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Homocysteine-induced acute excitotoxicity in cerebellar granule cells in vitro is accompanied by PP2A-mediated dephosphorylation of tau
Neurochemistry International 55 (2009) 174–180
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Homocysteine-induced acute excitotoxicity in cerebellar granule cells in vitro is accompanied by PP2A-mediated dephosphorylation of tau Magdalena Kuszczyk, Wanda Gordon-Krajcer, Jerzy W. Lazarewicz * Department of Neurochemistry, Medical Research Centre, Polish Academy of Sciences, 5 Pawin´skiego Street, 02-106 Warsaw, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 September 2008 Received in revised form 27 January 2009 Accepted 17 February 2009 Available online 24 February 2009
Our results demonstrate that acute exposure of primary rat cerebellar granule cell cultures to homocysteine at millimolar concentrations induces a glutamate receptor-mediated decrease in tau protein phosphorylation, which is accompanied by excitotoxic neuronal damage. This contrasts with the previously reported hyperphosphorylation of tau in homocysteine-treated neurons, and with the assumption that hyperhomocysteinemia is one of the risk factors in Alzheimer disease, in which abnormal hyperphosphorylation of tau protein leads to neurofibrillary degeneration. The mechanisms of homocysteine neurotoxicity have been explained mainly either by disturbances in methylation processes, that may also lead to the accumulation of phosphorylated tau due to reduced activity of tauselective protein phosphatase 2A, or by excitotoxicity. Since the relationships between homocysteine excitotoxicity and tau phosphorylation are unclear, the aim of this study was to characterize these processes in neurons acutely treated with homocysteine at neurotoxic concentrations, and to link them to the activities of glutamate receptors and protein phosphatase 2A. Within 24 h following a 30 min exposure of neuronal cultures to 20 mM D,L-homocysteine, significant neurotoxicity was induced. This could be reduced by treatment with an uncompetitive NMDA receptor antagonist, MK-801 (0.5 mM), or by mGlu1 and mGlu5 receptor antagonists, LY367385 and MPEP, respectively (both at 25 mM). Western blot analysis showed a rapid decrease in immunostaining of phospho-tau, 2 h after incubation of cell cultures with 15 mM D,L-homocysteine, which persisted for 6 h after the insult. Application of MK-801, LY367385 or okadaic acid (100 nM), an inhibitor of protein phosphatases 1 and 2A, significantly prevented dephosphorylation of tau, implying a role for the activation of glutamate receptors and protein phosphatase 2A. The phosphorylation of tau may be increased or reduced by treatment with homocysteine, and the nature of the cellular response to this sulfur-containing amino acid depends on the neuronal phenotype. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Cerebellar granule cells Excitotoxicity Homocysteine mGluRs NMDA receptors Phosphorylation Tau protein PP2A
1. Introduction Alzheimer’s disease (AD) is a late-onset progressive neurodegenerative disorder that has been linked to genetic as well as acquired factors (Reddy, 2006). Apart from deposits of b-amyloid proteins, one of the main features of AD is progressive, intraneuronal accumulation of fibers referred to as paired helical filaments (PHF), which is followed by the generation of neurofibrillary tangles (NFT). It is believed that PHF formation is a late event in AD progression and is related to extensive losses of neurons and synapses in selected areas of the brain (Alonso et al., 2001). The major component of PHF is abnormally hyperphosphorylated full-length tau. Tau is a member of the microtubule-associated phosphoprotein (MAP) family that
* Corresponding author. Tel.: +48 22 608 65 28; fax: +48 22 668 54 23. E-mail address: [email protected] (J.W. Lazarewicz). 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.02.010
plays an essential role in the regulation and stabilization of neuronal microtubule assembly in the central nervous system and is involved in axonal transport (Terry, 1998). The degree of tau phosphorylation modulates its affinity for microtubule binding sites and regulates the dynamics of microtubules. Hyperphosphorylated tau protein isolated from the brains of AD patients lacks the ability to promote microtubule assembly (Iqbal et al., 1986). Abnormal phosphorylation of tau protein, which leads to PHF and subsequently NFT formation observed in AD brains (Iqbal et al., 1994), might result from multiple metabolic abnormalities. The pivotal dysfunction appears to be an imbalance in kinase and phosphatase activities resulting from dysregulation of the phosphorylation/dephosphorylation system (Gong et al., 2006). Several studies have shown attenuated expression and/or activity of protein phosphatases, particularly protein phosphatase 2A (PP2A), in brain tissues from patients with AD, and the critical role of decreased PP2A activity in tau hyperphosphorylation has been demonstrated in model
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experiments (Gordon-Krajcer et al., 2000; Vogelsberg-Ragaglia et al., 2001; Sontag et al., 2004). Homocysteine (Hcy) is a sulfur-containing amino acid, which is an endogenous product of methionine metabolism in the course of the transmethylation pathway. Methionine is metabolized to S-adenosylmethionine (SAM), which serves as the proximal methyl donor for most methylation reactions (Kruman et al., 2000; Shi et al., 2003). SAM-dependent methylation reactions result in the production of S-adenosylhomocysteine (SAH), which is a potent inhibitor of methyltransferase enzymes. In the presence of high concentrations of Hcy, SAH accumulates in cells, leading to global inhibition of methylation processes (Medina et al., 2001; Obeid and Herrmann, 2006). In adults, the normal range for total plasma Hcy is 5–15 mM (Kang et al., 1992). High methionine intake, Hcy metabolism abnormalities (Medina et al., 2001) and/or dietary folate and vitamin B deficiencies (Mattson and Shea, 2003) can lead to hyperhomocysteinemia, where total plasma levels can reach 50–200 mM. Epidemiological studies have shown that elevated level of Hcy in tissue fluid is associated with cardiovascular diseases (Biasioli and Schiavon, 2000; Kang et al., 1992). It is also a risk factor in AD (Leblhuber et al., 2000; Miller, 2000; Morris, 2003; Shea et al., 2002). Data from recent studies link tau pathology with Hcy (Vafai and Stock, 2002; Morris, 2003; Sontag et al., 2007; Luo et al., 2007). Hcy-induced disturbances in methylation processes may lead to downregulation of PP2A, which in turn results in hyperphosphorylation of tau protein (Vafai and Stock, 2002; Obeid et al., 2007; Sontag et al., 2007). Alternatively, homocysteine-induced activation of excitatory amino acid receptors may influence the level of tau phosphorylation (Ho et al., 2002). The role of NMDA receptors and group I metabotropic glutamate receptors (mGluRs GI) in homocysteine-induced neurodegeneration has been repeatedly demonstrated (Lipton et al., 1997; Zieminska et al., 2003, 2006; Obeid and Herrmann, 2006; Zieminska and Lazarewicz, 2006). Excitotoxicity-evoked disturbances in calcium-mediated signaling, that may alter the equilibrium in protein phosphorylation and dephosphorylation systems are among the putative contributory factors involved in abnormal tau phosphorylation (Mattson, 2003). Thus, excitotoxicity triggered by Hcy might be among the mechanisms interfering with the phosphorylation of tau. Hcy-induced tau phosphorylation has been demonstrated in the studies of Ho et al. (2002) and Chan et al. (2008), using primary cultures of neurons submitted to a prolonged treatment with Hcy or a folate-deprived neuroblastoma cell line, respectively. Both investigations demonstrated the accumulation of phosphorylated tau protein linked to Hcy-mediated activation of NMDA receptors and mitogen-activated protein kinase (MAPK). However, the increased tau phosphorylation evoked by prolonged exposure to Hcy may result not only from NMDA receptor- and calcium-mediated activation of kinases. Also SAM deficit and a reduction in phosphatase activity may be involved. Acute exposure of neurons to Hcy under conditions promoting excitotoxicity, but minimizing its interference with methylation processes, might distinctly affect phosphorylation of tau. The role of PP2A activity in Hcy-induced effects also remains unclear. The aim of the present study was to characterize changes in tau phosphorylation in neurons acutely treated with Hcy at neurotoxic concentrations, and to relate these changes to NMDA receptor and mGluRs GI activation, and to the activity of PP2A. Primary cultures of rat cerebellar granule cells were acutely exposed to D,Lhomocysteine in the absence or presence of NMDAR and mGluR antagonists, and these were examined for neuronal viability and changes in tau protein phosphorylation.
