Most cellular responses to extracellular stimuli are mediated by kinase and phosphatase cascades. One of the most important kinase families in inflammatory cells is mitogen-activated protein kinases (MAPKs). They are strongly conserved through evolution, suggesting their vital role in intracellular signaling (Marshall, 1994, 1995; Cobb and Goldsmith, 1995; Sugden and Clerk, 1997). The MAPK family comprises four subfamilies in mammalian cells. These include (1) extracellular signal-regulated kinases (ERK1/2; also known as p44/42 MAPK); (2) c-jun N-terminal kinases (JNKs), also called stress-activated protein kinases (SAPKs); (3) ERK5/big MAP kinase 1 (BMK1); and (4) p38 MAPKs. Traditionally, the p44/42 MAPK pathway has been connected with the regulation of cell growth and differentiation (Cobb et al., 1991; Sugden and Clerk, 1997; Ono and Han, 2000) but, at least in the brain, p44/42 MAPK is also involved in cellular responses to stress stimuli, including oxidative stress, glutamate receptor stimulation, or increases in intracellular calcium levels (Fiore et al., 1993; Rosen et al., 1994; Aikawa et al., 1997). The JNK group of MAPKs is activated by various stressful stimuli, including oxidative stress, deprivation of trophic factors, and activation of death domain receptors by extracellular tumor necrosis factor-α (TNF-α) or FasL (Ip and Davis, 1998; Mielke and Herdegen, 2000). The BMK1 MAPK signaling pathway has been shown to regulate serum-induced early gene expression through the transcription factor MEF2C (Kato et al., 1997). The p38 MAPK family is associated with signaling pathways that are activated by inflammatory molecules, ultraviolet (UV) shock, osmotic changes, and withdrawal of trophic factors, but the p38 MAPK pathway is also involved in cell cycle, cell growth, and differentiation (Kyriakis and Avruch, 1996; Herlaar and Brown, 1999; Mielke and Herdegen, 2000; Ono and Han, 2000).
P38 AND P44/42 SUBFAMILIES OF MAPKS
p38 and p44/42 MAPKs are serine threonine kinases that are localized in the cytoplasm until activated by dual phosphorylation on both Tyr and Thr residues (Chen et al., 1992; Sanghera et al., 1992; Seth et al., 1992; Gonzalez et al., 1993; Raingeaud et al., 1995). p38 MAPKs have the Thr-Gly-Tyr dual phosphorylation motif, and p44/42 MAPKs are phosphorylated at Thr-Glu-Tyr motif (Hanks and Hunter, 1995).
An overview of the elements involved in the phosphorylation cascade leading to p38 and p44/42 MAPK activation is presented in Figure 1. p38 MAPK is selectively phosphorylated by MAPK kinases (MAPKKs) MEK3 or MEK6 (Cuenda et al., 1996, 1997), whereas p44/42 MAPK is activated by MEK1 and MEK2 (Crews et al., 1992). MEKs are regulated by phosphorylation on serine and threonine residues. The kinase cascade that takes part in the phosphorylation of p38 MAPK activators MEK3 and MEK6 is diverse and complex. The most well known players in this pathway are apoptosis signal-regulating kinase 1 (ASK1), TGFβ-activated kinase 1 (TAK1), and thousand and one kinases (TAOs) (reviewed in Kyriakis and Avruch, 2001). Upstream of these MAPK kinase kinases (MAPKKKs) are adapter proteins that couple the p38 MAPK pathway to tumor necrosis factor (TNF) family receptors. These adapters include TNF receptor-associated factor 2 (TRAF2) and receptor interacting kinase (RIP) (Yuasa et al., 1998). The kinases involved in MEK1/2 activation are Raf-proteins (protooncoprotein c-Raf, A-Raf, and B-Raf) which are phosphorylated by various kinases, including at least Ras.GTP and c-Src protein tyrosine kinase (PTK), and possibly protein kinase C (PKC) and 14-3-3 proteins as well (reviewed in Sugden and Clerk, 1997).
