Neuronal p38 MAPK signalling: an emerging regulator of cell fate and function in the nervous system

p38 mitogen-activated protein kinases (MAPKs), together with extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs), constitute the MAPK family. Multiple intracellular signalling pathways that converge on MAPKs exist in all eukaryotic cells and play pivotal roles in a wide variety of cellular functions. p38 MAPKs and JNKs, also termed stress-activated protein kinases (SAPKs), are preferentially activated by various cytotoxic stresses and cytokines and appear to be potent regulators of stress-induced apoptosis. Whereas JNKs have been shown to play pivotal roles in the regulation of neuronal apoptosis, the role of p38 MAPKs in the nervous system is poorly understood. However, accumulating evidence from mammalian cell culture systems and the strong genetic tool C. elegans suggests that neuronal p38 signalling has diverse functions beyond the control of cell death and survival. This review focuses on possible roles for the p38 pathway in the nervous system, with principal emphasis placed on the roles in neuronal cell fate decision and function.     

Activation of c-Jun N-terminal kinases by ribotoxic stresses

The c-Jun N-terminal kinases （JNKs） are classic stress-activated protein kinases. Many cellular stresses have been shown to stimulate JNK activation. In this review, we focus on ribotoxic stresses based on their multiple biological potencies including anti-HIV-1 activity. Some of the functions of ribotoxins and the signaling transduction pathway that mediated are mentioned. Different from other stimulators, ribotoxic stresses act on special motifs of 28S rRNA in translationally active mammal ribosomes. Binding and damaging on the motif leads to JNK activation and subsequently biological response to the signal initiator, which is named ribotoxic stress response. Cellular ＆ Molecular Immunology. 2005;2（6）：419-425.     

c-Jun N-terminal kinase signaling regulates events associated with both health and degeneration in motoneurons

The c-Jun N-terminal kinases (JNKs) are activated by various stimuli and are critical for neuronal development as well as for death following a stressful stimulus. Here, we have evaluated JNK activity in both healthy and dying motoneurons from developing chick embryos and found no apparent difference in overall JNK activity between the conditions, suggesting that this pathway maybe critical in both circumstances. Pharmacological inhibition of JNK in healthy motoneurons supplied with trophic support resulted in decreased mitochondrial membrane potential, neurite outgrowth, and phosphorylation of microtubule-associated protein 1B. On the other hand, in motoneurons deprived of trophic support, inhibition of JNK attenuated caspase activation, and nuclear condensation. We also examined the role of JNK's downstream substrate c-Jun in mediating these events. While c-Jun expression and phosphorylation were greater in cells supplied with trophic support as compared with those deprived, inhibition of c-Jun had no effect on nuclear condensation in dying cells or neurite outgrowth in healthy cells, suggesting that JNK's role in these events is independent of c-Jun. Together, our data underscore the dualistic nature of JNK signaling that is critical for both survival and degenerative changes in motoneurons.     

Age-specific changes in expression, activity, and activation of the c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinases by methyl methanesulfonate in rats

The stress-activated protein kinases (SAPKs), c-Jun NH(2)-terminal kinases (JNKs) and p38 mitogen-activated protein kinases, were evaluated in the liver and brain of young and old rats in response to a direct-acting alkylating agent, methyl methanesulfonate (MMS). A slight but statistically significant increase in the baseline expression levels of JNK isoforms was detected in both the liver and brain of old as compared with young rats. In the liver of both young and old rats, no basal activities of JNKs were detected. In the brain, JNK activities were constitutively high and significantly increased in old rats compared with their young counterparts. Upon MMS treatment, JNKs were strongly activated in the liver, but not in the brain, of both young and old animals. The basal activity of p38 significantly increased in both the liver and brain of old rats as compared with young rats. An increase in the basal expression of p38 was detected in the brain but not in the liver of old rats. Upon treatment with MMS, p38 was activated in the liver of both young and old rats. In the brain, p38 was only activated in young but not in old rats. Taken together, these results demonstrate age-specific as well as organ-specific SAPKs signaling pathways in the rat in vivo. The possible implications of these findings in terms of resistance to endogenous and environmentally induced genotoxic stress during aging are discussed.     