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2. Experimental procedures 2.1. Materials D,L-homocysteine (Hcy), N-methyl- D-aspartate (NMDA), okadaic acid (OA) and materials for cell culture were purchased from Sigma (St. Louis, MO, USA). (S)(+)-a-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385) and 2-methyl6-(phenylethynyl)pyridine hydrochloride (MPEP) were obtained from Tocris Neuramin Ltd. (Bristol, UK). (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) was obtained from RBA (Natic, MA, USA).
2.2. Cell cultures Primary cultures of granule neurons were prepared from cerebella of 8-day-old Wistar rats as described by Schousboe et al. (1985). Use of the rat pups was in accordance with Polish and international regulations. The procedure was approved by the First Local Ethical Committee in Warsaw. All efforts were made to reduce the number of animals used and minimize their suffering. Briefly, the brains were removed from decapitated heads, cerebella were dissected and meninges with the vessels were stripped off. Then the cerebella were chopped into 400 mm cubes. The tissue was then incubated for 15 min at 37 8C in the medium containing 120 mM NaCl, 5 mM KCl, 25 mM HEPES (pH 7.4) and 9.1 mM glucose supplemented with 0.025% trypsin and 0.05% DNase I. The incubation was terminated by the addition of trypsin inhibitor (0.04%) and the mixture centrifuged for 1.5 min at 480 g and 4 8C. The pellet was triturated, and after further centrifugation the cells were suspended in basal Eagle’s medium supplemented with heat-inactivated 10% fetal calf serum (Sigma), 25 mM KCl, 4 mM glutamine, streptomycin (50 mg/ml) and penicillin (50 U/ml). Granule neurons were seeded at a density of 2 106 cells per well in 12well plates (NUNC) or at a density of 15 106 cells per poly-L-lysine-coated culture bottle. Cultures were supplemented with 7.5 mM cytosine arabinofuranoside 36 h after plating. The cells were used for experiments after 7 days in culture at 37 8C in a humidified atmosphere containing a 5% CO2. 2.3. Experimental procedure and sample preparation Acute Hcy-induced neurotoxic damage was evoked in vitro as described by Ankarcrona et al. (1995). The growth medium was replaced by Locke 25 incubation buffer (134 mM NaCl, 25 mM KCl, 4 mM NaHCO3, 5 mM HEPES, pH 7.4, 2.3 mM CaCl2, 5 mM glucose). Glutamate receptor antagonists were added for 5 min before 30-min incubation with Hcy. The Locke medium was then removed and the cells returned to the original growth medium and cultured for a further 24 h under standard conditions. The viability of cultures was estimated morphologically using 5% propidium iodide staining and live/dead neurons were counted using a Zeiss Axiovert 25 fluorescence microscope. The proportion of live cells was expressed as a percentage of the total cell number. To study effects of Hcy on tau phosphorylation, the cells were treated as described above, using Locke 25 incubation buffer. To examine the role of PP2A in alterations of the phosphorylation state of tau, neurons in Locke 25 buffer were pretreated with okadaic acid for 1 h before Hcy administration. Control cells were incubated in Locke 25 buffer without additions. Thereafter, the Locke 25 buffer was removed and the cells were cultured in the original growth medium. The cells were harvested just after incubation (0 h), and 2, 4 or 6 h after removing the pharmacological agents. According to the method of Valerio et al. (1995), the cells were collected in 300 ml of ice-cold lysis buffer containing 50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA, plus 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5% Protease Inhibitor Cocktail, 0.5% nonidet-P40 and 0.5% Na-deoxycholate (all from Sigma). The suspension was homogenized and centrifuged at 10,000 g for 20 min at 4 8C. The resulting supernatant was used for further analysis. 2.4. Immunochemical analysis of tau protein abundance and phosphorylation For Western immunoblot analysis, monoclonal antibody AT-8, purchased from Innogenetics Laboratories (Ghent, Belgium, dilution 1:400) or monoclonal antibody Tau 46 obtained from Zymed Laboratories (CA, USA, dilution 1:500) were used. Secondary antibody (GAM) conjugated to horseradish peroxidase (Vector Laboratories, Burlingame, CA, USA) was used at 1:1000 dilution. Electrophoresis of protein lysates (70 mg) was performed on 10% SDS-polyacrylamide gels (SDS-PAGE). The separated proteins were electrophoretically transferred onto nitrocellulose membranes and these were incubated with 5% non-fat dried milk in 10 mM Tris–HCl, pH 7.4, 150 mM NaCl (TBS) for 1 h at room temperature to block non-specific binding sites. The blots were then incubated overnight with primary antibodies at 4 8C. After washing in TBS containing 0.05% Tween-20, the membranes were incubated with secondary antibody for 2 h at room temperature. Both the primary and secondary antibodies were diluted in 5% dried milk-TBS. Specific immunoreactivity was detected by enhanced chemiluminescence staining (ECL), placing the blots processed with Amersham ECL reagents against Amersham Hyperfilm ECL. The intensity of immunostained bands was quantified by densitometric scanning of the autoradiographs using an Image Scanner III with LabScan 6.0 software (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
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2.5. Data analysis The results were expressed as the mean S.D. (n = 4–5). The statistical significance of the differences was determined by analysis of variance (ANOVA) followed by Dunnett’s test. A value of P < 0.05 was regarded as statistically significant.
3. Results 3.1. Neurotoxic ability of Hcy: role of NMDAR and mGluRs GI It has been previously demonstrated that acute exposure of cerebellar granule cells to millimolar concentrations of Hcy induces neuronal death, which may be significantly reduced by antagonists of NMDA receptors and of both subtypes of mGluRs GI, mGlu1 and mGlu5 (Zieminska et al., 2003). As the first step in this study we re-examined these characteristics of Hcy neurotoxicity. The results shown in Fig. 1 confirmed that a 30 min exposure of neuronal cultures to high concentrations of Hcy resulted in a dosedependent decrease in cell survival, visualized 24 h after the toxin treatment by staining with propidium iodide. The EC50 for Hcy was 16.23 mM, which is in agreement with our previous findings (Zieminska et al., 2003). These data confirmed that comparing to glutamate which is known to induce excitotoxicity at 10 4 M concentrations, Hcy is a relatively weak neurotoxin and millimolar concentrations are required to induce significant neuronal damage after acute exposure of cells in culture. To test the hypothesis that acute Hcy-induced neurotoxicity might be mediated by NMDA receptors and mGluRs GI, the effects of NMDA channel blocker MK-801, mGlu1 antagonist LY367385 and mGlu5 antagonist MPEP were tested. Application of 0.5 mM MK-801 greatly inhibited the acute neurotoxicity of 20 mM Hcy, while 25 mM LY367385 or 25 mM MPEP only slightly reduced this harmful effect (Fig. 2). Simultaneous treatment of cerebellar granule cells with MK-801 and mGluRs GI antagonists did not increase the neuroprotection provided by MK-801 alone. Our data indicate a key role for NMDA receptors in the mechanism of acute Hcy neurotoxicity.
Fig. 2. Inhibition of acute homocysteine-induced neurotoxicity by administration of selective glutamate receptor antagonists. The NMDA receptor antagonist MK-801 (0.5 mM), and antagonists of mGlu1 and mGlu5, LY367385 and MPEP, respectively (both 25 mM) were added 5 min before 30 min incubation of cell cultures with 20 mM D,L-homocysteine (Hcy). Data represent the percentage of live cells compared with the total number of neurons. Results are the mean S.D. (n = 5). * Means significantly different from results obtained with 20 mM Hcy added alone (P < 0.05).
3.2. Hcy induces alterations in the phosphorylation state of tau protein To try and relate our findings on acute Hcy neurotoxicity to its effect on the phosphorylation state of tau protein, we used the phosphorylation-dependent anti-tau antibody AT-8, which recognizes phospho-Ser202/Thr205. As shown in Fig. 3A and B, no significant changes in tau phosphorylation were observed immediately after 30 min incubation of cultured cells with 15 mM Hcy (lane 2). However, 2 h after the exposure to Hcy a significant decrease in immunolabeling by AT-8 was noted, indicating a rapid reduction in the level of the phosphorylated form of tau. This effect persisted at 4 and 6 h after exposure of cells to Hcy (Fig. 3A and B). To exclude the possibility that the Hcyevoked decrease in the immunoreactivity of the phosphorylated form of tau might reflect a reduction in the level of tau protein, the phosphate-independent tau antibody Tau 46 was used. Fig. 3C shows a Western blot of total cell proteins from untreated cultures (lane 1) and from cultures recovering for 2, 4 and 6 h after 30 min exposure to 15 mM Hcy, immunostained with Tau 46. Quantitative densitometry showed no significant alterations in tau protein levels in cells collected at any time after incubation with this toxic agent (Fig. 3D). 3.3. Role of glutamate receptors in Hcy-evoked dephosphorylation of tau protein
Fig. 1. Concentration-dependent homocysteine-evoked neurotoxicity in cultured cerebellar granule neurons. The number of cells surviving 24 h after a 30 min treatment with different concentrations of D,L-homocysteine (Hcy) was determined by propidium iodide staining. Data represent the proportion of live cells compared with the total cell number. Results are the mean S.D. (n = 5). * Means significantly different from control (P < 0.05).
