoxicity were not related to a direct free radical scavenging effect. Because GSK-3β inhibitor VIII did not affect Aβ42-induced free radical production, the neuroprotective effect of GSK-3β inhibition is not likely related to the inhibition of free radical production. It is well known that free radical production is one of the mechanisms of Aβ42-induced neurotoxicity (Yatin et al., 1998). According to the present study, GSK-3β inhibition did not affect Aβ42-induced free radical production but
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –2 62
protected primary cortical neurons against Aβ42. This finding suggests that GSK-3β inhibition does not have an antioxidant effect and that other neuroprotective mechanisms might play important roles. Therefore, other mechanisms involved in the GSK-3β pathway were investigated. GSK-3β is well known to promote cell death, especially neuronal cell death (Kirschenbaum et al., 2001; Koh et al., 2003, 2004, 2005; Lee et al., 2007; Martinez et al., 2002; Pap and Cooper, 2002), by both enhancing the expression of pro-apoptotic proteins and inhibiting the activity of anti-apoptotic proteins (Kirschenbaum et al., 2001; Koh et al., 2003, 2004, 2005; Lee et al., 2007; Martinez et al., 2002; Pap and Cooper, 2002). GSK-3β directly affects CCAAT/enhancer binding protein, nuclear factor of activated T cells, Myc, HSTF-1, Tau, cyclic adenosine mono phosphate (AMP) response element binding protein, Activator protein-1, β-catenin, NFkB, and p53, promotes the release of cytochrome c, activates caspase-3, and induces neuronal cell death (Bijur and Jope, 2000, 2001; Grimes and Jope, 2001; Koh et al., 2003, 2004, 2005; Lee et al., 2007; Takadera and Ohyashiki, 2004; Watcharasit et al., 2003). In this study, Aβ42 treatment significantly increased GSK-3β activity, similar to a previous finding (Takashima et al., 1996). Aβ42 also increased pro-apoptotic signals such as phosphorylated Tau(Thr235), cytosolic cytochrome c, and cleaved caspase3 and reduced survival signals including HSTF-1. GSK-3β inhibition restored cell viability and survival signals and prevented pro-apoptotic signals (Fig. 6). These data suggest that GSK-3β activation by Aβ might be a mechanism for Aβ neurotoxic effects. This hypothesis was confirmed by the finding that a GSK-3β inhibitor blocked GSK-3β activity and inhibited the neuronal cell death induced by Aβ42. One limitation to the interpretation of these results, however, is that this study was performed under in vitro conditions, and the results could be different in an in vivo situation, which involves more complicated factors.
4.
Conclusion
It can be suggested that Aβ42 neurotoxicity is closely linked to GSK-3β activation and that inhibition of GSK-3β could be a new promising strategy for preventing Aβ42 neurotoxicity.
5.
Experimental procedures
5.1.
Materials
Dulbecco's Modified Eagle's Medium (DMEM, high glucose) was purchased from GIBCO. The sources of drugs were as follows: amyloid beta-protein (1–42) (Aβ42), lithium chloride, trypan blue solution, Insulin, and DNase I were purchased from Sigma. GSK-3β inhibitor VIII {N-(4-Methoxybenzyl)-N′-(5nitro-1,3-thiazol-2-yl) urea, IC50 = 104 nM, C12H12N4O4S; molecular weight: 308.3 (Noble et al., 2005)}, a direct and highly selective GSK-3β inhibitor (Bhat et al., 2003; Gould et al., 2004; Vasdev et al., 2005), which effectively inhibited GSK-3β activity in a concentration-dependent manner in a previous study (Lee et al., 2007), was purchased from Calbiochem (La Jolla, CA, USA). Before use, these drugs were dissolved in distilled water
259
and then further diluted with culture medium to yield the desired final concentrations.
5.2.
Cell culture and treatment
All animal procedures were performed in accordance with the Hanyang University guidelines for the care and use of laboratory animals and were approved by the Institutional Review Board of Hanyang University. Primary cultures were obtained from the cerebral cortex of fetal Sprague Dawley rats (16 days of gestation). Briefly, rat embryos were decapitated, and the brains were rapidly removed and placed in a Petri dish half-filled with ice-cold Hank's balanced salt solution (HBSS; 137 mM NaCl, 5.4 mM KCl, 0.3 mM Na2HPO4, 0.4 mM KH2PO4, 5.6 mM glucose, and 2.5 mM HEPES) (Gibco BRL, NY, USA). Single cells dissociated from the whole cerebral cortex of fetal rats were plated on poly-L-lysine (Sigma, Saint Louis, MO, USA) pre-coated 100-mm corning dishes (5 × 106 cells/cm2) or glass coverslips and placed in Nunc 6- or 24-well plates (5 × 105, 2.5 × 106 cells/cm2). Cultures were incubated in Dulbecco's Modified Eagle Medium (high glucose) and supplemented with 10% heat-inactivated fetal bovine serum (1.7 days after plating), 1% penicillin streptomycin, 3.7 g/ L NaHCO3, 5 μg/mL insulin, and p-aminobenzoic acid. Cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere. Two days after plating, non-neuronal cells were removed by adding 5 μM cytosine arabinoside for 24 h. Only mature cultures (7 days in vitro) were used for experiments.
5.3.
Oligomeric Aβ42 preparation and treatment
Synthetic Aβ42 peptide with N95% purity by RPHPLC chromatography was used (Sigma). Oligomeric Aβ42 peptides were prepared as described previously (Dahlgren et al., 2002). Briefly, Aβ42 peptide was dissolved to 1 mmol/L in 100% hexafluoroisopropanol (Sigma), the hexafluoroisopropanol was removed under vacuum, and the peptide was stored at ¡©20 °C. For the aggregation, the peptide was first resuspended in dry dimethylsulfoxide (Me2SO; Sigma) to 5 mmol/L. For oligomeric conditions, culture medium was added to bring the peptide to a final concentration of 100 μmol/L, and the peptide was incubated at 4 °C for 24 h. Different concentrations of oligomeric Aβ42 were tested to assess its effect on neuronal cell viability. After primary cultured cortical neuronal cells were incubated with oligomeric Aβ42, an aggregate form [0 (control), 5, 10, 20, or 50 μM] (Sigma) for 72 h (Nakagawa et al., 2000), each plate was washed, and cell viability was measured using the MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] assay and trypan blue stain. To evaluate the role of GSK-3β in Aβ42-induced neurotoxicity, cortical cells were incubated in the presence of 20 μM Aβ42 together with several concentrations of GSK-3β inhibitor VIII (0, 25, 50, or 100 nM) or lithium chloride (0, 1, 5, 10, 100 mM) for 72 h. To estimate the alteration in intracellular signals, such as heat shock transcription factor-1 (HSTF-1), phosphorylated tau (pThr231), Tau, cytosolic cytochrome c, or caspase-3, cortical cells were harvested 72 h after treatment under various conditions and then immediately used for immunoblotting.
260 5.4.
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –26 2
MTT assay and trypan blue staining
MTT is absorbed into cells and transformed into formazan by mitochondrial succinate dehydrogenase. Accumulation of formazan directly reflects the activity of mitochondria, which functions as an indirect measurement of cell viability. Cells were plated at a density of 1 × 104 cells/well in a 96-well plate, cultured, and differentiated prior to adding 50 μL of 2 mg/mL MTT (Sigma) to 200 μL of medium present in each well. An aliquot (220 μL) of the resulting solution was removed from each well followed by the addition of 150 μL dimethyl sulfoxide. After the precipitate in each well was resuspended on a microplate mixer for 10 min, an optical density (OD) reading at 540 nm was measured using an ELISA plate reader. All results were normalized to OD values measured from an identically conditioned well without cell culture. For trypan blue staining, 10 μL of trypan blue solution was incubated for 2 min with 10 μL of cells from each sample. Unstained live cells were counted on a hemocytometer.
5.5.
DAPI staining
10 mM DTT, 0.1 μM okadaic acid, 0.5 mM Na3VO4, 1 mM benzamidine, and 1 mM PMSF) and sonicated on ice using a Vibratome probe sonicator (setting 4). The lysates were cleared by centrifugation at 10,000×g for 30 min. The supernatant was then assayed for GSK-3β activity.