Secreted beta-amyloid precursor protein activates microglia via JNK and p38-MAPK

Reactive microglia are thought to play a role in the pathogenesis of Alzheimer's disease (AD) and are localized to the senile plaques that are associated with cognitive decline. The beta-amyloid precursor protein (betaAPP) is over-expressed in the dystrophic neurites near such plaques, and secreted forms of betaAPP (sAPPalpha) activate inflammatory responses in microglia. To characterize the mechanisms by which sAPPalpha activates microglia, we assayed its effects on MAP kinases, including c-Jun N-terminal kinases (JNK), extracellular signal-regulated protein kinases (ERK), and p38-MAPK. sAPPalpha was found to rapidly activate JNKs, ERKs and p38-MAPK in a dose-dependent manner. The JNK inhibitor SP600125 and the p38 inhibitor SB203580 independently reduced both nitrite accumulation and induction of inflammatory nitric oxide synthase (iNOS). By contrast, inhibition of the ERK pathway with U0126 did not appreciably affect either outcome measure. These findings suggest that sAPP activates the ERK, JNK and p38 classes of MAP kinases but that only JNK and p38-MAPK are critical for activation of microglia by sAPPalpha, a process that compromises neuronal function and survival.     

MKK7 deficiency in mature neurons impairs parental behavior in mice

c-Jun N-terminal kinases (JNKs) are constitutively activated in mammalian brains and are indispensable for their development and neural functions. MKK7 is an upstream activator of all JNKs. However, whether the common JNK signaling pathway regulates the brain's control of social behavior remains unclear. Here, we show that female mice in which Mkk7 is deleted specifically in mature neurons (Mkk7^flox/floxSyn-Cre mice) give birth to a normal number of pups but fail to raise them due to a defect in pup retrieval. To explore the mechanism underlying this abnormality, we performed comprehensive behavioral tests. Mkk7^flox/floxSyn-Cre mice showed normal locomotor functions and cognitive ability but exhibited depression-like behavior. cDNA microarray analysis of mutant brain revealed an altered gene expression pattern. Quantitative RT-PCR analysis demonstrated that mRNA expression levels of genes related to neural signaling pathways and a calcium channel were significantly different from controls. In addition, loss of neural MKK7 had unexpected regulatory effects on gene expression patterns in oligodendrocytes. These findings indicate that MKK7 has an important role in regulating the gene expression patterns responsible for promoting normal social behavior and staving off depression.     

Suppression of survival kinases and activation of JNK mediate ethanol-induced cell death in the developing rat brain

Administration of ethanol to immature rat pups during the period in which synaptogenesis occurs triggers extensive apoptotic cell death in the brain. This ethanol-induced cell death is known to be mediated by Bax activation, which is caused by mitochondrial dysfunction. However, little data is available regarding the regulation of survival signaling pathways and their downstream events that lead to Bax activation. Thus, in the present study, we aimed to investigate the effect of ethanol on survival signaling pathways and their downstream events that lead to cell death in the rat brain during the brain developmental period. Ethanol (3 g/kg, 20% in saline) was administered subcutaneously to post-natal 7-day-old rat pups twice at 2-h intervals and the pups were sacrificed at 4 h following the first ethanol injection. Ethanol treatment suppressed the activation of survival kinases, particularly Akt, Erk1/2 and PKAalpha, whereas it increased the activation of JNK. Moreover, dissociation of dephosphorylated Bad from 14-3-3 and the interaction of activated JNK with Bcl-2 were elevated by ethanol treatment. The present study demonstrated that ethanol treatment during the brain developmental period induced mitochondrial dysfunction, which led to cell death by the suppression of survival kinases, Bad release from 14-3-3 and inactivation of Bcl-2 by activated JNK.     