To determine whether the Hcy-induced decrease in the phosphorylation state of tau was mediated by glutamate receptors, we tested the effects of the NMDA channel blocker MK-801 and mGlu1 and mGlu5 antagonists. Cells were preincubated with the
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Fig. 3. Effects of homocysteine on the phosphorylation state of tau and the level of tau protein in cultured cerebellar granule neurons. The cells were incubated with 15 mM D,L-homocysteine (Hcy) for 30 min. Samples of cell lysates were collected from cultures at different times following incubation with Hcy: after 0, 2, 4, and 6 h. Phosphorylated forms of tau were detected by immunoblotting with phospho-tau specific antibody AT-8 (A); the level of tau protein was detected with phosphate-independent tau-specific antibody Tau 46 (B). The autoradiographs visualize two detected isoforms of tau protein of 64 kDa and 58 kDa MW. Data are presented as a percentage of the control value (untreated cultures). Results are the mean S.D. (n = 4). * Means significantly different from control (P < 0.05).
specific antagonists for 5 min before their 30 min exposure to 15 mM Hcy. Samples were collected 2 h after termination of the challenge with Hcy. Administration of the individual antagonists modified Hcy-evoked dephosphorylation of the tau protein in different ways. Application of MK-801 completely prevented the effect of Hcy and even evoked an increase in tau phosphorylation of about 40%, whereas mGlu1 antagonist LY367385 reversed the effect of Hcy, while mGlu5 antagonist MPEP had only a very slight impact on the level of phosphorylated tau (Fig. 4A and B). The combination of MK-801 with the mGluRs GI antagonists did not significantly alter the effects of MK-801 alone. These results demonstrate that NMDA receptors, and to a lesser extent mGlu1 play an essential role in mediating Hcy-evoked dephosphorylation of tau protein. 3.4. Role of protein phosphatase 2A in Hcy-evoked dephosphorylation of tau protein To determinate the role of PP2A in Hcy-induced tau dephosphorylation, the selective inhibitor of PP1 and PP2A okadaic acid was used. In agreement with previous studies, our preliminary results demonstrated that OA at high concentration of 250 mM induced neurodegeneration (data not shown). To reduce the cytotoxic effect of this drug, while still obtaining significant inhibition of phosphatase activity, cell cultures were preincubated with 100 nM OA for 1 h. Incubation of cell cultures with 100 nM OA alone caused no changes in the phosphorylation state of tau at any analyzed time point, and no significant alterations in neuronal viability were observed (results not shown). However, as shown in Fig. 5A and B, this treatment of rat cerebellar granule neurons followed by administration of 15 mM Hcy for 30 min reversed Hcy-induced tau dephosphorylation. Instead, it resulted in a gradual increase in labeling of the phosphorylated form of tau by AT-8, which reached its highest level 6 h after Hcy treatment. This effect of OA, which is an inhibitor of tau-selective PPA2 indicated that Hcy simultaneously induces phosphorylation and dephosphorylation of tau protein. The latter PP2A-mediated process predominates, however, its inhibition by OA unmasks Hcy-induced phosphorylation of tau protein.
Fig. 4. Alterations in the homocysteine-induced phosphorylation state of tau after administration of the selective glutamate receptor antagonists. The antagonist of NMDA receptors, MK-801 (0.5 mM) and antagonists of mGlu1, LY367385 (25 mM) and of mGlu5, MPEP (25 mM), were applied 5 min before 30 min incubation with 15 mM D,L-homocysteine (Hcy). Samples were collected 2 h after Hcy treatment. Densitometric analysis of the tau protein phosphorylation are presented as a percentage of the control value (untreated cultures). Data are the mean S.D. (n = 4). * Means significantly different from control (P < 0.05).
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Fig. 5. Effect of okadaic acid (OA) on homocysteine-induced changes in the phosphorylation state of tau. The cells were pre-incubated with 100 nM OA for 1 h before adding 15 mM D,L-homocysteine (Hcy) for 30 min. Samples were collected from neuronal cultures after times 0, 2, 4, and 6 h following Hcy treatment. The intensity of the autoradiographic signal was regarded as the degree of tau phosphorylation and is presented as a percentage of the control cultures treated with OA alone. Two or three forms of tau were identified in Hcy- and OA-treated cultures. Data are the mean S.D. (n = 4). * Means significantly different from control + OA (P < 0.05).
4. Discussion In this study we investigated the effect of acute challenge of rat cerebellar granule cells in primary culture with Hcy on the phosphorylation state of tau protein in parallel with Hcy-induced neurotoxicity. The experiments were focused on the role of excitatory amino acid receptors in both processes. We confirmed that Hcy at millimolar concentrations induces acute excitotoxicity mediated by NMDAR and mGluRs GI. A new and important finding of this study is that instead of the expected Hcy-evoked hyperphosphorylation of tau protein, we observed dephosphorylation of tau lasting several hours after acute exposure to Hcy. This effect was inhibited by NMDA and mGlu1 receptor antagonists. Our results also indicate that protein phosphatase PP2A is instrumental in Hcy-evoked tau dephosphorylation. Below we will discuss possible mechanisms of these phenomena. Our results confirmed that the acute treatment with Hcy at millimolar concentrations leads to neuronal cell death (Lipton et al., 1997; Zieminska et al., 2003). In the present work, as in several previous studies, we used the racemic form of Hcy (D,L-Hcy) instead of the physiologically active L-form of homocysteine which is not commercially available. Moreover, it is known that Hcy interferes in a complex way with NMDA receptors, activating the glutamate binding site and inhibiting the glycine co-agonist site (Lipton et al., 1997). This also explains why acutely administered Hcy is a weak neurotoxic agent comparing to glutamate. In conformity with previous data (Lipton et al., 1997; Mattson and Shea, 2003; Zieminska et al., 2003) we found that the neurodegeneration evoked by Hcy was effectively blocked by an
uncompetitive antagonist of NMDA receptors, MK-801. In addition, the mGlu1 and mGlu5 antagonists, LY367385 and MPEP, known as effective neuroprotective agents in in vitro models of excitotoxicity (Bruno et al., 2000), had a moderate ability to prevent the loss of neurons induced by exposure to Hcy, which is in accordance with our earlier findings (Zieminska et al., 2003, 2006; Zieminska and Lazarewicz, 2006). In the present study we focused on the effect of acute Hcy application on the phosphorylation state of tau protein in primary cultures of cerebellar granule neurons. We found that in parallel with development of neuronal injury a significant decrease in the immunoreactivity of phosphorylated tau protein arose. This effect, detected using AT-8 antibody, persisted up to 6 h after incubation with this toxic agent. Under our experimental conditions we did not observe any alterations in the total level of tau protein immunoreactivity (detected with Tau 46 antibody). This indicates that exposure of neurons to Hcy suppresses tau phosphorylation in a way related to excitotoxicity. Our results of experiments using glutamate receptor antagonists confirm this assumption. Hcyevoked dephosphorylation of tau protein, as well as Hcy neurotoxicity was completely reversed by MK-801, an uncompetitive NMDA channel blocker. Such a treatment even enhanced tau phosphorylation. Administration of the mGlu1 receptor antagonist LY367385 less effectively prevented Hcy-induced dephosphorylation of tau, while mGlu5 antagonist MPEP had only a negligible effect. Hcy-evoked dephosphorylation of tau was an unexpected finding, since recently Luo et al. (2007) demonstrated that acute intracerebroventricular injections of Hcy in rats induced tau hyperphosphorylation in the brain in vivo. Others have also observed Hcy-evoked tau hyperphosphorylation which they linked with NMDA receptor-mediated activation of calcium-dependent kinase pathways in neurons (Ho et al., 2002; Chan et al., 2008). The majority of previous studies demonstrating Hcy-induced hyperphosphorylation of tau protein were conducted under conditions of chronic or subchronic exposure to this amino acid, promoting inhibition of methylation processes (Vafai and Stock, 2002; Sontag et al., 2007; Obeid et al., 2007). We believe that acute 30-min exposure of cell cultures to Hcy, which we used in the present study, minimized such an effect, and thus we are able to ascribe the phenomena observed in this study mainly to glutamate receptor-mediated processes. Nevertheless, there are discrepancies in the literature concerning the effects of acute glutamateinduced excitotoxicity and the phosphorylation of tau protein. Davis et al. (1995) demonstrated that treatment of primary rat cortical