5.6.2.
Assay conditions
GSK-3β activity was measured by the transfer of 32P from [γ-32P] ATP to the eIF2B peptide substrate. Per assay, 12.5 μL of extract (the supernatant from above) was mixed with 6.25 μL substrate mix (4 mg/mL eIF2B peptide with or without 200 mM lithium chloride). Assays were initiated by the addition of 6.25 mL ATP mix [200 mM HEPES, pH 7.5, 50 mM MgCl2, 8 mM DTT, 400 μM ATP, 0.125 μCi/μΛ (γ-32P) ATP (3000 Ci/mL)]. The reactions were incubated at room temperature and terminated after 8 min by spotting 20 μL onto P81 ion-exchange paper (Whatman). The paper was washed three times in 100 mM phosphoric acid, and the bound radioactivity was quantified by scintillation counting (Ryves et al., 1998). All assays were run in duplicate with the means and standard error (SEM) of at least two separate experiments shown in each figure.
5.7.
Determination of free radical production
DAPI staining was performed to evaluate apoptosis as follows: cortical cells were incubated for 72 h with one of three treatments: (1) without Aβ42 or GSK-3β inhibitor VIII, (2) with 20 μM Aβ42, and (3) with both 20 μM Aβ42 and 50 nM GSK-3β inhibitor VIII. All samples were then centrifuged at 2000×g for 2 min, at which point the supernatants were discarded and 4% neutral buffered formalin (100 μL) was added to each cell pellet. A 50-μL aliquot of the cell suspension was applied to a glass slide and dried at room temperature. The fixed cells were then washed with PBS, air dried, and stained with the DNAspecific fluorochrome 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma, St. Louis, MO, USA) for 20 min. The adherent cells were rinsed with PBS, air dried, and mounted with 90% glycerol. The slides were observed under an Olympus fluorescence microscope ( Jiang et al., 2000). The percentage of apoptotic cells was determined, which in turn coincided with the proportion of cells exhibiting morphological hallmarks of apoptosis, such as DNA fragmentation, nuclear condensation, and segmentation.
To measure free radical production, cortical cells were treated as follows: (1) with 0 (control), (2) with 20 μM Aβ42, and (3) with both 20 μM Aβ42 and 50 nM GSK-3β inhibitor VIII for 30 min. Treated cells were incubated with the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Molecular Probes Inc, Eugene, OR, USA), which freely crosses the cell membranes and is hydrolyzed by cellular esterases to 2′,7′-dichlorodihydrofluorescein (DCFH2). Though DSFH2 is a non-fluorescent molecule, it is oxidized to the fluorescent 2′ 7′-dichlorofluorescein (DCF) in the presence of peroxides. Accumulation of DCF in cells was measured as a change in fluorescence at 525 nm when the sample was excited at 488 nm by a Microplate Fluorescence Reader (FL600) (D.I Biotech Ltd., Seoul, Korea) (Tammariello et al., 2000). The time point of 30 min of incubation was chosen because maximal effects were shown at this time point according to a report by Filomeni et al. (2003).
5.6.
5.8.
GSK-3 activity assay
The following peptide substrates were purchased from Peptron Inc. (Daejeon, Korea): Eukaryotic translation initiation factor 2B peptide substrate, eIF2B-SP: RRAAEELDSRAG-pSPQL (positive control substrate); eIF2BA: RRAAEELDSRAGAPQL (negative control substrate) (Ryves et al., 1998). A 10 mg/mL peptide stock solution was prepared in deionized water and stored at −20 °C. These purified peptide stocks were assumed to be 100% pure for subsequent calculations. Escherichia coliexpressed mammalian GSK-3β (mGSK-3β), encoded by rabbit skeletal muscle cDNA, was purchased from New England Biolabs (USA) and stored at −20 °C.
5.6.1.
Preparation of lysates
1 × 108 cortical cells incubated for 72 h were washed twice in cold PBS, resuspended in 1 mL lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1% Triton X-100,
Western blot analysis
Heat shock transcription factor-1 (HSTF-1), phosphorylated tau (pThr231), cytosolic cytochrome c, and the cleaved form of caspase-3 were analyzed by western blot. Briefly, 5 × 106 cells were collected after incubation for 72 h, washed twice in cold PBS, and incubated for 10 min on ice in lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.2% SDS, 100 μg/ mL phenylmethylsulfonyl fluoride (PMSF), 50 μl/mL aprotinin, 1% igepal 630, 100 mM NaF, 0.5% sodium deoxycholate, 0.5 mM EDTA, 0.1 mM EGTA]. Cell lysates were centrifuged at 10,000×g for 20 min at 4 °C, and the supernatants were used to evaluate HSTF-1, phosphorylated tau (pThr231), Tau, and the cleaved form of caspase-3. For evaluation of cytosolic cytochrome c levels, the washed cells were suspended in sucrose-supplemented cell extract buffer (SCEB, 300 mM sucrose, 10 mM HEPES at pH 7.4, 50 mM KCl, 5 mM EGTA, 5 mM MgCl2, 1 mM DTT, 10 μM cytochalasin B, 1 mM PMSF), left on ice for 30 min,
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –2 62
and then homogenized by 50 strokes in an ice-cold Dounce homogenizer. Unbroken cells and nuclei were pelleted by centrifugation at 2000×g for 10 min and discarded. Mitochondria were collected from the resulting supernatant by further centrifugation at 13,000×g for 10 min. The resulting supernatant containing the post-mitochondrial fraction was used for immunoblotting. Protein concentrations of the cell lysates were determined using a Bio-Rad protein assay kit. For western blot analysis, 20 μg of total protein from each sample was resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membranes were blocked with 5% skim milk and incubated with specific antibodies for HSTF-1 (1:1000, Santa Cruz Biotech, Delaware, CA, USA), phospho-tau (pThr231) (1:1000, Calbiochem), cytochrome c (1:500, Santa Cruz Biotech, Delaware, CA, USA), and cleaved (activated form) caspase-3 (Asp 175) (1:1000, Cell signaling, Beverly, MA, USA). The membranes were washed with Tris buffered saline containing 0.05% Tween-20 (TBST) and processed with an HRP-conjugated anti-rabbit antibody or anti-mouse antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA) for ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The results of several western blots were quantified on a Quantity One-4,2,0 image analyzer (Bio-Rad, Hercules, CA) and were normalized to actin immunostaining (Koh et al., 2003, 2004, 2005; Lee et al., 2007).
5.9.
Statistical analysis
All data are presented as means ± SEM from five or more independent experiments. Statistical comparisons between different treatment groups were conducted with Duncan's Multiple Range Test after One-Way ANOVA. Differences in p-values of less than 0.05 were considered statistically significant.
Acknowledgments This work was supported by a grant from Hanyang University (2007-000-0000-4823) and the Laboratory for Clinical Investigation, College of Medicine, Hanyang University, Seoul, Korea.