Development of a pentylenetetrazole-induced seizure model to evaluate kinase inhibitor efficacy in the central nervous system

c-Jun N-terminal kinases (JNKs) are implicated in cell death in neurodegenerative disorders. Therefore, JNK inhibitors could act as neuroprotective agents. To evaluate potential candidates, reproducible and quantitative CNS in vivo models are required. To that end, a pentylenetetrazole-induced seizure model was explored. c-Jun phosphorylation was detected in hippocampal extracts by blotting c-Jun immunoprecipitates with phosphorylation-specific antibodies. Pentylenetetrazole administration induced rapid and reproducible increases in c-Jun phosphorylation. However, special attention had to be paid to the composition of the extraction buffer to ensure stabilization of protein phosphorylation, as demonstrated using internal standards of phosphorylated recombinant c-Jun. As JNK and its upstream activator MKK4 are activated by phosphorylation, these events were also evaluated. In principle, kinase inhibitors could act at the level of JNK or upstream kinases to inhibit c-Jun phosphorylation. MKK4 phosphorylation was dramatically increased in response to pentylenetetrazole but, again, only when appropriate phosphatase inhibitors were in the extraction buffer. In contrast, JNK was found to be constitutively phosphorylated and unaltered upon pentylenetetrazole treatment. The JNK inhibitor SP600125 was shown to inhibit c-Jun phosphorylation without affecting MKK4 phosphorylation. Our procedures enable analysis of JNK pathway signalling in a CNS model and, also, should be applicable to that of other protein phosphorylation events in vivo.     

Kinases and kinase signaling pathways: potential therapeutic targets in Parkinson's disease

Complex molecular mechanisms underlying the pathogenesis of Parkinson's disease (PD) are gradually being elucidated. Accumulating genetic evidence implicates dysfunction of kinase activities and phosphorylation pathways in the pathogenesis of PD. Causative and risk gene products associated with PD include protein kinases (such as PINK1, LRRK2 and GAK) and proteins related phosphorylation signaling pathways (such as SNCA, DJ-1). PINK1, LRRK2 and several PD gene products have been associated with mitogen-activated protein (MAP) and protein kinase B (AKT) kinase signaling pathways. C-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK) and p38, signaling pathways downstream of MAP, are particularly important in PD. JNK and p38 play an integral role in neuronal death. Targeting JNK or p38 signaling may offer an effective therapy for PD. Inhibitors of the ERK signaling pathway, which plays an important role in the development of l-DOPA-induced dyskinesia (LID), have been shown to attenuate this condition in animal models. In this review, we summarize experimental evidence gathered over the last decade on the role of PINK1, LRRK2 and GAK and their related phosphorylation signaling pathways (JNK, ERK, p38 and PI3K/AKT) in PD. It is speculated that improvement or modulation of these signaling pathways will reveal potential therapeutic targets for attenuation of the cardinal symptoms and motor complications in patients with PD in the future.     

Activation of mitogen-activated protein kinases in gerbil hippocampus with ischemic tolerance induced by 3-nitropropionic acid

The present study investigated the activation of c-Jun NH2-terminal kinases (JNK), p38 mitogen-activated protein kinases (p38) and extracellular signal-regulated kinases (ERK) in the gerbil hippocampus by immunohistochemistry to clarify the role of these kinases in ischemic tolerance induced by3-NP. Intraperitoneal administration of 3-NP (3 or 10 mg/kg) caused the activation of JNK in CA1 subfield, which induced tolerance to subsequent ischemia and prevented delayed neuronal death (DND). As concerns p38 and ERK, no activation was induced by intoxication of 3-NP. Our results show the activation of JNK following chemical preconditioning with low dose of 3-NP is closely related to the acquisition of resistance to DND.     

Controversies surrounding glucocorticoid-mediated cell death in the hippocampus

The adrenal gland releases mineralocorticoids (MCs) and glucocorticoids (GCs) in response to a variety of stimuli, including stress. Once released, these adrenal steroids mediate a plethora of physiological responses in both the periphery and the central nervous system. The collective actions of GCs in the brain are paradoxical, however, in that basal levels of GCs are essential for neuronal development, plasticity and survival, while stress levels of GCs produce neuronal loss. Aging represents another contradictory function of GCs in the brain, since lifelong exposure to GCs has been implicated as a causative factor in senescent neuronal loss. In addition, glucocorticoids have also been shown to intensify neuronal damage in the hippocampus during ischemia and excitotoxicity through mechanisms that modulate synaptic glutamate concentrations. Conversely, the absence of adrenal steroids has been shown to regulate both neurogenesis and neuronal loss in the dentate gyrus of the hippocampus. Evidence continues to accumulate which suggests that GC-induced neuronal death in all these physiological and pathophysiological settings occurs by apoptosis. Accordingly, this review will examine the pharmacological, cellular and molecular mechanisms through which glucocorticoids mediate or contribute to neuronal remodeling and, ultimately, neuronal death.     