neuronal cultures with high concentrations of glutamate leads to decreased immunolabeling by phospho-tau specific AT-8 antibody. However, other in vitro studies have shown intense AT-8 immunostaining after glutamate or NMDA administration (Sindou et al., 1994; Couratier et al., 1996; Li et al., 2004). Some authors have reported that glutamate triggers notable tau phosphorylation only during the initial phase of glutamate toxicity (Sindou et al., 1992; Davis et al., 1995). Also, previous in vivo studies demonstrated that intracortical perfusion of glutamate results in the rise in tau phosphorylation in specific brain regions (Irving et al., 1996). These differences may reflect altered regulation of the activity of specific protein kinases and phosphatases, resulting in diverse responses of tau phosphorylation to the activation of glutamate receptors. A crucial role of NMDA receptors in Hcy-evoked changes in the phosphorylation state of tau which was detected in this study strongly suggests involvement of calcium. As it is known that regulation of the phosphorylation/dephosphorylation system is dependent on intracellular calcium levels (Sindou et al., 1994). In several studies an experimentally induced increase in intracellular calcium in cultures of hippocampal and human cortical neurons produced tau hyperphosphorylation as well as ultrastructural and
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antigenic changes similar to NFT (Mattson et al., 1991; Ho et al., 2002; Chan et al., 2008). However, Fleming and Johnson (1995) and Adamec et al. (1997) demonstrated that the NMDA receptormediated increase of intracellular calcium level resulted in the activation of calcium-dependent proteins responsible for the dephosphorylation of tau in vitro. Even more mysterious is the role of calcium in the mechanism of the acute neurotoxic effects of Hcy and eo ipso in changes of tau phosphorylation. Previous studies on Hcy neurotoxicity in cultured neurons described significant intracellular calcium transients accompanying Hcy-induced neurotoxicity and hyperphosphorylation of tau (Lipton et al., 1997; Kim, 1999; Ho et al., 2002; Chan et al., 2008). However, our previous experiments demonstrating homocysteine-evoked neurodegeneration in primary cultures of cerebellar granule cells, failed to show any pronounced activation of 45Ca uptake by homocysteine-treated neurons or intracellular calcium transients comparable to the effects of NMDA or glutamate (Zieminska et al., 2003, 2006). Shea and Ekinci (1999) demonstrated in a neuroblastoma cell line biphasic effects of calcium influx on tau phosphorylation. Experimental conditions promoting a reduced calcium load depleted phospho-tau levels, while enhanced calcium influx was accompanied by the rapid accumulation of phosphorylated tau. In conformity with these data, also our results show that the very low level of calcium influx into Hcy-treated cerebellar granule cells and the minor calcium transients (Zieminska et al., 2003, 2006) are accompanied by a decrease in tau phosphorylation. Our unpublished data demonstrated that in spite of maintaining cerebellar granule cells under standard culture conditions, these cells sporadically altered their phenotype, showing drastic increases in sensitivity to Hcy neurotoxicity. In these cells the concentration of Hcy that normally induced moderate neurotoxicity triggered extensive neuronal loss after 24 h. We noticed that this effect was usually accompanied by an initial temporary increase in tau phosphorylation after Hcy treatment. Presumably these cells may also develop more pronounced Hcy-evoked calcium transients. This possibility will be a subject of further investigation. Previous studies which demonstrated glutamate- or Hcyevoked increases in phosphorylation of tau protein focused on protein kinases possibly involved in this effect. Ho et al. (2002) and Chan et al. (2008) suggested that the Hcy-evoked NMDA receptormediated rise in intracellular calcium may activate calciumdependent protein kinases including MAPK and protein kinase C (PKC). In turn Li et al. (2004) using hippocampal organotypic cultures, demonstrated that glutamate-induced activation of NMDA receptors resulting in a rise in intracellular calcium levels in neurons had no apparent effect on PP2A activity in brain slices, whereas a marked increase in the phosphorylation of tau protein and activation of the calcium/calmodulin-dependent protein kinase II (CaMKII) was observed. However, our results which show dephosphorylation of tau suggest that protein phosphatases rather than kinases may be predominantly activated. PP2A, a trimeric complex is believed to be the most efficient enzyme in dephosphorylating tau in vitro and in vivo (Sontag et al., 2004; Liu et al., 2005). The results of Chan and Sucher (2001) indicate that stimulation of the NMDA receptor in cerebrocortical neuronal cultures results in dissociation of its complex with PP2A protein, leading to decreased phosphatase activity. In the present study, okadaic acid, an inhibitor of PPA1 and PP2A activity (Adamec et al., 1997; Li et al., 2004) has been used in order to examine the role of protein phosphatases in the dephosphorylation of tau following exposure of cerebellar granule cells to Hcy. Treatment of the cultures with nanomolar concentrations of OA, simultaneously with Hcy, completely blocked Hcy-induced dephosphorylation of tau and triggered a progressive, time-dependent increase in AT-8 immunoreactivity. Since PP2A rather than PP1 participates in tau
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dephosphorylation, this finding indicates that activation of the protein phosphatase PP2A is directly responsible for Hcy-induced dephosphorylation of tau protein. Moreover these results visualized the potential of Hcy for activation of simultaneous phosphorylation of tau protein. Inhibition of PP2A by OA in the Hcy-treated cells disturbed equilibrium between tau phosphorylation and dephosphorylation and disclosed tau phosphorylation by protein kinases. Our study was focused on acute effects of Hcy mediated by excitatory amino acid receptors. Excitotoxic challenge of cerebellar granule cells with this compound resulted in significant neuronal damage preceded by dephosphorylation of tau protein. This contrasts with the hyperphosphorylation of tau characteristically found in Alzheimer’s patients with hyperhomocysteinemia (Obeid et al., 2007), and also with the results of several in vivo and in vitro studies demonstrating increased tau phosphorylation in Hcytreated neurons (Ho et al., 2002; Sontag et al., 2007; Luo et al., 2007; Chan et al., 2008). We believe that this variation in the responses of the tau phosphorylation/dephosphorylation system of neurons to Hcy challenge results from the various models used in these studies and from the diversity of calcium-dependent and calcium-independent mechanisms regulating protein kinases and phosphatases. It seems that in chronic experiments, the Hcyevoked alterations in tau phosphorylation compatible with Alzheimer’s pathology mainly result from disturbances in methylation processes in the presence of Hcy (Sontag et al., 2007). However, Hcy-evoked glutamate receptor-mediated regulation of tau phosphorylation represents another signaling pathway that may be active under acute and prolonged experimental conditions (Lipton et al., 1997; Zieminska et al., 2003; Zieminska and Lazarewicz, 2006). It is possible that hyperhomocysteinemia in vivo may activate several such mechanisms in parallel. Our findings indicate that Hcy-induced hyperphosphorylation of tau is not an inherent feature of this sulfur-containing amino acid, but that the nature of the cellular response depends on the neuronal phenotype. Hcy may interact with glutamate receptors resulting in activation of both protein kinases and protein phosphatases, which regulate the level of tau phosphorylation. The final outcome depends on predominance of one of these systems. Our observations also suggest that the overall effect of Hcy on tau phosphorylation may depend on the degree of neuronal sensitivity to Hcy toxicity, which is probably related to Hcy-evoked calcium imbalance. 5. Conclusions The results of this study demonstrate that acute exposure of cerebellar granule cells in primary culture to a sufficient dose of Hcy causes excitotoxicity and leads to a rapid, long-lasting decrease in tau protein phosphorylation, without changing the total level of tau. Both effects are mediated mainly by NMDA receptors, with additional involvement of mGluRs GI. PP2A is the main protein phosphatase responsible for Hcy-induced dephosphorylation of tau. Acknowledgements The authors thank Dr. John Gittins for constructive comments on the manuscript. This study was supported by the Medical Research Centre PAS, Warsaw. References Adamec, E., Mercken, M., Beermann, M.L., Didier, M., Nixon, R.A., 1997. Acute rise in the concentration of free cytoplasmic calcium leads to dephosphorylation of the microtubule-associated protein tau. Brain Res. 757, 93–101.