REFERENCES
Bhat, R., Xue, Y., Berg, S., Hellberg, S., Ormo, M., Nilsson, Y., Radesater, A.C., Jerning, E., Markgren, P.O., Borgegard, T., Nylof, M., Gimenez-Cassina, A., Hernandez, F., Lucas, J.J., Diaz-Nido, J., Avila, J., 2003. Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A014418. J. Biol. Chem. 278, 45937–45945. Bijur, G.N., Jope, R.S., 2000. Opposing actions of phosphatidylinositol 3-kinase and glycogen synthase kinase-3beta in the regulation of HSTF-1 activity. J. Neurochem. 75, 2401–2408. Bijur, G.N., Jope, R.S., 2001. Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3 beta. J. Biol. Chem. 276, 37436–37442. Dahlgren, K.N., Manelli, A.M., Stine Jr., W.B., Baker, L.K., Krafft, G.A., LaDu, M.J., 2002. Oligomeric and fibrillar species of amyloidbeta
261
peptides differentially affect neuronal viability. J. Biol. Chem. 277, 32046–32053. Embi, N., Rylatt, D.B., Cohen, P., 1980. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur. J. Biochem. 107, 519–527. Filomeni, G., Aquilano, K., Rotilio, G., Ciriolo, M.R., 2003. Reactive oxygen species-dependent c-Jun NH2-terminal kinase/c-Jun signaling cascade mediates neuroblastoma cell death induced by diallyl disulfide. Cancer Res. 63, 5940–5949. Ghribi, O., Herman, M.M., Savory, J., 2003. Lithium inhibits Abeta-induced stress in endoplasmic reticulum of rabbit hippocampus but does not prevent oxidative damage and tau phosphorylation. J. Neurosci. Res. 71, 853–862. Gould, T.D., Einat, H., Bhat, R., Manji, H.K., 2004. AR-A014418, a selective GSK-3 inhibitor, produces antidepressant-like effects in the forced swim test. Int. J. Neuropsychopharmacol. 7, 387–390. Grimes, C.A., Jope, R.S., 2001. The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog. Neurobiol. 65, 391–426. Jellinger, K.A., 2002. Alzheimer disease and cerebrovascular pathology: an update. J. Neural Trans. 109, 813–836. Jellinger, K.A., Bancher, C., 1998. Neuropathology of Alzheimer's disease: a critical update. J. Neural Transm., Suppl. 54, 77–95. Jiang, Q., Gu, Z., Zhang, G., Jin, G., 2000. Diphosphorylation and involvement of extracellular signal-regulated kinases (ERK1/2) in glutamate-induced apoptotic-like death in cultured rat cortical neurons. Brain Res. 857, 71–77. Jope, R.S., Johnson, G.V., 2004. The glamour and gloom of glycogen synthase kinase-3. Trends Biochem. Sci. 29, 95–102. Kirschenbaum, F., Hsu, S.C., Cordell, B., McCarthy, J.V., 2001. Glycogen synthase kinase-3beta regulates presenilin 1 C-terminal fragment levels. J. Biol. Chem. 276, 30701–30707. Koh, S.H., Kim, S.H., Kwon, H., Park, Y., Kim, K.S., Song, C.W., Kim, J., Kim, M.H., Yu, H.J., Henkel, J.S., Jung, H.K., 2003. Epigallocatechin gallate protects nerve growth factor differentiated PC12 cells from oxidative-radical-stress-induced apoptosis through its effect on phosphoinositide 3-kinase/Akt and glycogen synthase kinase-3. Brain Res. Mol. Brain Res. 118, 72–81. Koh, S.H., Kim, S.H., Kwon, H., Kim, J.G., Kim, J.H., Yang, K.H., Kim, J., Kim, S.U., Yu, H.J., Do, B.R., Kim, K.S., Jung, H.K., 2004a. Phosphatidylinositol-3 kinase/Akt and GSK-3 mediated cytoprotective effect of epigallocatechin gallate on oxidative stress-injured neuronal-differentiated N18D3 cells. Neurotoxicology 25, 793–802. Koh, S.H., Kwon, H., Park, Y., Kim, J.G., Kim, K.S., Song, C.W., Kim, J.H., Kim, J., Kim, M.H., Kim, K.S., Yu, H.J., Jung, H.G., Kim, S.H., 2004b. Diallyl disulfide inhibits cytochrome c-mediated apoptosis in H2O2 induced death of neuronal-differentiated PC12 cells. J. Korean Neurol. Assoc. 22, 375–381. Koh, S.H., Lee, Y.B., Kim, K.S., Kim, H.J., Kim, M., Lee, Y.J., Kim, J., Lee, K.W., Kim, S.H., 2005. Role of GSK-3beta activity in motor neuronal cell death induced by G93A or A4V mutant hSOD1 gene. Eur. J. Neurosci. 22, 301–309. Lee, K.Y., Koh, S.H., Noh, M.Y., Park, K.W., Lee, Y.J., Kim, S.H., 2007. Glycogen synthase kinase-3β activity plays very important roles in determining the fate of oxidative stress-inflicted neuronal cells. Brain Res. 1129, 89–99. Martinez, A., Castro, A., Dorronsoro, I., Alonso, M., 2002. Glycogen synthase kinase 3 (GSK-3) inhibitors as new promising drugs for diabetes, neurodegeneration, cancer, and inflammation. Med. Res. Rev. 22, 373–384. Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B.A., Yuan, J., 2000. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 403, 98–103.
262
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –26 2
Noble, W., Planel, E., Zehr, C., Olm, V., Meyerson, J., Suleman, F., Gaynor, K., Wang, L., LaFrancois, J., Feinstein, B., Burns, M., Krishnamurthy, P., Wen, Y., Bhat, R., Lewis, J., Dickson, D., Duff, K., 2005. Inhibition of glycogen synthase kinase-3 by lithium correlates with reduced tauopathy and degeneration in vivo. Proc. Natl. Acad. Sci. U. S. A. 102, 6990–6995. Pap, M., Cooper, G.M., 2002. Role of translation initiation factor 2B in control of cell survival by the phosphatidylinositol 3-kinase/ Akt/glycogen synthase kinase 3beta signaling pathway. Mol. Cell. Biol. 22, 578–586. Robinson, S.R., Bishop, G.M., 2002. Abeta as a bioflocculant: implications for the amyloid hypothesis of Alzheimer's disease. Neurobiol. Aging 23, 1051–1072. Rockenstein, E., Torrance, M., Adame, A., Mante, M., Bar-on, P., Rose, J.B., Crews, L., Masliah, E., 2007. Neuroprotective effects of regulators of the glycogen synthase kinase-3beta signaling pathway in a transgenic model of Alzheimer's disease are associated with reduced amyloid precursor protein phosphorylation. J. Neurosci. 27, 1981–1991. Ryves, W.J., Fryer, L., Dale, T., Harwood, A.J., 1998. An assay for glycogen synthase kinase 3 (GSK-3) for use in crude cell extracts. Anal. Biochem. 264, 124–127. Tammariello, S.P., Quinn, M.T., Estus, S., 2000. NADPH oxidase contributes directly to oxidative stress and apoptosis in nerve growth factor-deprived sympathetic neurons. J. Neurosci. 20, 1–5. Takadera, T., Ohyashiki, T., 2004. Glycogen synthase kinase-3 inhibitors prevent caspase-dependent apoptosis induced by ethanol in cultured rat cortical neurons. Eur. J. Pharmacol. 499, 239–245.
Takashima, A., Noguchi, K., Michel, G., Mercken, M., Hoshi, M., Ishiguro, K., Imahori, K., 1996. Exposure of rat hippocampal neurons to amyloid h peptide (25–35) induces the inactivation of phosphatidyl inositol-3 kinase and the activation of tau protein kinase I/glycogen synthase kinase-3h. Neurosci. Lett. 203, 33–36. Trojanowski, J.Q., Lee, V.M., 2000. “Fatal attractions” of proteins. A comprehensive hypothetical mechanism underlying Alzheimer's disease and other neurodegenerative disorders. Ann. N. Y. Acad. Sci. 924, 62–67. Vasdev, N., Natesan, S., Galineau, L., Garcia, A., Stableford, W.T., McCormick, P., Seeman, P., Houle, S., Wilson, A.A., 2005. Synthesis and ex vivo evaluation of carbon-11 labelled N-(4-methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea ([11C] AR-A014418): a radiolabelled glycogen synthase kinase-3beta specific inhibitor for PET studies. Bioorg. Med. Chem. Lett. 15, 5270–5273. Verdile, G., Fuller, S., Atwood, C.S., Laws, S.M., Gandy, S.E., Martins, R.N., 2004. The role of beta amyloid in Alzheimer's disease: still a cause of everything or the only one who got caught? Pharmacol. Res. 50, 397–409. Watcharasit, P., Bijur, G.N., Song, L., Zhu, J., Chen, X., Jope, R.S., 2003. Glycogen synthase kinase-3beta (GSK3beta) binds to and promotes the actions of p53. J. Biol. Chem. 278, 48872–48879. Yatin, S.M., Aksenova, M., Aksenov, M., Markesbery, W.R., Aulick, T., Butterfield, D.A., 1998. Temporal relations among beta-peptide-induced free-radical oxidative stress, neuronal toxicity, and neuronal defensive responses. J. Mol. Neurosci. 11, 183–197.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Amyloid-beta-induced neurotoxicity is reduced by inhibition of glycogen synthase kinase-3 Seong-Ho Koh, Min Young Noh, Seung Hyun Kim⁎ Department of Neurology, College of Medicine, Hanyang University, Seoul, Republic of Korea
A R T I C LE I N FO
AB S T R A C T
Article history:
Deposition of amyloid-β protein (Aβ) is one of the most important pathologic features in
Accepted 18 October 2007
Alzheimer's disease. It is well known that Aβ induces neuronal cell death through several
Available online 1 November 2007
pathogenic mechanisms. Although the role of glycogen synthase kinase (GSK)-3β in the
Keywords:
direct GSK-3β inhibition on Aβ-induced neurotoxicity. Thus, in this study, the relationship
Amyloid beta
between GSK-3β activity and Aβ-induced neurotoxicity was explored. To investigate the
Glycogen synthase kinase-3
role of GSK-3β in Aβ-induced neurotoxicity, neurons were treated with amyloid beta-
Neurotoxicity
protein (1–42) (Aβ42) oligomers with or without the addition of a GSK-3β inhibitor for 72 h.
