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Hyperhomocysteinemia is an independent risk factor for both acute and chronic neurological disorders, but little is known about the underlying mechanisms by which elevated homocysteine can promote neuronal cell death. We recently established a role for NMDA receptor-mediated activation of extracellular signal-regulated kinase (ERK)-MAPK in homocysteine-induced neuronal cell death. In this study, we examined the involvement of the stress-induced MAPK, p38 in homocysteine-induced neuronal cell death, and further explored the relationship between the two MAPKs, ERK and p38, in triggering cell death. Homocysteine-mediated NMDA receptor stimulation and subsequent Ca2+ influx led to a biphasic activation of p38 MAPK characterized by an initial rapid, but transient activation followed by a delayed and more prolonged response. Selective inhibition of the delayed p38 MAPK activity was sufficient to attenuate homocysteine-induced neuronal cell death. Using pharmacological and RNAi approaches, we further demonstrated that both the initial and delayed activation of p38 MAPK is downstream of, and dependent on activation of ERK MAPK. Our findings highlight a novel interplay between ERK and p38 MAPK in homocysteine-NMDA receptor-induced neuronal cell death.
Hyperhomocysteinemia, a common metabolic disorder of the methionine cycle, is an independent risk factor for multiple neurodegenerative disorders such as cerebral stroke, age-associated dementia, Alzheimer's disease, Parkinson's disease, cerebral atrophy, and seizures (Sacco et al. 1998; Miller 1999; Seshadri et al. 2002; Obeid and Herrmann 2006; Zoccolella et al. 2006). In vivo studies using an animal model of Parkinson's disease have shown that elevated levels of homocysteine increases the vulnerability of neurons to dysfunction and death in these animals (Duan et al. 2002). Studies using cultured neurons have also shown that elevated homocysteine can sensitize neurons to increased cellular injury in response to excitotoxic or oxidative insult, amyloid ß-peptide, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Kruman et al. 2000, 2002; Duan et al. 2002). In addition, prolonged exposure to increased levels of homocysteine alone has been shown to induce cell death in cultured neurons (Lipton et al. 1997; Kruman et al. 2000; Ho et al. 2002; Mattson and Shea 2003; Poddar and Paul 2009). Despite the general recognition of the possible role of homocysteine in the etiology of multiple neurodegenerative disorders, relatively little is known about the underlying molecular mechanisms involved in homocysteine-induced neuronal death.
It has been shown that oxidative injury and neuronal cell death associated with elevated levels of extracellular homocysteine involves stimulation of the NMDA subtype of ionotropic glutamate receptors (Lipton et al. 1997; Kruman et al. 2000, 2002; Jara-Prado et al. 2003; Mattson and Shea 2003; Poddar and Paul 2009). As overactivation of NMDA receptors is known to be involved in glutamate-mediated excitotoxic cell death, it has been assumed that homocysteine activates NMDA receptors analogous to glutamate, limiting studies in this area. Contrary to this notion, our recent findings (Poddar and Paul 2009) suggest that the effects of homocysteine and glutamate on NMDA receptor activation are quite different. Glutamate-mediated excitotoxic cell death has been primarily attributed to activation of NR1/NR2B subunit-containing NMDA receptors (NR2B-NMDAR), (Hardingham et al. 2002; Li et al. 2002; Riccio and Ginty 2002; Kim et al. 2005; Liu et al. 2007; Zhang et al. 2007). In contrast, our findings show that homocysteine-mediated neuronal cell death involves stimulation of NR1/NR2A-containing NMDAR (NR2A-NMDAR) (Poddar and Paul 2009), the pool of NMDAR that is generally thought to be involved in cell survival (Hetman and Kharebava 2006; Liu et al. 2007). Our findings further show that homocysteine–NMDAR-induced neuronal cell death involves sustained activation of extracellular signal-regulated kinase (ERK) MAPK, which also differs from glutamate–NMDAR-mediated transient activation of ERK MAPK (Paul et al. 2003; Mao et al. 2004). These findings raise the possibility that homocysteine-induced neurotoxicity involves unique signaling pathways that are different from glutamate–NMDAR-mediated excitotoxic cell death.
The aim of this study was to determine the relative contribution of p38 stress-activated kinase (Ono and Han 2000; Cuadrado and Nebreda 2010), another member of the MAPK family that is up-regulated following NMDAR stimulation (Waxman and Lynch 2005; Poddar et al. 2010), in mediating homocysteine-dependent neuronal cell death. Our results show that homocysteine leads to biphasic activation of p38 MAPK, where the initial rise is rapid but transient, and the delayed increase is more sustained. We also show that homocysteine–NMDAR-mediated sustained activation ERK MAPK follows a two-tier pattern. Both the initial and delayed activation of p38 MAPK are dependent on ERK MAPK activity. This novel interplay between ERK and p38 MAPK facilitates homocysteine-induced neuronal cell death.