p38 MAPK exists in four isoforms that are differentially synthesized and regulated in various cell types (Lee et al., 1994; Herlaar and Brown, 1999). The genes encoding p38α and p38β isoforms are expressed ubiquitously (Jiang et al., 1996; Herlaar and Brown, 1999). p38γ is particularly enriched in skeletal muscle (Lechner et al., 1996), whereas p38δ predominates in lung, kidney, testis, pancreas, and small intestine (Kumar et al., 1997). Inflammatory cell lineages show differential expression and activation of p38 MAPK isoforms. In monocytes and macrophages, p38α is abundant, but p38β is undetectable (Hale et al., 1999). In contrast, p38β is the dominant form in endothelial cells. Bacterial lipopolysaccharide (LPS) stimulation of macrophages results in strong dual phosphorylation of only p38α isoform (Hale et al., 1999), suggesting that the α-isoform plays a central role in the inflammatory response of p38 MAPK pathway in microglia as well. The widely used pyridinyl imidazole inhibitors of p38 MAPK, SB205380, and SB202190 are specific for p38α and p38β and do not inhibit the δ- and γ-isoforms (Jiang et al., 1996; Kumar et al., 1997; Wang et al., 1997). These compounds do not inhibit the dual phosphorylation of p38 MAPKs, but rather bind to the ATP pocket in these enzymes thus inhibiting their enzymatic activity (Tong et al., 1997).
The two related ERKs, p44 MAPK (ERK1) and p42 MAPK (ERK2), are distributed ubiquitously and expressed in brain and spinal cord at high levels (Boulton and Cobb, 1991; Boulton et al., 1990, 1991). Chemical inhibitors of p44/42 MAPKs include PD98059 [2-(2′-amino-3′-methoxyphenyl)oxanaphthalen-4-one], which inhibits the activation of p44/42 upstream kinase MEK1 (Alessi et al., 1995; Dudley et al., 1995) and UO126, which acts on MEK1/2 activities, preventing the phosphorylation of p44/42 MAPKs (Favata et al., 1998).
Activation of these MAPK pathways leads to phosphorylation of nuclear transcription factors and other protein kinases located in the cytoplasm (Fig. 1). p38 MAPK is capable of phosphorylating MAPK-activated protein kinases (MAPKAP Ks) 2 and 3, which, in turn, phosphorylate the small heat shock protein HSP27 (Freshney et al., 1994; Rouse et al., 1994; Huot et al., 1997; Lambert et al., 1999). p38-regulated/activated kinase (PRAK) is structurally and functionally related to MAPKAP K2/3 and is also phosphorylated by p38 MAPK (New et al., 1998). Nuclear targets of p38 MAPK include ATF2, MEF2A/C, and SAP1a, all of which are involved in the formation of AP-1 transcription complex (Kyriakis and Avruch, 2001). A novel function of p38 MAPK is phosphorylation and phosphoacetylation of histone H3 in response to inflammatory stimuli (Saccani et al., 2001). The p38 MAPK-dependent H3 phosphorylation modifies promoter regions of specific genes, such as interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP 1), resulting in opening of NF-κB binding sites and thereby promoting transcriptional activation. p38 MAPK activation through various pathways has been demonstrated to be essential for IL-1, IL-6, TNF-α, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) expression (Lee et al., 1994; Da Silva et al., 1997; Ridley et al., 1997; Bhat et al., 1998; Guan et al., 1998; Miyazawa et al., 1998). In some cell lines, the p38 MAPK pathway increases COX-2 and TNF-α expression by increasing mRNA stability and protein translation, but the mechanism of these actions are still unclear (Ridley et al., 1998; Lee et al., 2000).
The downstream targets of activated p44/42 include cytoskeletal, nuclear and signaling proteins. Most of the known cytoskeletal proteins are expressed in neurons (e.g., MAP-2, tau, neurofilaments, synapsin-1) (Adams and Sweatt, 2002). The best-characterized signaling protein targets include MAPKAP K1, MAPK-interacting kinases (MNKs) involved in translation, and mitogen and stress-activated protein kinases (MSKs) involved in transcription and chromatin remodeling (Hsiao et al., 1994; Fukunaga and Hunter, 1997; Deak et al., 1998; Frodin and Gammeltoft, 1999; Smith et al., 1999; Waskiewicz et al., 1997, 1999; Kyriakis and Avruch, 2001). One important signaling molecule in inflammation is phospholipase A2, which is also phosphorylated by p44/42 MAPK. The main nuclear target of p44/42 MAPK is transcription factor Elk1, leading to activation of serum response element on fos promoter, and thus c-fos induction (Marais et al., 1993; Whitmarsh et al., 1995; Kyriakis and Avruch, 2001). Other downstream targets of nuclear proteins include c-Myc, c-Jun, ATF-2, and CREB (Adams and Sweatt, 2002).