Alzheimer's dementia
An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, trypan blue
neurotoxicity of Aβ has been highlighted, there has been no report evaluating the effect of
staining, and DAPI staining all showed that Aβ42 treatment alone resulted in decreased neuronal cell viability in a concentration-dependent manner. Aβ42 treatment significantly increased the activity of GSK-3β and cell death signals such as phosphorylated Tau (pThr231), cytosolic cytochrome c, and activated caspase-3. Aβ42 treatment also resulted in decreased survival signals, including that of heat shock transcription factor-1. Treatment with a GSK-3β inhibitor prevented Aβ-induced cell death. These results suggest that the neurotoxic effect of Aβ42 is mediated by GSK-3β activation and that inhibition of GSK-3β can reduce Aβ42-induced neurotoxicity. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Alzheimer's disease (AD), in which patients show a progressive decline in multiple cognitive functions, is characterized by widespread neurodegeneration, deposition of amyloid beta-protein (Aβ), and formation of intraneuronal neurofibrillary tangles (NFTs) in the brain ( Jellinger and Bancher, 1998; Jellinger, 2002; Trojanowski and Lee, 2000). Although several pathogenic mechanisms are proposed to be related to AD, Aβ is thought to be a key molecule in the pathogenesis of AD (Verdile et al., 2004). According to the amyloid cascade hypothesis (Robinson and Bishop, 2002; Verdile et al., 2004),
an increase in amyloid beta-protein fragment (1–42) (Aβ42) levels induces accumulation, oligomerization, deposition, and aggregation of amyloid beta. Aggregated Aβ42 elicits an inflammatory response, causes oxidative stress, alters homeostasis of the central nervous system, and gives rise to neuronal cell death, which is linked to dementia. Glycogen synthase kinase (GSK)-3β has been suggested to play important roles in several proposed pathogenic mechanisms of Aβinduced neuronal cell death (Takashima et al., 1996). Glycogen synthase kinase (GSK)-3, originally identified as a regulator of glycogen synthesis (Embi et al., 1980), is now known to be a multifaceted enzyme, affecting a diverse range
⁎ Corresponding author. Department of Neurology, College of Medicine, Hanyang University, #17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, Republic of Korea. Fax: +82 2 2296 8370. E-mail address: [email protected] (S.H. Kim). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.10.064
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –2 62
of biological functions including gene expression, cellular architecture, and apoptosis ( Jope and Johnson, 2004). Of the two closely related isoforms, GSK-3α and GSK-3β, GSK-3β is known to play critical roles in oxidative stress-induced neuronal cell death mechanisms (Bijur and Jope, 2000, 2001; Grimes and Jope, 2001; Koh et al., 2003, 2004a,b; Takadera and Ohyashiki, 2004; Watcharasit et al., 2003). However, it has not been reported whether direct inhibition of GSK-3β can prevent Aβ-induced neuronal cell death. This study investigated the role of GSK-3β in Aβ42-induced primary cultured cortical neuron death. The effects of GSK-3β inhibition on Aβ42-induced neurotoxicity were also examined.
2.
Results
2.1.
Role of GSK-3β in Aβ42-induced neurotoxicity
To evaluate the viability of cells following Aβ42 treatment, cortical cells were incubated with Aβ42 [0 (control), 5, 10, 20, or
255
50 μM] for 72 h. After incubation, cell viability was measured with an MTT assay, and surviving cells were counted using trypan blue stain. Following Aβ42 treatment, cell viability was significantly decreased in a concentration-dependent pattern (Fig. 1A). Based on these results, 20 μM was selected as an optimal Aβ42 concentration for subsequent experiments as cell viability was approximately 60% at that concentration (Fig. 1A). Additionally, cell death at 20 μM Aβ42 appeared to be due mainly to apoptosis (Fig. 2). To investigate the effects of GSK-3β inhibitor VIII itself, cortical cells were treated with several concentrations of GSK3β inhibitor VIII (0, 10, 25, 50, 100, 200, 500, and 1000 nM) for 24 h. Compared with the control, cell viability was not decreased at the 100 nM treatment level (97.9 ± 6.2% in TBS and 97.8 ± 3.9% in MTT, p N 0.10), but was significantly decreased at treatment levels of 200 nM or greater (89.8 ± 3.2% in TBS and 90.1 ± 4.5% in MTT, p < 0.01) (Fig. 1B). These results suggest that excessive inhibition of GSK-3β induces neuronal cell death. To investigate the importance of GSK-3β in Aβ42-induced neurotoxicity, cortical cells were incubated with both 20 μM
Fig. 1 – Measurement of primary cultured cortical neuronal cell viability by MTT assay and trypan blue stain. All data are presented as means (% of control) ± SEM from five or more independent experiments. Each treatment group was compared with the other groups using Duncan's Multiple Range Test after One-Way ANOVA. *p < 0.05 when compared with the control group. # p < 0.05 when compared with the group treated only with 20 μM Aβ42.
256
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –26 2
Fig. 2 – Detection of apoptosis in cortical cells by DAPI staining. The percent of apoptosis was increased to 34.0 ± 2.9% in cortical cells treated only with 20 μM Aβ42, but decreased to 13.3 ± 2.6% in cells treated with both 20 μM Aβ42 and 50 nM GSK-3β inhibitor VIII (p < 0.01). The data are represented as means (% of control) ± SEM. Each treatment group was compared with the other groups using Duncan's Multiple Range Test after One-Way ANOVA. *p < 0.01 when compared with the control group. #p < 0.01 when compared with the group treated only with 20 μM Aβ42.
Aβ42 and several concentrations of either GSK-3β inhibitor VIII (0, 25, 50, or 100 nM) or lithium chloride (0, 1, 5, 10, or 100 mM) for 72 h. Compared with the control, cell viability was significantly decreased following 20 μM Aβ42 treatment (59.0 ± 9.0% in MTT and 59.6 ± 5.8% in TBS, p < 0.01). However, cotreatment with 50 nM GSK-3β inhibitor VIII increased cell viability (81.5 ± 5.3% in MTT and 83.4 ± 5.6% in PBS, p < 0.01) (Fig. 1C). Co-treatment with 5 mM lithium chloride increased cell viability approximately 23% (p < 0.05, Fig. 1D). These results suggest that GSK-3β might play very important roles in Aβ42induced neuronal cell death. DAPI staining demonstrated that the percent of apoptotic cells was significantly increased with 20 μM Aβ42 treatment (34.0 ± 2.9%) compared with control (4.5 ± 1.3%). The percent of apoptotic cells was markedly reduced by treatment with 50 nM GSK-3β inhibitor VIII (13.3 ± 2.6%) ( p < 0.01) (Fig. 2).
2.2.
Fig. 3 – GSK-3β enzyme activity assay. Kinase activity was measured in cortical cell lysates 72 h after the initial treatment under several different conditions. Treatment with 20 μM Aβ42 increased kinase activity, but 50 nM GSK-3β inhibitor VIII treatment effectively inhibited the increase in kinase activity from 20 μM Aβ42 treatment. The data are represented as means (% of control) ± SEM. Each treatment group was compared with the other groups using Duncan's Multiple Range Test after One-Way ANOVA. *p < 0.05 when compared with the control group. #p < 0.05 when compared with the group treated with only 20 μM Aβ42.