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- Experimental procedures
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This study provides the first evidence that homocysteine–NMDAR-induced activation of p38 MAPK plays a crucial role in neuronal cell death. Activation of p38 MAPK shows a biphasic response, characterized by an initial rapid but transient induction followed by a delayed and more prolonged secondary increase. Although the earlier peak of p38 MAPK activation is part of homocysteine–NMDAR-induced downstream signaling, the later peak is primarily involved in mediating homocysteine-induced neuronal cell death. The time and duration of the secondary activation of p38 MAPK correlates with that of ERK MAPK activation, which also plays a role in facilitating homocysteine-induced neuronal cell death. Another key finding of this study is that activation of p38 MAPK is downstream of and dependent on ERK MAPK activity establishing a novel interaction between the two MAPK pathways that facilitate homocysteine-induced neuronal cell death. These results suggest that homocysteine–NMDAR-induced neuronal cell death involves a unique cascade of events that is different from downstream signaling pathways triggered by other NMDAR agonists (Fig. 9).
Figure 9. Schematic representation of the crosstalk between extracellular signal-regulated kinase (ERK) and p38 MAPKs in homocysteine–NMDAR-induced neuronal cell death. (a) Homocysteine–NMDAR stimulation leads to sustained two-tier activation of ERK MAPK and biphasic activation of p38 MAPK, where p38 MAPK activation is downstream and dependent on ERK MAPK. (b) Glutamate–NMDAR stimulation leads to transient activation of ERK and p38 MAPKs that are independent of each other.
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Extracellular signal-regulated kinase and p38 MAPKs are ubiquitously expressed signaling proteins that regulate neuronal functions as diverse as cell survival and cell death (Shaul and Seger 2007; Cuadrado and Nebreda 2010). They are serine/threonine kinases that are activated by dual phosphorylation of the regulatory tyrosine and threonine residues in the catalytic domain (Seth et al. 1992; Raingeaud et al. 1995). A three-tiered kinase cascade regulates the activation of these kinases (Ono and Han 2000). The first member of this cascade is MAP kinase kinase kinase (MAPKK or MEKK family), which phosphorylates and activates MAP kinase kinase (MEK 1/2 for ERK; MEK 3/6 for p38) that in turn phosphorylates and activates ERK and p38 MAPKs. In general, transient activation of ERK MAPK by neurotrophic factors and neurotransmitters plays an important role in neuronal survival and long-term potentiation (Boulton et al. 1991; Xia et al. 1995; Segal and Greenberg 1996). In contrast, p38 MAPK is activated in response to a multitude of stress stimuli and inflammatory cytokines and can contribute to neuronal cell death (Raingeaud et al. 1995; Zhu et al. 2000, 2003; Daniels et al. 2001; Choi et al. 2004; Waxman and Lynch 2005; Poddar et al. 2010). Both ERK and p38 MAPKs are activated following NMDAR stimulation by multiple agonists (Sato et al. 2001; Paul et al. 2003; Rakhit et al. 2005; Waxman and Lynch 2005; Poddar and Paul 2009; Poddar et al. 2010). Glutamate is one such agonist that leads to transient activation of both ERK and p38 MAPKs, where activation of ERK MAPK promotes neuronal cell survival and p38 MAPK promotes injury (Kim et al. 2005; Waxman and Lynch 2005; Balazs 2006; Poddar et al. 2010). In contrast, this study demonstrates that homocysteine–NMDAR-mediated sustained activation of ERK as well as p38 MAPKs facilitate neuronal cell death.
The opposing effects of ERK MAPK on neuronal survival and death following exposure to glutamate and homocysteine could be attributed to the differential activation of the NMDAR subunits. Glutamate leads to sequential activation of both the stimulatory and inhibitory (NR2B-NMDAR) pathways to limit the duration of ERK activation (Chandler et al. 2001; Paul et al. 2003; Kim et al. 2005; Poddar and Paul 2009). In contrast, homocysteine activates only the stimulatory pathway (NR2A-NMDAR) resulting in prolonged activation of ERK MAPK (Poddar and Paul 2009). This study also emphasizes the difference between the two NMDAR agonists, glutamate and homocysteine, by demonstrating that glutamate–NMDAR-mediated activation of ERK and p38 MAPK pathways are parallel and independent of each other, whereas homocysteine–NMDAR-mediated activation of p38 MAPK is downstream of and dependent on ERK MAPK activity. Taken together, the study suggests that the view that p38 promotes apoptosis, whereas ERK opposes apoptosis in neurons is not generalizable and detailed analysis of these two pathways could be useful in other models of neurotoxicity.