ACTIVATION of P38 AND P44/42 MAPK IN CHRONIC NEURODEGENERATIVE DISEASES
Both microglial activation and activation of p38 MAPK or p44/42 MAPK pathways occur in most chronic neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease, and amyotrophic lateral sclerosis, and their animal models. Even though both MAPKs and microglia may play a dual role in the etiology of neurodegenerative disease, all the information that is currently available on MAPKs in microglia is related to the aggravation of the inflammatory processes. In this review, we concentrate on AD, which is the most common chronic neurodegenerative disease in elderly.
AD is a dementing disorder characterized by selective neuron loss in specific brain regions, the presence of neuritic plaques composed of extracellular aggregates β-amyloid (Aβ) fibrils, and neurofibrillary tangles that are intracellular inclusions of hyperphosphorylated tau protein (Wisniewski and Wegiel, 1992; Iqbal and Grundke-Iqbal, 1996). AD brain also shows signs of chronic inflammation, because activated microglia and pro-inflammatory molecules are present at the sites of extracellular lesions (reviewed in McGeer and McGeer, 1999; Akiyama et al., 2000; Halliday et al., 2000). Further evidence for the involvement of inflammation in the pathogenesis of AD is offered by epidemiological findings showing delayed onset and slowed progression of AD among long-term nonsteroidal antiinflammatory drug (NSAID) users (McGeer et al., 1996). Immunohistochemical analysis of postmortem AD brains revealed increased levels of activated p38 MAPK compared with age-matched control brains (Hensley et al., 1999). p38 MAPK activity was associated with the hallmark lesions of AD, the neuritic plaques, neurofibrillary tangle-bearing neurons, and neuropil threads. Within plaques, numerous astrocytes and microglia were also stained. In addition to AD, p38 MAPK immunoreactivity in neurons and glial cells has been demonstrated in many neurodegenerative disorders characterized by intracellular deposits of hyperphosphorylated tau, including Pick's disease, progressive supranuclear palsy, corticobasal degeneration, and frontotemporal dementia with parkinsonism linked to chromosome 17 (Atzori et al., 2001). As phosphorylation of p38 MAPK is a response to cellular stress, such as neuritic plaques and inflammatory molecules, it is not likely that p38 MAPK activation is an initiating step in the disease cascade. However, activation of the p38 MAPK pathway in neurons and glial cells may stimulate the production of inflammatory mediators, thereby contributing to the degeneration or further activation of these cells.
Transgenic mice overexpressing human β-amyloid precursor protein (APP) develop early AD-like changes, including diffuse, extracellular Aβ deposits in brain parenchyma, as well as specific spatial learning and memory deficit (Quon et al., 1991; Higgins et al., 1994, 1995; Koistinaho et al., 2001). Interestingly, these mice showed approximately threefold elevation in the number of activated p38 MAPK-positive cells in the brain (Koistinaho et al., 2002). Phosphorylated p38 MAPK immunoreactivity was detected solely in microglial cells, and no colocalization with a neuronal or astrocytic marker was seen. The finding of increased activation of p38 MAPK was confirmed by measuring the activity of MAPKAP K2, a specific substrate of p38 MAPK. Similar finding of increased p38 MAPK activation is also seen in a mouse model of AD with more advanced amyloid pathology (Lappalainen U, Goldsteins G, Koistinaho M, Puoliväli J, Tanila H, Koistinaho J, unpublished data, 2002). Altogether, the observation that p38 MAPK is activated in microglia of APP transgenic mice suggests that plaque-associated Aβ is not necessary for MAPK activation in microglia, and that secreted forms APP or soluble β amyloid peptides (Aβ) may be sufficient. Numerous in vitro studies have demonstrated that while fibrillar Aβ peptides induce rapid activation of both p38 and p44/42 MAPKs, resulting in increased TNF-α and NO release (McDonald et al., 1998; Pyo et al., 1998; Combs et al., 1999, 2001) (Table 1), fresh Aβ peptides activate p44/42 MAPK in microglia only when CD45 receptor is inactivated with tyrosine phosphatase inhibitors (Tan et al., 2000a). It should be noted that in the AD brain a complex inflammatory process, involving also neurons and astrocytes, is activated. It is possible that the interaction of activated astrocytes and microglia together with the neurons stressed by Aβ peptides and neurofibrillary tangles sensitize microglia to extracellular stimuli, which then trigger MAPK pathway and cytokine release. Further studies are required to demonstrate the relevance of Aβ-induced MAPK activation in microglia in neurodegenerative diseases.