DA (Fig. 4). The level of free radicals was significantly increased in cells treated with 20 μM Aβ42 (197.35 ± 22.66%), compared with the control (100.00 ± 30.93%, p < 0.01). However, co-treatment with GSK-3β inhibitor VIII did not decrease the amounts of free radicals (189.47 ± 37.55%). These findings suggest that Aβ42 induces the production of free radicals and that GSK-3β inhibitor VIII does not reduce
Effects of Aβ42 on GSK-3 enzyme activity
Based on the viability results, GSK-3β enzyme activity was evaluated in the cultured cells. Twenty micromolar Aβ42 treatment for 72 h increased GSK-3β enzyme activity to 210.7 ± 30.0% (p < 0.001) compared with control cortical cells without any treatment (100.0 ± 10.2%), while the combined treatment of 20 μM Aβ42 and 50 nM GSK-3β inhibitor VIII for 72 h successfully inhibited the activity increase (127.3 ± 15.8%, p < 0.001) (Fig. 3). These findings suggest that Aβ42 treatment increased GSK-3β enzyme activity and GSK-3β inhibitor VIII effectively inhibited the activity.
2.3. Effects of Aβ42 and GSK-3β inhibitor VIII on free radical formation To appreciate the oxidant effects of Aβ42, the amounts of free radicals in the cortical cells treated only with 0 or 20 μM Aβ42 for 30 min were measured using the fluorescent probe DCFH-
Fig. 4 – Measurement of free radical production. Data are represented as means ± SEM. Each treatment group was compared with the other groups using Duncan's Multiple Range Test. Cortical cells were treated with 20 μM Aβ42 for 30 min to measure free radicals generated in the presence of Aβ42 using the fluorescent probe DCFH-DA (see Materials and methods). The amount of free radicals was significantly different in the cells treated with 20 μM Aβ42. However, this change was not blocked by combined treatment with 50 nM GSK-3β inhibitor VIII. * = p < 0.05 (level of free radicals compared with that of the control group).
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –2 62
the production of free radicals induced by Aβ42. Thus, the blocking of Aβ42-induced neurotoxic effects by GSK-3β inhibitor VIII is not related to the inhibition of free radical production by Aβ42.
2.4.
257
Effect of Aβ42 on intracellular signal levels
To ascertain the effects of Aβ42 treatment on intracellular signal levels, cortical cell homogenates from 72-hour post-
Fig. 5 – Immunoreactivity (IR) of heat shock transcription factor-1, phosphorylated Tau (Thr231), Tau, cytosolic cytochrome c, and activated caspase-3 in cortical cells 72 h after the initial treatment with 20 μM Aβ42. Quantitative data from cortical cells are expressed in arbitrary units normalized to the simultaneously assayed control group value. Each treatment group was compared with the other groups using Duncan's Multiple Range Test. Representative ECL radiographs of immunoblots, showing the IRs of heat shock transcription factor-1 (HSTF-1) (A), phosphorylated Tau (Thr231) and Tau (B), cytosolic cytochrome c (C), and activated caspase-3 (D). Quantitative data expressing the IR ratios of HSTF-1 (A), phosphorylated Tau (Thr231)/Tau (B), cytosolic cytochrome c (C), and activated caspase-3 (D) normalized to actin immunostaining in the control group, the 20 μM Aβ42-treated group, and groups simultaneously treated with 20 μM Aβ42 and 50 nM GSK-3β inhibitor VIII. The data are represented as means (% of control) ± SEM. Each treatment group was compared with the other groups using Duncan's Multiple Range Test after One-Way ANOVA. *p < 0.05 when compared with the control group. #p < 0.05 when compared with the group treated with only 20 μM Aβ42.
258
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –26 2
Fig. 6 – Schematic diagram of a model for the prevention of Aβ42-induced neurotoxicity by GSK-3β inhibition. treatment samples were immunoblotted to examine levels of heat shock transcription factor-1 (HSTF-1), phosphorylated Tau (Thr231), Tau, cytosolic cytochrome c, and activated caspase-3. Compared with non-treated cells (1.00 ± 0.17), the immunoreactivity (IR) of HSTF-1 was significantly decreased in 20 μM Aβ42-treated cells (0.10 ± 0.08, p < 0.01) at 72 h (Fig. 5). However, this decrease was recovered with a combined treatment of Aβ42 and 50 nM GSK-3β inhibitor VIII (0.71 ±0.12, p < 0.01). In contrast, treatment with 20 μM Aβ42 increased the IRs of phosphorylated Tau (Thr231)/Tau, cytosolic cytochrome c, and activated caspase-3 (4.69 ± 0.82, 7.32 ±0.78, and 3.77 ± 0.54, respectively), compared with non-treated cells (1.00 ± 0.31, 1.00 ± 0.26, and 1.00 ± 0.15, respectively) (p < 0.01) (Fig. 5). The combined treatment with 50 nM GSK-3β inhibitor VIII resulted in decreased expression levels of these proteins (1.71 ± 0.57, 1.79 ± 0.43, and 1.50 ± 0.49, respectively) (p < 0.01). These findings suggest that Aβ42 decreases survival signals, including HSTF-1, and increases cell death signals including phosphorylated Tau (Thr231), cytochrome c, and activated caspase-3 and that these cytotoxic effects of Aβ42 can be blocked by GSK-3β inhibition.
3.
Discussion
In recent years, numerous studies have suggested that neurotoxicity of amyloid beta (Aβ) is one of the most important pathogenic factors in Alzheimer's disease (Robinson and Bishop, 2002; Verdile et al., 2004). With the recent emphasis on the role of GSK-3β in neurodegenerative diseases, it has been reported that treatment of neuronal cells with Aβ increased GSK-3β activity (Takashima et al., 1996). Lithium, a GSK-3β inhibitor, reduced production of Aβ in an animal model of AD (Rockenstein et al., 2007) and inhibited Aβ-induced stress in the endoplasmic reticulum of rabbit hippocampus (Ghribi et al., 2003). However, there has been no report of the effects of the direct inhibition of GSK-3β, GSK-3β activity, or the GSK-3β signaling pathway on Aβ-induced neuronal cell death.