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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint
Homocysteine-induced acute excitotoxicity in cerebellar granule cells in vitro is accompanied by PP2A-mediated dephosphorylation of tau Magdalena Kuszczyk, Wanda Gordon-Krajcer, Jerzy W. Lazarewicz * Department of Neurochemistry, Medical Research Centre, Polish Academy of Sciences, 5 Pawin´skiego Street, 02-106 Warsaw, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 September 2008 Received in revised form 27 January 2009 Accepted 17 February 2009 Available online 24 February 2009
Our results demonstrate that acute exposure of primary rat cerebellar granule cell cultures to homocysteine at millimolar concentrations induces a glutamate receptor-mediated decrease in tau protein phosphorylation, which is accompanied by excitotoxic neuronal damage. This contrasts with the previously reported hyperphosphorylation of tau in homocysteine-treated neurons, and with the assumption that hyperhomocysteinemia is one of the risk factors in Alzheimer disease, in which abnormal hyperphosphorylation of tau protein leads to neurofibrillary degeneration. The mechanisms of homocysteine neurotoxicity have been explained mainly either by disturbances in methylation processes, that may also lead to the accumulation of phosphorylated tau due to reduced activity of tauselective protein phosphatase 2A, or by excitotoxicity. Since the relationships between homocysteine excitotoxicity and tau phosphorylation are unclear, the aim of this study was to characterize these processes in neurons acutely treated with homocysteine at neurotoxic concentrations, and to link them to the activities of glutamate receptors and protein phosphatase 2A. Within 24 h following a 30 min exposure of neuronal cultures to 20 mM D,L-homocysteine, significant neurotoxicity was induced. This could be reduced by treatment with an uncompetitive NMDA receptor antagonist, MK-801 (0.5 mM), or by mGlu1 and mGlu5 receptor antagonists, LY367385 and MPEP, respectively (both at 25 mM). Western blot analysis showed a rapid decrease in immunostaining of phospho-tau, 2 h after incubation of cell cultures with 15 mM D,L-homocysteine, which persisted for 6 h after the insult. Application of MK-801, LY367385 or okadaic acid (100 nM), an inhibitor of protein phosphatases 1 and 2A, significantly prevented dephosphorylation of tau, implying a role for the activation of glutamate receptors and protein phosphatase 2A. The phosphorylation of tau may be increased or reduced by treatment with homocysteine, and the nature of the cellular response to this sulfur-containing amino acid depends on the neuronal phenotype. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Cerebellar granule cells Excitotoxicity Homocysteine mGluRs NMDA receptors Phosphorylation Tau protein PP2A
1. Introduction Alzheimer’s disease (AD) is a late-onset progressive neurodegenerative disorder that has been linked to genetic as well as acquired factors (Reddy, 2006). Apart from deposits of b-amyloid proteins, one of the main features of AD is progressive, intraneuronal accumulation of fibers referred to as paired helical filaments (PHF), which is followed by the generation of neurofibrillary tangles (NFT). It is believed that PHF formation is a late event in AD progression and is related to extensive losses of neurons and synapses in selected areas of the brain (Alonso et al., 2001). The major component of PHF is abnormally hyperphosphorylated full-length tau. Tau is a member of the microtubule-associated phosphoprotein (MAP) family that
* Corresponding author. Tel.: +48 22 608 65 28; fax: +48 22 668 54 23. E-mail address: [email protected] (J.W. Lazarewicz). 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.02.010
plays an essential role in the regulation and stabilization of neuronal microtubule assembly in the central nervous system and is involved in axonal transport (Terry, 1998). The degree of tau phosphorylation modulates its affinity for microtubule binding sites and regulates the dynamics of microtubules. Hyperphosphorylated tau protein isolated from the brains of AD patients lacks the ability to promote microtubule assembly (Iqbal et al., 1986). Abnormal phosphorylation of tau protein, which leads to PHF and subsequently NFT formation observed in AD brains (Iqbal et al., 1994), might result from multiple metabolic abnormalities. The pivotal dysfunction appears to be an imbalance in kinase and phosphatase activities resulting from dysregulation of the phosphorylation/dephosphorylation system (Gong et al., 2006). Several studies have shown attenuated expression and/or activity of protein phosphatases, particularly protein phosphatase 2A (PP2A), in brain tissues from patients with AD, and the critical role of decreased PP2A activity in tau hyperphosphorylation has been demonstrated in model
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experiments (Gordon-Krajcer et al., 2000; Vogelsberg-Ragaglia et al., 2001; Sontag et al., 2004). Homocysteine (Hcy) is a sulfur-containing amino acid, which is an endogenous product of methionine metabolism in the course of the transmethylation pathway. Methionine is metabolized to S-adenosylmethionine (SAM), which serves as the proximal methyl donor for most methylation reactions (Kruman et al., 2000; Shi et al., 2003). SAM-dependent methylation reactions result in the production of S-adenosylhomocysteine (SAH), which is a potent inhibitor of methyltransferase enzymes. In the presence of high concentrations of Hcy, SAH accumulates in cells, leading to global inhibition of methylation processes (Medina et al., 2001; Obeid and Herrmann, 2006). In adults, the normal range for total plasma Hcy is 5–15 mM (Kang et al., 1992). High methionine intake, Hcy metabolism abnormalities (Medina et al., 2001) and/or dietary folate and vitamin B deficiencies (Mattson and Shea, 2003) can lead to hyperhomocysteinemia, where total plasma levels can reach 50–200 mM. Epidemiological studies have shown that elevated level of Hcy in tissue fluid is associated with cardiovascular diseases (Biasioli and Schiavon, 2000; Kang et al., 1992). It is also a risk factor in AD (Leblhuber et al., 2000; Miller, 2000; Morris, 2003; Shea et al., 2002). Data from recent studies link tau pathology with Hcy (Vafai and Stock, 2002; Morris, 2003; Sontag et al., 2007; Luo et al., 2007). Hcy-induced disturbances in methylation processes may lead to downregulation of PP2A, which in turn results in hyperphosphorylation of tau protein (Vafai and Stock, 2002; Obeid et al., 2007; Sontag et al., 2007). Alternatively, homocysteine-induced activation of excitatory amino acid receptors may influence the level of tau phosphorylation (Ho et al., 2002). The role of NMDA receptors and group I metabotropic glutamate receptors (mGluRs GI) in homocysteine-induced neurodegeneration has been repeatedly demonstrated (Lipton et al., 1997; Zieminska et al., 2003, 2006; Obeid and Herrmann, 2006; Zieminska and Lazarewicz, 2006). Excitotoxicity-evoked disturbances in calcium-mediated signaling, that may alter the equilibrium in protein phosphorylation and dephosphorylation systems are among the putative contributory factors involved in abnormal tau phosphorylation (Mattson, 2003). Thus, excitotoxicity triggered by Hcy might be among the mechanisms interfering with the phosphorylation of tau. Hcy-induced tau phosphorylation has been demonstrated in the studies of Ho et al. (2002) and Chan et al. (2008), using primary cultures of neurons submitted to a prolonged treatment with Hcy or a folate-deprived neuroblastoma cell line, respectively. Both investigations demonstrated the accumulation of phosphorylated tau protein linked to Hcy-mediated activation of NMDA receptors and mitogen-activated protein kinase (MAPK). However, the increased tau phosphorylation evoked by prolonged exposure to Hcy may result not only from NMDA receptor- and calcium-mediated activation of kinases. Also SAM deficit and a reduction in phosphatase activity may be involved. Acute exposure of neurons to Hcy under conditions promoting excitotoxicity, but minimizing its interference with methylation processes, might distinctly affect phosphorylation of tau. The role of PP2A activity in Hcy-induced effects also remains unclear. The aim of the present study was to characterize changes in tau phosphorylation in neurons acutely treated with Hcy at neurotoxic concentrations, and to relate these changes to NMDA receptor and mGluRs GI activation, and to the activity of PP2A. Primary cultures of rat cerebellar granule cells were acutely exposed to D,Lhomocysteine in the absence or presence of NMDAR and mGluR antagonists, and these were examined for neuronal viability and changes in tau protein phosphorylation.
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2. Experimental procedures 2.1. Materials D,L-homocysteine (Hcy), N-methyl- D-aspartate (NMDA), okadaic acid (OA) and materials for cell culture were purchased from Sigma (St. Louis, MO, USA). (S)(+)-a-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385) and 2-methyl6-(phenylethynyl)pyridine hydrochloride (MPEP) were obtained from Tocris Neuramin Ltd. (Bristol, UK). (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) was obtained from RBA (Natic, MA, USA).