Table 1. Stimuli and Responses of the Activation of p38 and p44/42 MAPK Pathways in Microglia In Vitro
ACTIVATION OF P38 AND P44/42 MAPK IN ACUTE BRAIN INJURIES
Acute brain injuries, such as stroke and brain trauma, elicit a strong inflammatory reaction in response to inflammatory mediators released by the affected cells and activated inflammatory cells recruited from the peripheral circulation into the injury site. p38 and p44/42 MAPKs most likely play a significant role in initiating and sustaining the inflammatory reaction in the brain. Detailed analysis of p38 MAPK phosphorylation in different experimental animal models of acute collapse of cerebral blood flow has demonstrated that p38 MAPK is involved in microglial stress response. Activated form of p38 MAPK was found to be increased in microglial cells adjacent to dying CA1 neurons in gerbil transient forebrain ischemia model 2–4 days after the insult (Walton et al., 1998).
Permanent occlusion of the middle cerebral artery (MCA) induced a transient activation of p38 MAPK in cortical neurons shortly after the onset of ischemia in mice (Koistinaho et al., 2002). At 1 day the neuronal induction was completely over. In contrast, activated p38 MAPK was detected in microglial cells in periinfarct areas. Interestingly, not only was the microgliosis more severe in APP overexpressing mice, but the microglial cells of these mice showed an even more dramatic increase in p38 MAPK phosphorylation compared with wild-type mice. APP overexpressing mice showed 4.3-fold the amount of activated p38 MAPK-positive microglia in areas next to the infarcted core compared with wild-type littermates (Koistinaho et al., 2002), suggesting that either full-length APP, or its proteolytic fragments, stimulate microglial cells, leading to activation of p38 MAPK. Consequently, the APP overexpressing mice developed 35–40% larger infarcts in response to permanent occlusion of the MCA than nontransgenic control mice (Koistinaho et al., 2002).
A similar two-phase activation of p38 MAPK was demonstrated by Tian et al. (2000) in response to transient MCA occlusion in rats. The first phase occurred within 1 h in cortical neurons suffering from the lack of oxygen and nutrients, whereas the second phase of p38 MAPK activation was localized in ameboid microglial cells 1–3 days after the MCA occlusion.
These observations suggest that p38 MAPK is involved in the activation of microglial cells that reach the fully activated state over the first 1–2 days after the ischemic insult (Morioka et al., 1993; Rupalla et al., 1998; Davies et al., 1998, 1999), and maintenance of the activated state of microglial cells, further contributing to a self-propagating cycle of cytokine production after brain ischemia. Indeed, the inhibition of p38 MAPK in microglia by a selective, novel inhibitor, SD-282, decreased the number of activated microglia in ischemic brain and completely abolished the APP overexpression-induced ischemic sensitivity in transgenic mice (Koistinaho et al., 2002).
The extracellular stimuli, which may trigger p38 MAPK in microglia after ischemic brain injury, are numerous. First, after the blood-brain barrier becomes leaky, thrombin, some cytokines, and CD40 ligands reach the microglia. In vitro studies have demonstrated that nanomolar concentrations of thrombin induce rapid activation of p38 MAPK in microglia, possibly through protease-activated receptor-1, resulting in NO release and expression of CD40 receptor (Moller et al., 2000; Ryu et al., 2000; Suo et al., 2002) (Table 1). CD40 ligands, in turn, may sensitize microglia to interferon-γ (IFN-γ), facilitating p44/42 MAPK-mediated cytokine induction (D'Aversa et al., 2002). Second, ischemic neuronal injury results in increased extracellular concentrations of glutamate and ATP. Cultured microglia respond to stimulation of N-methyl-D-aspartate (NMDA) and kainate receptors by p38 MAPK activation, which mediates microglial proliferation and release of neurotoxic molecules (Tikka and Koistinaho, 2001; Tikka et al., 2001). In contrast, ATP stimulation can trigger IL-6 and TNF-α release by a p38 MAPK-dependent mechanism (Hide et al., 2000; Shigemoto-Mogami et al., 2001). Whether ischemia can directly activate MAPK pathways in microglia is unknown.