In the present study, we aimed to investigate what mechanisms, in addition to the reduction of phosphorylation of Tau (Bhat et al., 2003), may contribute to the protective effects of GSK-3β inhibition against Aβ-induced neurotoxicity. Therefore, we studied whether GSK-3β inhibition could have an antioxidant effect or could alter other cellular signals such as HSTF-1 and caspase-3. This study is an extension of similar studies that have used highly selective GSK-3β inhibitors to define the role of GSK-3β in cellular signaling in the pathogenesis of Alzheimer's disease (Bhat et al., 2003). Our results showed that Aβ42 treatment induced neuronal cell death, and this neurotoxic effect was blocked by treatment with either GSK-3β inhibitor VIII, a direct and highly selective GSK-3β inhibitor (Bhat et al., 2003; Gould et al., 2004; Vasdev et al., 2005) which effectively inhibited GSK-3β activity in a concentration-dependent manner in a previous study (Lee et al., 2007), or lithium chloride, another GSK-3β inhibitor. Aβ42 treatment induced neuronal cell death, and this neurotoxicity was associated with GSK-3β activation and decreased survival signals, including HSTF-1, which is a direct substrate of GSK-3β and an important survival signal (Bijur and Jope, 2000). Aβ42 treatment also resulted in increased cell death signals, including phosphorylated Tau (Thr231), cytosolic cytochrome c, and activated caspase-3. These neurotoxic effects of Aβ42 were ameliorated by combined treatment with the specific GSK-3β inhibitor. The GSK-3β inhibitor inhibited the activation of GSK-3β, the reduction of HSTF-1, the increase of phosphorylated Tau (Thr231) and cytosolic cytochrome c, the activation of caspase-3, and the neuronal cell death induced by Aβ42. These effects of the GSK-3β inhibitor on Aβ42-induced neurotoxicity were not related to a direct free radical scavenging effect. Because GSK-3β inhibitor VIII did not affect Aβ42-induced free radical production, the neuroprotective effect of GSK-3β inhibition is not likely related to the inhibition of free radical production. It is well known that free radical production is one of the mechanisms of Aβ42-induced neurotoxicity (Yatin et al., 1998). According to the present study, GSK-3β inhibition did not affect Aβ42-induced free radical production but
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –2 62
protected primary cortical neurons against Aβ42. This finding suggests that GSK-3β inhibition does not have an antioxidant effect and that other neuroprotective mechanisms might play important roles. Therefore, other mechanisms involved in the GSK-3β pathway were investigated. GSK-3β is well known to promote cell death, especially neuronal cell death (Kirschenbaum et al., 2001; Koh et al., 2003, 2004, 2005; Lee et al., 2007; Martinez et al., 2002; Pap and Cooper, 2002), by both enhancing the expression of pro-apoptotic proteins and inhibiting the activity of anti-apoptotic proteins (Kirschenbaum et al., 2001; Koh et al., 2003, 2004, 2005; Lee et al., 2007; Martinez et al., 2002; Pap and Cooper, 2002). GSK-3β directly affects CCAAT/enhancer binding protein, nuclear factor of activated T cells, Myc, HSTF-1, Tau, cyclic adenosine mono phosphate (AMP) response element binding protein, Activator protein-1, β-catenin, NFkB, and p53, promotes the release of cytochrome c, activates caspase-3, and induces neuronal cell death (Bijur and Jope, 2000, 2001; Grimes and Jope, 2001; Koh et al., 2003, 2004, 2005; Lee et al., 2007; Takadera and Ohyashiki, 2004; Watcharasit et al., 2003). In this study, Aβ42 treatment significantly increased GSK-3β activity, similar to a previous finding (Takashima et al., 1996). Aβ42 also increased pro-apoptotic signals such as phosphorylated Tau(Thr235), cytosolic cytochrome c, and cleaved caspase3 and reduced survival signals including HSTF-1. GSK-3β inhibition restored cell viability and survival signals and prevented pro-apoptotic signals (Fig. 6). These data suggest that GSK-3β activation by Aβ might be a mechanism for Aβ neurotoxic effects. This hypothesis was confirmed by the finding that a GSK-3β inhibitor blocked GSK-3β activity and inhibited the neuronal cell death induced by Aβ42. One limitation to the interpretation of these results, however, is that this study was performed under in vitro conditions, and the results could be different in an in vivo situation, which involves more complicated factors.
4.
Conclusion
It can be suggested that Aβ42 neurotoxicity is closely linked to GSK-3β activation and that inhibition of GSK-3β could be a new promising strategy for preventing Aβ42 neurotoxicity.
5.
Experimental procedures
5.1.
Materials
Dulbecco's Modified Eagle's Medium (DMEM, high glucose) was purchased from GIBCO. The sources of drugs were as follows: amyloid beta-protein (1–42) (Aβ42), lithium chloride, trypan blue solution, Insulin, and DNase I were purchased from Sigma. GSK-3β inhibitor VIII {N-(4-Methoxybenzyl)-N′-(5nitro-1,3-thiazol-2-yl) urea, IC50 = 104 nM, C12H12N4O4S; molecular weight: 308.3 (Noble et al., 2005)}, a direct and highly selective GSK-3β inhibitor (Bhat et al., 2003; Gould et al., 2004; Vasdev et al., 2005), which effectively inhibited GSK-3β activity in a concentration-dependent manner in a previous study (Lee et al., 2007), was purchased from Calbiochem (La Jolla, CA, USA). Before use, these drugs were dissolved in distilled water
259
and then further diluted with culture medium to yield the desired final concentrations.
5.2.
Cell culture and treatment
All animal procedures were performed in accordance with the Hanyang University guidelines for the care and use of laboratory animals and were approved by the Institutional Review Board of Hanyang University. Primary cultures were obtained from the cerebral cortex of fetal Sprague Dawley rats (16 days of gestation). Briefly, rat embryos were decapitated, and the brains were rapidly removed and placed in a Petri dish half-filled with ice-cold Hank's balanced salt solution (HBSS; 137 mM NaCl, 5.4 mM KCl, 0.3 mM Na2HPO4, 0.4 mM KH2PO4, 5.6 mM glucose, and 2.5 mM HEPES) (Gibco BRL, NY, USA). Single cells dissociated from the whole cerebral cortex of fetal rats were plated on poly-L-lysine (Sigma, Saint Louis, MO, USA) pre-coated 100-mm corning dishes (5 × 106 cells/cm2) or glass coverslips and placed in Nunc 6- or 24-well plates (5 × 105, 2.5 × 106 cells/cm2). Cultures were incubated in Dulbecco's Modified Eagle Medium (high glucose) and supplemented with 10% heat-inactivated fetal bovine serum (1.7 days after plating), 1% penicillin streptomycin, 3.7 g/ L NaHCO3, 5 μg/mL insulin, and p-aminobenzoic acid. Cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere. Two days after plating, non-neuronal cells were removed by adding 5 μM cytosine arabinoside for 24 h. Only mature cultures (7 days in vitro) were used for experiments.
5.3.
Oligomeric Aβ42 preparation and treatment
Synthetic Aβ42 peptide with N95% purity by RPHPLC chromatography was used (Sigma). Oligomeric Aβ42 peptides were prepared as described previously (Dahlgren et al., 2002). Briefly, Aβ42 peptide was dissolved to 1 mmol/L in 100% hexafluoroisopropanol (Sigma), the hexafluoroisopropanol was removed under vacuum, and the peptide was stored at ¡©20 °C. For the aggregation, the peptide was first resuspended in dry dimethylsulfoxide (Me2SO; Sigma) to 5 mmol/L. For oligomeric conditions, culture medium was added to bring the peptide to a final concentration of 100 μmol/L, and the peptide was incubated at 4 °C for 24 h. Different concentrations of oligomeric Aβ42 were tested to assess its effect on neuronal cell viability. After primary cultured cortical neuronal cells were incubated with oligomeric Aβ42, an aggregate form [0 (control), 5, 10, 20, or 50 μM] (Sigma) for 72 h (Nakagawa et al., 2000), each plate was washed, and cell viability was measured using the MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] assay and trypan blue stain. To evaluate the role of GSK-3β in Aβ42-induced neurotoxicity, cortical cells were incubated in the presence of 20 μM Aβ42 together with several concentrations of GSK-3β inhibitor VIII (0, 25, 50, or 100 nM) or lithium chloride (0, 1, 5, 10, 100 mM) for 72 h. To estimate the alteration in intracellular signals, such as heat shock transcription factor-1 (HSTF-1), phosphorylated tau (pThr231), Tau, cytosolic cytochrome c, or caspase-3, cortical cells were harvested 72 h after treatment under various conditions and then immediately used for immunoblotting.
260 5.4.
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –26 2
MTT assay and trypan blue staining
MTT is absorbed into cells and transformed into formazan by mitochondrial succinate dehydrogenase. Accumulation of formazan directly reflects the activity of mitochondria, which functions as an indirect measurement of cell viability. Cells were plated at a density of 1 × 104 cells/well in a 96-well plate, cultured, and differentiated prior to adding 50 μL of 2 mg/mL MTT (Sigma) to 200 μL of medium present in each well. An aliquot (220 μL) of the resulting solution was removed from each well followed by the addition of 150 μL dimethyl sulfoxide. After the precipitate in each well was resuspended on a microplate mixer for 10 min, an optical density (OD) reading at 540 nm was measured using an ELISA plate reader. All results were normalized to OD values measured from an identically conditioned well without cell culture. For trypan blue staining, 10 μL of trypan blue solution was incubated for 2 min with 10 μL of cells from each sample. Unstained live cells were counted on a hemocytometer.
5.5.
DAPI staining
10 mM DTT, 0.1 μM okadaic acid, 0.5 mM Na3VO4, 1 mM benzamidine, and 1 mM PMSF) and sonicated on ice using a Vibratome probe sonicator (setting 4). The lysates were cleared by centrifugation at 10,000×g for 30 min. The supernatant was then assayed for GSK-3β activity.