2.2. Cell cultures Primary cultures of granule neurons were prepared from cerebella of 8-day-old Wistar rats as described by Schousboe et al. (1985). Use of the rat pups was in accordance with Polish and international regulations. The procedure was approved by the First Local Ethical Committee in Warsaw. All efforts were made to reduce the number of animals used and minimize their suffering. Briefly, the brains were removed from decapitated heads, cerebella were dissected and meninges with the vessels were stripped off. Then the cerebella were chopped into 400 mm cubes. The tissue was then incubated for 15 min at 37 8C in the medium containing 120 mM NaCl, 5 mM KCl, 25 mM HEPES (pH 7.4) and 9.1 mM glucose supplemented with 0.025% trypsin and 0.05% DNase I. The incubation was terminated by the addition of trypsin inhibitor (0.04%) and the mixture centrifuged for 1.5 min at 480 g and 4 8C. The pellet was triturated, and after further centrifugation the cells were suspended in basal Eagle’s medium supplemented with heat-inactivated 10% fetal calf serum (Sigma), 25 mM KCl, 4 mM glutamine, streptomycin (50 mg/ml) and penicillin (50 U/ml). Granule neurons were seeded at a density of 2 106 cells per well in 12well plates (NUNC) or at a density of 15 106 cells per poly-L-lysine-coated culture bottle. Cultures were supplemented with 7.5 mM cytosine arabinofuranoside 36 h after plating. The cells were used for experiments after 7 days in culture at 37 8C in a humidified atmosphere containing a 5% CO2. 2.3. Experimental procedure and sample preparation Acute Hcy-induced neurotoxic damage was evoked in vitro as described by Ankarcrona et al. (1995). The growth medium was replaced by Locke 25 incubation buffer (134 mM NaCl, 25 mM KCl, 4 mM NaHCO3, 5 mM HEPES, pH 7.4, 2.3 mM CaCl2, 5 mM glucose). Glutamate receptor antagonists were added for 5 min before 30-min incubation with Hcy. The Locke medium was then removed and the cells returned to the original growth medium and cultured for a further 24 h under standard conditions. The viability of cultures was estimated morphologically using 5% propidium iodide staining and live/dead neurons were counted using a Zeiss Axiovert 25 fluorescence microscope. The proportion of live cells was expressed as a percentage of the total cell number. To study effects of Hcy on tau phosphorylation, the cells were treated as described above, using Locke 25 incubation buffer. To examine the role of PP2A in alterations of the phosphorylation state of tau, neurons in Locke 25 buffer were pretreated with okadaic acid for 1 h before Hcy administration. Control cells were incubated in Locke 25 buffer without additions. Thereafter, the Locke 25 buffer was removed and the cells were cultured in the original growth medium. The cells were harvested just after incubation (0 h), and 2, 4 or 6 h after removing the pharmacological agents. According to the method of Valerio et al. (1995), the cells were collected in 300 ml of ice-cold lysis buffer containing 50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA, plus 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5% Protease Inhibitor Cocktail, 0.5% nonidet-P40 and 0.5% Na-deoxycholate (all from Sigma). The suspension was homogenized and centrifuged at 10,000 g for 20 min at 4 8C. The resulting supernatant was used for further analysis. 2.4. Immunochemical analysis of tau protein abundance and phosphorylation For Western immunoblot analysis, monoclonal antibody AT-8, purchased from Innogenetics Laboratories (Ghent, Belgium, dilution 1:400) or monoclonal antibody Tau 46 obtained from Zymed Laboratories (CA, USA, dilution 1:500) were used. Secondary antibody (GAM) conjugated to horseradish peroxidase (Vector Laboratories, Burlingame, CA, USA) was used at 1:1000 dilution. Electrophoresis of protein lysates (70 mg) was performed on 10% SDS-polyacrylamide gels (SDS-PAGE). The separated proteins were electrophoretically transferred onto nitrocellulose membranes and these were incubated with 5% non-fat dried milk in 10 mM Tris–HCl, pH 7.4, 150 mM NaCl (TBS) for 1 h at room temperature to block non-specific binding sites. The blots were then incubated overnight with primary antibodies at 4 8C. After washing in TBS containing 0.05% Tween-20, the membranes were incubated with secondary antibody for 2 h at room temperature. Both the primary and secondary antibodies were diluted in 5% dried milk-TBS. Specific immunoreactivity was detected by enhanced chemiluminescence staining (ECL), placing the blots processed with Amersham ECL reagents against Amersham Hyperfilm ECL. The intensity of immunostained bands was quantified by densitometric scanning of the autoradiographs using an Image Scanner III with LabScan 6.0 software (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
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2.5. Data analysis The results were expressed as the mean S.D. (n = 4–5). The statistical significance of the differences was determined by analysis of variance (ANOVA) followed by Dunnett’s test. A value of P < 0.05 was regarded as statistically significant.
3. Results 3.1. Neurotoxic ability of Hcy: role of NMDAR and mGluRs GI It has been previously demonstrated that acute exposure of cerebellar granule cells to millimolar concentrations of Hcy induces neuronal death, which may be significantly reduced by antagonists of NMDA receptors and of both subtypes of mGluRs GI, mGlu1 and mGlu5 (Zieminska et al., 2003). As the first step in this study we re-examined these characteristics of Hcy neurotoxicity. The results shown in Fig. 1 confirmed that a 30 min exposure of neuronal cultures to high concentrations of Hcy resulted in a dosedependent decrease in cell survival, visualized 24 h after the toxin treatment by staining with propidium iodide. The EC50 for Hcy was 16.23 mM, which is in agreement with our previous findings (Zieminska et al., 2003). These data confirmed that comparing to glutamate which is known to induce excitotoxicity at 10 4 M concentrations, Hcy is a relatively weak neurotoxin and millimolar concentrations are required to induce significant neuronal damage after acute exposure of cells in culture. To test the hypothesis that acute Hcy-induced neurotoxicity might be mediated by NMDA receptors and mGluRs GI, the effects of NMDA channel blocker MK-801, mGlu1 antagonist LY367385 and mGlu5 antagonist MPEP were tested. Application of 0.5 mM MK-801 greatly inhibited the acute neurotoxicity of 20 mM Hcy, while 25 mM LY367385 or 25 mM MPEP only slightly reduced this harmful effect (Fig. 2). Simultaneous treatment of cerebellar granule cells with MK-801 and mGluRs GI antagonists did not increase the neuroprotection provided by MK-801 alone. Our data indicate a key role for NMDA receptors in the mechanism of acute Hcy neurotoxicity.
Fig. 2. Inhibition of acute homocysteine-induced neurotoxicity by administration of selective glutamate receptor antagonists. The NMDA receptor antagonist MK-801 (0.5 mM), and antagonists of mGlu1 and mGlu5, LY367385 and MPEP, respectively (both 25 mM) were added 5 min before 30 min incubation of cell cultures with 20 mM D,L-homocysteine (Hcy). Data represent the percentage of live cells compared with the total number of neurons. Results are the mean S.D. (n = 5). * Means significantly different from results obtained with 20 mM Hcy added alone (P < 0.05).
3.2. Hcy induces alterations in the phosphorylation state of tau protein To try and relate our findings on acute Hcy neurotoxicity to its effect on the phosphorylation state of tau protein, we used the phosphorylation-dependent anti-tau antibody AT-8, which recognizes phospho-Ser202/Thr205. As shown in Fig. 3A and B, no significant changes in tau phosphorylation were observed immediately after 30 min incubation of cultured cells with 15 mM Hcy (lane 2). However, 2 h after the exposure to Hcy a significant decrease in immunolabeling by AT-8 was noted, indicating a rapid reduction in the level of the phosphorylated form of tau. This effect persisted at 4 and 6 h after exposure of cells to Hcy (Fig. 3A and B). To exclude the possibility that the Hcyevoked decrease in the immunoreactivity of the phosphorylated form of tau might reflect a reduction in the level of tau protein, the phosphate-independent tau antibody Tau 46 was used. Fig. 3C shows a Western blot of total cell proteins from untreated cultures (lane 1) and from cultures recovering for 2, 4 and 6 h after 30 min exposure to 15 mM Hcy, immunostained with Tau 46. Quantitative densitometry showed no significant alterations in tau protein levels in cells collected at any time after incubation with this toxic agent (Fig. 3D). 3.3. Role of glutamate receptors in Hcy-evoked dephosphorylation of tau protein
Fig. 1. Concentration-dependent homocysteine-evoked neurotoxicity in cultured cerebellar granule neurons. The number of cells surviving 24 h after a 30 min treatment with different concentrations of D,L-homocysteine (Hcy) was determined by propidium iodide staining. Data represent the proportion of live cells compared with the total cell number. Results are the mean S.D. (n = 5). * Means significantly different from control (P < 0.05).