The role of p44/42 MAPK in microglial response to acute brain injuries remains unclear. One report described increased p44/42 MAPK phosphorylation in neurons, astrocytes, endothelial cells as well as in activated microglial cells in areas surrounding cerebral infarcts in patients who died 1–44 days after acute ischemic attack (Slevin et al., 2000). We were unable to detect phosphorylated p44/42 MAPK in microglial cells in mouse permanent MCA occlusion model at 1 day after the onset of ischemia (M. Koistinaho and J. Koistinaho, unpublished data, 2001). Similarly, two other reports show that activated p44/42 MAPK is not present in microglial cells at the time p38 MAPK phosphorylation is detected (Walton et al., 1998; Tian et al., 2000).
Excitotoxicity, the excessive release of excitatory amino acids such as glutamate, has been considered as a major mechanism underlying neuronal cell death in brain ischemia (Choi, 1988, 1992). Intracerebral injections of glutamate analogue, quinolinic acid, produces a limited and reproducible lesion around the injection site. Phosphorylated p44/42 MAPK was detected in neurons and glial cells, including activated microglia in areas surrounding the excitotoxic lesion core, whereas the activated form of p38 MAPK was increased in the core after injection of quinolinic acid (Ferrer et al., 2001). However, our in vitro studies failed to demonstrate direct activation of p44/42 MAPK by excitotoxins in microglia (Tikka and Koistinaho, 2001; Tikka et al., 2001).
Studies on cultured microglia have demonstrated a clear role for p44/42 MAPK in those microglial functions, which are potentially important for acute brain injury (Table 1). Similar to p38 MAPK, p44/42 MAPK activation has been reported to occur in response to CD40 ligand (Tan et al., 2000b; D'Aversa et al., 2002), ATP (Hide et al., 2000; Shigemoto-Mogamiet al., 2001) and thrombin (Ryu et al., 2000; Suo et al., 2002). In addition, TGF-β, which may become released from astrocytes upon acute brain injury, induces p44/42 pathway (Schlapbach et al., 2000). Similarly, receptors of glial derived neurotrophic factor (GDNF) are expressed in microglia and stimulation of these receptors activated p44/42 MAPK pathway in vitro (Honda et al., 1999). Also, mitotic stimuli, such as granulocyte-macrophage-colony-stimulating factor (GM-CSF), triggers p44/42 MAPK in microglia, suggesting a role of this pathway in microglial proliferation (Liva et al., 1999).
BACTERIAL COMPONENTS AND MAPK ACTIVATION
Even though there are no in vivo experiments indicating that bacterial infections induce p38 or p44/42 MAPK pathways in microglia, several in vitro studies have demonstrated that LPS, a cell wall component of gram-negative bacteria thought to be responsible for pathophysiological sequelae of bacterial infections, is a strong stimulant of both p38 and p44/42 MAPK pathways. The LPS-induced NO and TNF-α release appears to require both p38 and p44/42 MAPK activity (Bhat et al., 1998; Pyo et al., 1998; Lee et al., 2000). Cell wall components of gram-positive bacteria also induce biosynthesis of pro-inflammatory cytokines in microglia through p38 and p44/42 MAPK pathways (Hanisch et al., 2001).
Numerous studies have demonstrated a prominent role for p38 MAPK and p44/42 MAPK pathways in the activation of cultured primary microglia or microglial cell lines. Figure 2 summarizes the molecules that have been shown to activate p38 or p44/42 MAPK pathways in microglia and provides an overview of the mechanisms and molecules that link p38 or p44/42 MAPK signaling cascades to harmful aspects of microglial activation. However, the evidence for a significant MAPK activation in microglia in animal models and especially in human brain diseases is still weak. In addition, expression patterns of p38 and p44/42 MAPK isoforms in different brain cells have not been well described. This information on MAPKs in the brain is crucial before we can conclude that their activation in microglia is involved in pathophysiological changes both in acute brain insults and in chronic neurodegenerative diseases. Although all the studies reviewed here on microglia suggest that the activation of p38 and p44/42 MAPKs mediates inflammation and release of neurotoxic molecules, it is obvious that, under certain circumstances, the activation of microglial cells may be beneficial for the plasticity of the CNS. Nevertheless, based on the few in vivo studies available so far, the kinases, such as p38 and p44/42 MAPK, which mediate activation of microglia with neurotoxic consequences, represent promising targets for pharmacotherapy for acute brain insults and neurodegenerative diseases.