The pharmacological inhibition of p38 MAPK activity using PD98059 and U0126 suggests an interaction between the ERK and p38 MAPK pathways, but does not clarify whether p38 MAPK is activated directly by ERK MAPK or by its upstream kinase MEK1/2. However, the ability of FR180204 to block p38 MAPK activity confirms a direct role of ERK MAPK in regulating p38 MAPK activity. This interpretation is based on earlier findings demonstrating that FR180204 is a potent ATP-competitive inhibitor of ERK1/2 activity that does not interfere with the MEK-dependent phosphorylation of ERK1/2 (Ohori et al. 2005). This is also consistent with our findings that suppression of ERK2 expression using shRNA significantly reduces homocysteine-induced phosphorylation of p38 MAPK. The direct regulatory role of ERK MAPK in p38 MAPK activation, as demonstrated in this study, reveals a previously unidentified cellular mechanism for neuronal cell death. There are some instances where a crosstalk between two MAPKs has been suggested in non-neuronal cells. In contrast to our findings, these studies show a reciprocal phosphorylation of MAPKs where activation of one MAPK pathway leads to inactivation of the other MAPKs (Houliston et al. 2001; Xiao et al. 2002; Wang et al. 2006; Junttila et al. 2008). In addition, these studies have emphasized the opposing effects of ERK and p38 MAPK on cell survival and cell death.
The precise mechanism by which ERK MAPK regulates the activity of p38 MAPK is yet to be determined. There is a growing body of evidence indicating that activation of ERK MAPK following stimulation of NR2A-NMDARs induces trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits to the surface, whereas inhibition of ERK MAPK following stimulation of NR2B-NMDARs inhibits AMPA receptor surface insertion (Ehlers 2000; Zhu et al. 2002; Kim et al. 2005; Keifer et al. 2007). Additional studies have also described a role of AMPA receptors in up-regulation of p38 MAPK activity (Krapivinsky et al. 2004). This raises the possibility that ERK-mediated trafficking of AMPA receptors may play a role in homocysteine-induced p38 MAPK activation and is an important topic for future studies.
Our study shows a sustained but two-tier pattern of ERK MAPK activation by homocysteine. A biphasic activation of p38 MAPK is also observed, where the initial phase is transient, while the secondary phase is more prolonged. It is difficult to explain why the initial ERK MAPK activation remains sustained, while p38 MAPK activation is transient. A probable explanation is that a phosphatase selective for p38 MAPK is activated as the cell's protective response to inhibit the pro-apoptotic p38 MAPK pathway. Such selective activation of dual-specificity phosphatases that inhibits either ERK or p38 MAPK resulting in preferential activation of the other MAPK has been reported in earlier studies (Muda et al. 1996; Xiao et al. 2002; Wang et al. 2006; Junttila et al. 2008). Regardless of this differential pattern of the initial ERK and p38 MAPK activation, it is the delayed larger increase in ERK MAPK activity and the secondary activation of p38 MAPK that is predominantly responsible for homocysteine-dependent neuronal cell death. Concerning this delayed large increase in ERK MAPK activity, evidences from earlier studies show that prolonged homocysteine exposure leads to a gradual increase in intracellular Ca2+, mitochondrial dysfunction, and reactive oxygen species (ROS) generation (Kruman et al. 2000; Zieminska et al. 2006; Ganapathy et al. 2011a,b). ROS-mediated activation of ERK MAPK has also been shown to play a detrimental role in oxidative neuronal injury (Stanciu et al. 2000; Stanciu and DeFranco 2002; Luo et al. 2007; Chen et al. 2009; Tuerxun et al. 2010). Thus, it appears that homocysteine–NMDAR-mediated intracellular Ca2+ accumulation over time may lead to mitochondrial dysfunction and subsequent ROS generation. This in turn could lead to the delayed large increase in ERK MAPK activity.
In conclusion, this study provides evidence for a novel signaling mechanism of neuronal injury involving a crosstalk between ERK and p38 MAPKs in response to elevated levels of homocysteine. Further evaluation of this molecular pathway is necessary to establish a role of hyperhomocysteinaemia in the progression of neurological disorders.