To determine whether the Hcy-induced decrease in the phosphorylation state of tau was mediated by glutamate receptors, we tested the effects of the NMDA channel blocker MK-801 and mGlu1 and mGlu5 antagonists. Cells were preincubated with the
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Fig. 3. Effects of homocysteine on the phosphorylation state of tau and the level of tau protein in cultured cerebellar granule neurons. The cells were incubated with 15 mM D,L-homocysteine (Hcy) for 30 min. Samples of cell lysates were collected from cultures at different times following incubation with Hcy: after 0, 2, 4, and 6 h. Phosphorylated forms of tau were detected by immunoblotting with phospho-tau specific antibody AT-8 (A); the level of tau protein was detected with phosphate-independent tau-specific antibody Tau 46 (B). The autoradiographs visualize two detected isoforms of tau protein of 64 kDa and 58 kDa MW. Data are presented as a percentage of the control value (untreated cultures). Results are the mean S.D. (n = 4). * Means significantly different from control (P < 0.05).
specific antagonists for 5 min before their 30 min exposure to 15 mM Hcy. Samples were collected 2 h after termination of the challenge with Hcy. Administration of the individual antagonists modified Hcy-evoked dephosphorylation of the tau protein in different ways. Application of MK-801 completely prevented the effect of Hcy and even evoked an increase in tau phosphorylation of about 40%, whereas mGlu1 antagonist LY367385 reversed the effect of Hcy, while mGlu5 antagonist MPEP had only a very slight impact on the level of phosphorylated tau (Fig. 4A and B). The combination of MK-801 with the mGluRs GI antagonists did not significantly alter the effects of MK-801 alone. These results demonstrate that NMDA receptors, and to a lesser extent mGlu1 play an essential role in mediating Hcy-evoked dephosphorylation of tau protein. 3.4. Role of protein phosphatase 2A in Hcy-evoked dephosphorylation of tau protein To determinate the role of PP2A in Hcy-induced tau dephosphorylation, the selective inhibitor of PP1 and PP2A okadaic acid was used. In agreement with previous studies, our preliminary results demonstrated that OA at high concentration of 250 mM induced neurodegeneration (data not shown). To reduce the cytotoxic effect of this drug, while still obtaining significant inhibition of phosphatase activity, cell cultures were preincubated with 100 nM OA for 1 h. Incubation of cell cultures with 100 nM OA alone caused no changes in the phosphorylation state of tau at any analyzed time point, and no significant alterations in neuronal viability were observed (results not shown). However, as shown in Fig. 5A and B, this treatment of rat cerebellar granule neurons followed by administration of 15 mM Hcy for 30 min reversed Hcy-induced tau dephosphorylation. Instead, it resulted in a gradual increase in labeling of the phosphorylated form of tau by AT-8, which reached its highest level 6 h after Hcy treatment. This effect of OA, which is an inhibitor of tau-selective PPA2 indicated that Hcy simultaneously induces phosphorylation and dephosphorylation of tau protein. The latter PP2A-mediated process predominates, however, its inhibition by OA unmasks Hcy-induced phosphorylation of tau protein.
Fig. 4. Alterations in the homocysteine-induced phosphorylation state of tau after administration of the selective glutamate receptor antagonists. The antagonist of NMDA receptors, MK-801 (0.5 mM) and antagonists of mGlu1, LY367385 (25 mM) and of mGlu5, MPEP (25 mM), were applied 5 min before 30 min incubation with 15 mM D,L-homocysteine (Hcy). Samples were collected 2 h after Hcy treatment. Densitometric analysis of the tau protein phosphorylation are presented as a percentage of the control value (untreated cultures). Data are the mean S.D. (n = 4). * Means significantly different from control (P < 0.05).
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Fig. 5. Effect of okadaic acid (OA) on homocysteine-induced changes in the phosphorylation state of tau. The cells were pre-incubated with 100 nM OA for 1 h before adding 15 mM D,L-homocysteine (Hcy) for 30 min. Samples were collected from neuronal cultures after times 0, 2, 4, and 6 h following Hcy treatment. The intensity of the autoradiographic signal was regarded as the degree of tau phosphorylation and is presented as a percentage of the control cultures treated with OA alone. Two or three forms of tau were identified in Hcy- and OA-treated cultures. Data are the mean S.D. (n = 4). * Means significantly different from control + OA (P < 0.05).
4. Discussion In this study we investigated the effect of acute challenge of rat cerebellar granule cells in primary culture with Hcy on the phosphorylation state of tau protein in parallel with Hcy-induced neurotoxicity. The experiments were focused on the role of excitatory amino acid receptors in both processes. We confirmed that Hcy at millimolar concentrations induces acute excitotoxicity mediated by NMDAR and mGluRs GI. A new and important finding of this study is that instead of the expected Hcy-evoked hyperphosphorylation of tau protein, we observed dephosphorylation of tau lasting several hours after acute exposure to Hcy. This effect was inhibited by NMDA and mGlu1 receptor antagonists. Our results also indicate that protein phosphatase PP2A is instrumental in Hcy-evoked tau dephosphorylation. Below we will discuss possible mechanisms of these phenomena. Our results confirmed that the acute treatment with Hcy at millimolar concentrations leads to neuronal cell death (Lipton et al., 1997; Zieminska et al., 2003). In the present work, as in several previous studies, we used the racemic form of Hcy (D,L-Hcy) instead of the physiologically active L-form of homocysteine which is not commercially available. Moreover, it is known that Hcy interferes in a complex way with NMDA receptors, activating the glutamate binding site and inhibiting the glycine co-agonist site (Lipton et al., 1997). This also explains why acutely administered Hcy is a weak neurotoxic agent comparing to glutamate. In conformity with previous data (Lipton et al., 1997; Mattson and Shea, 2003; Zieminska et al., 2003) we found that the neurodegeneration evoked by Hcy was effectively blocked by an
uncompetitive antagonist of NMDA receptors, MK-801. In addition, the mGlu1 and mGlu5 antagonists, LY367385 and MPEP, known as effective neuroprotective agents in in vitro models of excitotoxicity (Bruno et al., 2000), had a moderate ability to prevent the loss of neurons induced by exposure to Hcy, which is in accordance with our earlier findings (Zieminska et al., 2003, 2006; Zieminska and Lazarewicz, 2006). In the present study we focused on the effect of acute Hcy application on the phosphorylation state of tau protein in primary cultures of cerebellar granule neurons. We found that in parallel with development of neuronal injury a significant decrease in the immunoreactivity of phosphorylated tau protein arose. This effect, detected using AT-8 antibody, persisted up to 6 h after incubation with this toxic agent. Under our experimental conditions we did not observe any alterations in the total level of tau protein immunoreactivity (detected with Tau 46 antibody). This indicates that exposure of neurons to Hcy suppresses tau phosphorylation in a way related to excitotoxicity. Our results of experiments using glutamate receptor antagonists confirm this assumption. Hcyevoked dephosphorylation of tau protein, as well as Hcy neurotoxicity was completely reversed by MK-801, an uncompetitive NMDA channel blocker. Such a treatment even enhanced tau phosphorylation. Administration of the mGlu1 receptor antagonist LY367385 less effectively prevented Hcy-induced dephosphorylation of tau, while mGlu5 antagonist MPEP had only a negligible effect. Hcy-evoked dephosphorylation of tau was an unexpected finding, since recently Luo et al. (2007) demonstrated that acute intracerebroventricular injections of Hcy in rats induced tau hyperphosphorylation in the brain in vivo. Others have also observed Hcy-evoked tau hyperphosphorylation which they linked with NMDA receptor-mediated activation of calcium-dependent kinase pathways in neurons (Ho et al., 2002; Chan et al., 2008). The majority of previous studies demonstrating Hcy-induced hyperphosphorylation of tau protein were conducted under conditions of chronic or subchronic exposure to this amino acid, promoting inhibition of methylation processes (Vafai and Stock, 2002; Sontag et al., 2007; Obeid et al., 2007). We believe that acute 30-min exposure of cell cultures to Hcy, which we used in the present study, minimized such an effect, and thus we are able to ascribe the phenomena observed in this study mainly to glutamate receptor-mediated processes. Nevertheless, there are discrepancies in the literature concerning the effects of acute glutamateinduced excitotoxicity and the phosphorylation of tau protein. Davis et al. (1995) demonstrated that treatment of primary rat cortical neuronal cultures with high concentrations of glutamate leads to decreased immunolabeling by phospho-tau specific AT-8 antibody. However, other in vitro studies have shown intense AT-8 immunostaining after glutamate or NMDA administration (Sindou et al., 1994; Couratier et al., 1996; Li et al., 2004). Some authors have reported that glutamate triggers notable tau phosphorylation only during the initial phase of glutamate toxicity (Sindou et al., 1992; Davis et al., 1995). Also, previous in vivo studies demonstrated that intracortical perfusion of glutamate results in the rise in tau phosphorylation in specific brain regions (Irving et al., 1996). These differences may reflect altered regulation of the activity of specific protein kinases and phosphatases, resulting in diverse responses of tau phosphorylation to the activation of glutamate receptors. A crucial role of NMDA receptors in Hcy-evoked changes in the phosphorylation state of tau which was detected in this study strongly suggests involvement of calcium. As it is known that regulation of the phosphorylation/dephosphorylation system is dependent on intracellular calcium levels (Sindou et al., 1994). In several studies an experimentally induced increase in intracellular calcium in cultures of hippocampal and human cortical neurons produced tau hyperphosphorylation as well as ultrastructural and