5.6.2.
Assay conditions
GSK-3β activity was measured by the transfer of 32P from [γ-32P] ATP to the eIF2B peptide substrate. Per assay, 12.5 μL of extract (the supernatant from above) was mixed with 6.25 μL substrate mix (4 mg/mL eIF2B peptide with or without 200 mM lithium chloride). Assays were initiated by the addition of 6.25 mL ATP mix [200 mM HEPES, pH 7.5, 50 mM MgCl2, 8 mM DTT, 400 μM ATP, 0.125 μCi/μΛ (γ-32P) ATP (3000 Ci/mL)]. The reactions were incubated at room temperature and terminated after 8 min by spotting 20 μL onto P81 ion-exchange paper (Whatman). The paper was washed three times in 100 mM phosphoric acid, and the bound radioactivity was quantified by scintillation counting (Ryves et al., 1998). All assays were run in duplicate with the means and standard error (SEM) of at least two separate experiments shown in each figure.
5.7.
Determination of free radical production
DAPI staining was performed to evaluate apoptosis as follows: cortical cells were incubated for 72 h with one of three treatments: (1) without Aβ42 or GSK-3β inhibitor VIII, (2) with 20 μM Aβ42, and (3) with both 20 μM Aβ42 and 50 nM GSK-3β inhibitor VIII. All samples were then centrifuged at 2000×g for 2 min, at which point the supernatants were discarded and 4% neutral buffered formalin (100 μL) was added to each cell pellet. A 50-μL aliquot of the cell suspension was applied to a glass slide and dried at room temperature. The fixed cells were then washed with PBS, air dried, and stained with the DNAspecific fluorochrome 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma, St. Louis, MO, USA) for 20 min. The adherent cells were rinsed with PBS, air dried, and mounted with 90% glycerol. The slides were observed under an Olympus fluorescence microscope ( Jiang et al., 2000). The percentage of apoptotic cells was determined, which in turn coincided with the proportion of cells exhibiting morphological hallmarks of apoptosis, such as DNA fragmentation, nuclear condensation, and segmentation.
To measure free radical production, cortical cells were treated as follows: (1) with 0 (control), (2) with 20 μM Aβ42, and (3) with both 20 μM Aβ42 and 50 nM GSK-3β inhibitor VIII for 30 min. Treated cells were incubated with the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Molecular Probes Inc, Eugene, OR, USA), which freely crosses the cell membranes and is hydrolyzed by cellular esterases to 2′,7′-dichlorodihydrofluorescein (DCFH2). Though DSFH2 is a non-fluorescent molecule, it is oxidized to the fluorescent 2′ 7′-dichlorofluorescein (DCF) in the presence of peroxides. Accumulation of DCF in cells was measured as a change in fluorescence at 525 nm when the sample was excited at 488 nm by a Microplate Fluorescence Reader (FL600) (D.I Biotech Ltd., Seoul, Korea) (Tammariello et al., 2000). The time point of 30 min of incubation was chosen because maximal effects were shown at this time point according to a report by Filomeni et al. (2003).
5.6.
5.8.
GSK-3 activity assay
The following peptide substrates were purchased from Peptron Inc. (Daejeon, Korea): Eukaryotic translation initiation factor 2B peptide substrate, eIF2B-SP: RRAAEELDSRAG-pSPQL (positive control substrate); eIF2BA: RRAAEELDSRAGAPQL (negative control substrate) (Ryves et al., 1998). A 10 mg/mL peptide stock solution was prepared in deionized water and stored at −20 °C. These purified peptide stocks were assumed to be 100% pure for subsequent calculations. Escherichia coliexpressed mammalian GSK-3β (mGSK-3β), encoded by rabbit skeletal muscle cDNA, was purchased from New England Biolabs (USA) and stored at −20 °C.
5.6.1.
Preparation of lysates
1 × 108 cortical cells incubated for 72 h were washed twice in cold PBS, resuspended in 1 mL lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1% Triton X-100,
Western blot analysis
Heat shock transcription factor-1 (HSTF-1), phosphorylated tau (pThr231), cytosolic cytochrome c, and the cleaved form of caspase-3 were analyzed by western blot. Briefly, 5 × 106 cells were collected after incubation for 72 h, washed twice in cold PBS, and incubated for 10 min on ice in lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.2% SDS, 100 μg/ mL phenylmethylsulfonyl fluoride (PMSF), 50 μl/mL aprotinin, 1% igepal 630, 100 mM NaF, 0.5% sodium deoxycholate, 0.5 mM EDTA, 0.1 mM EGTA]. Cell lysates were centrifuged at 10,000×g for 20 min at 4 °C, and the supernatants were used to evaluate HSTF-1, phosphorylated tau (pThr231), Tau, and the cleaved form of caspase-3. For evaluation of cytosolic cytochrome c levels, the washed cells were suspended in sucrose-supplemented cell extract buffer (SCEB, 300 mM sucrose, 10 mM HEPES at pH 7.4, 50 mM KCl, 5 mM EGTA, 5 mM MgCl2, 1 mM DTT, 10 μM cytochalasin B, 1 mM PMSF), left on ice for 30 min,
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –2 62
and then homogenized by 50 strokes in an ice-cold Dounce homogenizer. Unbroken cells and nuclei were pelleted by centrifugation at 2000×g for 10 min and discarded. Mitochondria were collected from the resulting supernatant by further centrifugation at 13,000×g for 10 min. The resulting supernatant containing the post-mitochondrial fraction was used for immunoblotting. Protein concentrations of the cell lysates were determined using a Bio-Rad protein assay kit. For western blot analysis, 20 μg of total protein from each sample was resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membranes were blocked with 5% skim milk and incubated with specific antibodies for HSTF-1 (1:1000, Santa Cruz Biotech, Delaware, CA, USA), phospho-tau (pThr231) (1:1000, Calbiochem), cytochrome c (1:500, Santa Cruz Biotech, Delaware, CA, USA), and cleaved (activated form) caspase-3 (Asp 175) (1:1000, Cell signaling, Beverly, MA, USA). The membranes were washed with Tris buffered saline containing 0.05% Tween-20 (TBST) and processed with an HRP-conjugated anti-rabbit antibody or anti-mouse antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA) for ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The results of several western blots were quantified on a Quantity One-4,2,0 image analyzer (Bio-Rad, Hercules, CA) and were normalized to actin immunostaining (Koh et al., 2003, 2004, 2005; Lee et al., 2007).
5.9.
Statistical analysis
All data are presented as means ± SEM from five or more independent experiments. Statistical comparisons between different treatment groups were conducted with Duncan's Multiple Range Test after One-Way ANOVA. Differences in p-values of less than 0.05 were considered statistically significant.
Acknowledgments This work was supported by a grant from Hanyang University (2007-000-0000-4823) and the Laboratory for Clinical Investigation, College of Medicine, Hanyang University, Seoul, Korea.