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antigenic changes similar to NFT (Mattson et al., 1991; Ho et al., 2002; Chan et al., 2008). However, Fleming and Johnson (1995) and Adamec et al. (1997) demonstrated that the NMDA receptormediated increase of intracellular calcium level resulted in the activation of calcium-dependent proteins responsible for the dephosphorylation of tau in vitro. Even more mysterious is the role of calcium in the mechanism of the acute neurotoxic effects of Hcy and eo ipso in changes of tau phosphorylation. Previous studies on Hcy neurotoxicity in cultured neurons described significant intracellular calcium transients accompanying Hcy-induced neurotoxicity and hyperphosphorylation of tau (Lipton et al., 1997; Kim, 1999; Ho et al., 2002; Chan et al., 2008). However, our previous experiments demonstrating homocysteine-evoked neurodegeneration in primary cultures of cerebellar granule cells, failed to show any pronounced activation of 45Ca uptake by homocysteine-treated neurons or intracellular calcium transients comparable to the effects of NMDA or glutamate (Zieminska et al., 2003, 2006). Shea and Ekinci (1999) demonstrated in a neuroblastoma cell line biphasic effects of calcium influx on tau phosphorylation. Experimental conditions promoting a reduced calcium load depleted phospho-tau levels, while enhanced calcium influx was accompanied by the rapid accumulation of phosphorylated tau. In conformity with these data, also our results show that the very low level of calcium influx into Hcy-treated cerebellar granule cells and the minor calcium transients (Zieminska et al., 2003, 2006) are accompanied by a decrease in tau phosphorylation. Our unpublished data demonstrated that in spite of maintaining cerebellar granule cells under standard culture conditions, these cells sporadically altered their phenotype, showing drastic increases in sensitivity to Hcy neurotoxicity. In these cells the concentration of Hcy that normally induced moderate neurotoxicity triggered extensive neuronal loss after 24 h. We noticed that this effect was usually accompanied by an initial temporary increase in tau phosphorylation after Hcy treatment. Presumably these cells may also develop more pronounced Hcy-evoked calcium transients. This possibility will be a subject of further investigation. Previous studies which demonstrated glutamate- or Hcyevoked increases in phosphorylation of tau protein focused on protein kinases possibly involved in this effect. Ho et al. (2002) and Chan et al. (2008) suggested that the Hcy-evoked NMDA receptormediated rise in intracellular calcium may activate calciumdependent protein kinases including MAPK and protein kinase C (PKC). In turn Li et al. (2004) using hippocampal organotypic cultures, demonstrated that glutamate-induced activation of NMDA receptors resulting in a rise in intracellular calcium levels in neurons had no apparent effect on PP2A activity in brain slices, whereas a marked increase in the phosphorylation of tau protein and activation of the calcium/calmodulin-dependent protein kinase II (CaMKII) was observed. However, our results which show dephosphorylation of tau suggest that protein phosphatases rather than kinases may be predominantly activated. PP2A, a trimeric complex is believed to be the most efficient enzyme in dephosphorylating tau in vitro and in vivo (Sontag et al., 2004; Liu et al., 2005). The results of Chan and Sucher (2001) indicate that stimulation of the NMDA receptor in cerebrocortical neuronal cultures results in dissociation of its complex with PP2A protein, leading to decreased phosphatase activity. In the present study, okadaic acid, an inhibitor of PPA1 and PP2A activity (Adamec et al., 1997; Li et al., 2004) has been used in order to examine the role of protein phosphatases in the dephosphorylation of tau following exposure of cerebellar granule cells to Hcy. Treatment of the cultures with nanomolar concentrations of OA, simultaneously with Hcy, completely blocked Hcy-induced dephosphorylation of tau and triggered a progressive, time-dependent increase in AT-8 immunoreactivity. Since PP2A rather than PP1 participates in tau
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dephosphorylation, this finding indicates that activation of the protein phosphatase PP2A is directly responsible for Hcy-induced dephosphorylation of tau protein. Moreover these results visualized the potential of Hcy for activation of simultaneous phosphorylation of tau protein. Inhibition of PP2A by OA in the Hcy-treated cells disturbed equilibrium between tau phosphorylation and dephosphorylation and disclosed tau phosphorylation by protein kinases. Our study was focused on acute effects of Hcy mediated by excitatory amino acid receptors. Excitotoxic challenge of cerebellar granule cells with this compound resulted in significant neuronal damage preceded by dephosphorylation of tau protein. This contrasts with the hyperphosphorylation of tau characteristically found in Alzheimer’s patients with hyperhomocysteinemia (Obeid et al., 2007), and also with the results of several in vivo and in vitro studies demonstrating increased tau phosphorylation in Hcytreated neurons (Ho et al., 2002; Sontag et al., 2007; Luo et al., 2007; Chan et al., 2008). We believe that this variation in the responses of the tau phosphorylation/dephosphorylation system of neurons to Hcy challenge results from the various models used in these studies and from the diversity of calcium-dependent and calcium-independent mechanisms regulating protein kinases and phosphatases. It seems that in chronic experiments, the Hcyevoked alterations in tau phosphorylation compatible with Alzheimer’s pathology mainly result from disturbances in methylation processes in the presence of Hcy (Sontag et al., 2007). However, Hcy-evoked glutamate receptor-mediated regulation of tau phosphorylation represents another signaling pathway that may be active under acute and prolonged experimental conditions (Lipton et al., 1997; Zieminska et al., 2003; Zieminska and Lazarewicz, 2006). It is possible that hyperhomocysteinemia in vivo may activate several such mechanisms in parallel. Our findings indicate that Hcy-induced hyperphosphorylation of tau is not an inherent feature of this sulfur-containing amino acid, but that the nature of the cellular response depends on the neuronal phenotype. Hcy may interact with glutamate receptors resulting in activation of both protein kinases and protein phosphatases, which regulate the level of tau phosphorylation. The final outcome depends on predominance of one of these systems. Our observations also suggest that the overall effect of Hcy on tau phosphorylation may depend on the degree of neuronal sensitivity to Hcy toxicity, which is probably related to Hcy-evoked calcium imbalance. 5. Conclusions The results of this study demonstrate that acute exposure of cerebellar granule cells in primary culture to a sufficient dose of Hcy causes excitotoxicity and leads to a rapid, long-lasting decrease in tau protein phosphorylation, without changing the total level of tau. Both effects are mediated mainly by NMDA receptors, with additional involvement of mGluRs GI. PP2A is the main protein phosphatase responsible for Hcy-induced dephosphorylation of tau. Acknowledgements The authors thank Dr. John Gittins for constructive comments on the manuscript. This study was supported by the Medical Research Centre PAS, Warsaw. References Adamec, E., Mercken, M., Beermann, M.L., Didier, M., Nixon, R.A., 1997. Acute rise in the concentration of free cytoplasmic calcium leads to dephosphorylation of the microtubule-associated protein tau. Brain Res. 757, 93–101.
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