REFERENCES
Bhat, R., Xue, Y., Berg, S., Hellberg, S., Ormo, M., Nilsson, Y., Radesater, A.C., Jerning, E., Markgren, P.O., Borgegard, T., Nylof, M., Gimenez-Cassina, A., Hernandez, F., Lucas, J.J., Diaz-Nido, J., Avila, J., 2003. Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A014418. J. Biol. Chem. 278, 45937–45945. Bijur, G.N., Jope, R.S., 2000. Opposing actions of phosphatidylinositol 3-kinase and glycogen synthase kinase-3beta in the regulation of HSTF-1 activity. J. Neurochem. 75, 2401–2408. Bijur, G.N., Jope, R.S., 2001. Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3 beta. J. Biol. Chem. 276, 37436–37442. Dahlgren, K.N., Manelli, A.M., Stine Jr., W.B., Baker, L.K., Krafft, G.A., LaDu, M.J., 2002. Oligomeric and fibrillar species of amyloidbeta
261
peptides differentially affect neuronal viability. J. Biol. Chem. 277, 32046–32053. Embi, N., Rylatt, D.B., Cohen, P., 1980. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur. J. Biochem. 107, 519–527. Filomeni, G., Aquilano, K., Rotilio, G., Ciriolo, M.R., 2003. Reactive oxygen species-dependent c-Jun NH2-terminal kinase/c-Jun signaling cascade mediates neuroblastoma cell death induced by diallyl disulfide. Cancer Res. 63, 5940–5949. Ghribi, O., Herman, M.M., Savory, J., 2003. Lithium inhibits Abeta-induced stress in endoplasmic reticulum of rabbit hippocampus but does not prevent oxidative damage and tau phosphorylation. J. Neurosci. Res. 71, 853–862. Gould, T.D., Einat, H., Bhat, R., Manji, H.K., 2004. AR-A014418, a selective GSK-3 inhibitor, produces antidepressant-like effects in the forced swim test. Int. J. Neuropsychopharmacol. 7, 387–390. Grimes, C.A., Jope, R.S., 2001. The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog. Neurobiol. 65, 391–426. Jellinger, K.A., 2002. Alzheimer disease and cerebrovascular pathology: an update. J. Neural Trans. 109, 813–836. Jellinger, K.A., Bancher, C., 1998. Neuropathology of Alzheimer's disease: a critical update. J. Neural Transm., Suppl. 54, 77–95. Jiang, Q., Gu, Z., Zhang, G., Jin, G., 2000. Diphosphorylation and involvement of extracellular signal-regulated kinases (ERK1/2) in glutamate-induced apoptotic-like death in cultured rat cortical neurons. Brain Res. 857, 71–77. Jope, R.S., Johnson, G.V., 2004. The glamour and gloom of glycogen synthase kinase-3. Trends Biochem. Sci. 29, 95–102. Kirschenbaum, F., Hsu, S.C., Cordell, B., McCarthy, J.V., 2001. Glycogen synthase kinase-3beta regulates presenilin 1 C-terminal fragment levels. J. Biol. Chem. 276, 30701–30707. Koh, S.H., Kim, S.H., Kwon, H., Park, Y., Kim, K.S., Song, C.W., Kim, J., Kim, M.H., Yu, H.J., Henkel, J.S., Jung, H.K., 2003. Epigallocatechin gallate protects nerve growth factor differentiated PC12 cells from oxidative-radical-stress-induced apoptosis through its effect on phosphoinositide 3-kinase/Akt and glycogen synthase kinase-3. Brain Res. Mol. Brain Res. 118, 72–81. Koh, S.H., Kim, S.H., Kwon, H., Kim, J.G., Kim, J.H., Yang, K.H., Kim, J., Kim, S.U., Yu, H.J., Do, B.R., Kim, K.S., Jung, H.K., 2004a. Phosphatidylinositol-3 kinase/Akt and GSK-3 mediated cytoprotective effect of epigallocatechin gallate on oxidative stress-injured neuronal-differentiated N18D3 cells. Neurotoxicology 25, 793–802. Koh, S.H., Kwon, H., Park, Y., Kim, J.G., Kim, K.S., Song, C.W., Kim, J.H., Kim, J., Kim, M.H., Kim, K.S., Yu, H.J., Jung, H.G., Kim, S.H., 2004b. Diallyl disulfide inhibits cytochrome c-mediated apoptosis in H2O2 induced death of neuronal-differentiated PC12 cells. J. Korean Neurol. Assoc. 22, 375–381. Koh, S.H., Lee, Y.B., Kim, K.S., Kim, H.J., Kim, M., Lee, Y.J., Kim, J., Lee, K.W., Kim, S.H., 2005. Role of GSK-3beta activity in motor neuronal cell death induced by G93A or A4V mutant hSOD1 gene. Eur. J. Neurosci. 22, 301–309. Lee, K.Y., Koh, S.H., Noh, M.Y., Park, K.W., Lee, Y.J., Kim, S.H., 2007. Glycogen synthase kinase-3β activity plays very important roles in determining the fate of oxidative stress-inflicted neuronal cells. Brain Res. 1129, 89–99. Martinez, A., Castro, A., Dorronsoro, I., Alonso, M., 2002. Glycogen synthase kinase 3 (GSK-3) inhibitors as new promising drugs for diabetes, neurodegeneration, cancer, and inflammation. Med. Res. Rev. 22, 373–384. Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B.A., Yuan, J., 2000. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 403, 98–103.
262
BR A I N R ES E A RC H 1 1 8 8 ( 2 00 8 ) 2 5 4 –26 2
Noble, W., Planel, E., Zehr, C., Olm, V., Meyerson, J., Suleman, F., Gaynor, K., Wang, L., LaFrancois, J., Feinstein, B., Burns, M., Krishnamurthy, P., Wen, Y., Bhat, R., Lewis, J., Dickson, D., Duff, K., 2005. Inhibition of glycogen synthase kinase-3 by lithium correlates with reduced tauopathy and degeneration in vivo. Proc. Natl. Acad. Sci. U. S. A. 102, 6990–6995. Pap, M., Cooper, G.M., 2002. Role of translation initiation factor 2B in control of cell survival by the phosphatidylinositol 3-kinase/ Akt/glycogen synthase kinase 3beta signaling pathway. Mol. Cell. Biol. 22, 578–586. Robinson, S.R., Bishop, G.M., 2002. Abeta as a bioflocculant: implications for the amyloid hypothesis of Alzheimer's disease. Neurobiol. Aging 23, 1051–1072. Rockenstein, E., Torrance, M., Adame, A., Mante, M., Bar-on, P., Rose, J.B., Crews, L., Masliah, E., 2007. Neuroprotective effects of regulators of the glycogen synthase kinase-3beta signaling pathway in a transgenic model of Alzheimer's disease are associated with reduced amyloid precursor protein phosphorylation. J. Neurosci. 27, 1981–1991. Ryves, W.J., Fryer, L., Dale, T., Harwood, A.J., 1998. An assay for glycogen synthase kinase 3 (GSK-3) for use in crude cell extracts. Anal. Biochem. 264, 124–127. Tammariello, S.P., Quinn, M.T., Estus, S., 2000. NADPH oxidase contributes directly to oxidative stress and apoptosis in nerve growth factor-deprived sympathetic neurons. J. Neurosci. 20, 1–5. Takadera, T., Ohyashiki, T., 2004. Glycogen synthase kinase-3 inhibitors prevent caspase-dependent apoptosis induced by ethanol in cultured rat cortical neurons. Eur. J. Pharmacol. 499, 239–245.
Takashima, A., Noguchi, K., Michel, G., Mercken, M., Hoshi, M., Ishiguro, K., Imahori, K., 1996. Exposure of rat hippocampal neurons to amyloid h peptide (25–35) induces the inactivation of phosphatidyl inositol-3 kinase and the activation of tau protein kinase I/glycogen synthase kinase-3h. Neurosci. Lett. 203, 33–36. Trojanowski, J.Q., Lee, V.M., 2000. “Fatal attractions” of proteins. A comprehensive hypothetical mechanism underlying Alzheimer's disease and other neurodegenerative disorders. Ann. N. Y. Acad. Sci. 924, 62–67. Vasdev, N., Natesan, S., Galineau, L., Garcia, A., Stableford, W.T., McCormick, P., Seeman, P., Houle, S., Wilson, A.A., 2005. Synthesis and ex vivo evaluation of carbon-11 labelled N-(4-methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea ([11C] AR-A014418): a radiolabelled glycogen synthase kinase-3beta specific inhibitor for PET studies. Bioorg. Med. Chem. Lett. 15, 5270–5273. Verdile, G., Fuller, S., Atwood, C.S., Laws, S.M., Gandy, S.E., Martins, R.N., 2004. The role of beta amyloid in Alzheimer's disease: still a cause of everything or the only one who got caught? Pharmacol. Res. 50, 397–409. Watcharasit, P., Bijur, G.N., Song, L., Zhu, J., Chen, X., Jope, R.S., 2003. Glycogen synthase kinase-3beta (GSK3beta) binds to and promotes the actions of p53. J. Biol. Chem. 278, 48872–48879. Yatin, S.M., Aksenova, M., Aksenov, M., Markesbery, W.R., Aulick, T., Butterfield, D.A., 1998. Temporal relations among beta-peptide-induced free-radical oxidative stress, neuronal toxicity, and neuronal defensive responses. J. Mol. Neurosci. 11, 183–197.