Functional role of JNK in neuritogenesis of PC12-N1 cells

JNKs, also known as SAPKs, are activated in response to a wide variety of factors including growth factors, cytokines, UV radiation, and heat shock. In the rat pheochromocytoma PC12 cells, the JNK signaling pathway mediates diverse functions such as differentiation and apoptosis. We have previously shown that activated JNK is required for later stages of neuritogeneis induced by NGF in a variant PC12 cell line (N1). Here, the functional role of JNK in N1 cells is further investigated. We show that NGF treatment, which induces extensive neurite branching and cell soma enlargement in the N1 cells, stimulates a biphasic activation of JNK. The first phase of activation is rapid and transient, beginning at 15 min after NGF exposure and lasting approximately 45 min. The second phase of activation is sustained, beginning at 9-12 h of NGF treatment and lasting for at least 24 h. Similar biphasic pattern of JNK activation is also observed in the parental PC12 cells. Using the specific JNK inhibitor SP600125, we show that the biphasic activation is necessary for neurite outgrowth and branching, and that inhibition of either phase suppresses neuritogenesis in the N1 cells. These results suggest that dynamic JNK activation may play a key role in neurite outgrowth during neuronal development.     

Age-associated changes in SAPK/JNK and p38 MAPK signaling in response to the generation of ROS by 3-nitropropionic acid

Mitochondrial dysfunction has been identified as a major source of oxidative stress in aged tissues. In this study we asked whether activities of components of the SAPK/JNK and p38 MAPK stress response signaling pathways are indicative of oxidative stress in aged mouse livers and whether these pathways are responsive to oxidative stress generated by 3-nitropropionic acid (3-NPA), an inhibitor of complex II (succinic dehydrogenase). We asked whether (a) aging affects the basal activity of the SAPK/JNK stress signaling pathway; (b) specific isoforms of JNK, i.e. 46 or 54 kDa JNKs are activated by 3-NPA; (c) aging affects the response of this signaling pathway to 3-NPA; (d) there is a cross pathway activation of JNK or p38 MAPK by upstream activators. Our studies have shown that although their protein pool levels are not altered, the basal JNK activities using c-Jun as substrate is elevated. Furthermore, in aged livers, JNK activity is induced to a greater extent and takes longer to recover from 3-NPA treatment. The activities of the upstream activators of JNKs, MAP kinase kinase (MKK) 4 and 7, are also elevated in livers of aged C57BL/6 male mice. These activator kinases, which are induced (phosphorylated) by 3-NPA in young livers, are not inducible by this inhibitor in aged livers. In fact, these proteins are highly phosphorylated in the control aged livers and are dephosphorylated in response to 3-NPA. Finally, we demonstrate for the first time that MKK7 serves as an upstream activator of p38 MAPK and that MKK3 and MKK6 activates 54 kDa JNK2 in aged liver. Our studies suggest that failure to respond to 3-NPA may be indicative of the susceptibility of aged tissue to oxidative stress, supporting our hypothesis that aged tissues (especially liver) develop a state of chronic stress even in the absence of a challenge.     

Phosphorylation of microtubule-associated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function

Neurons, the basic information processing units of the nervous system, are characterized by a complex polar morphology which is essential for their function. To attain their precise morphology, neurons extend cytoplasmatic processes (axons and dendrites) and establish synaptic connections in a highly regulated way. Additionally, neurons are also subjected to small plastic changes at the adult stage which serve to regulate synaptic transmission. Every step of neuronal development is genetically controlled by endogenous determinants, as well as by environmental signals including intercellular contacts, extracellular matrix and diffusible signals. Cytoskeletal components are among the main protein targets modified in response to most of those extracellular signals which ultimately determine neuronal morphology. One of the major mechanisms controlling the neuronal cytoskeleton is the modification of the phosphorylation state of cytoskeletal proteins via changes in the relative activities of protein kinases and phosphatases within neurons. In particular, the microtubule-associated protein 2 (MAP2) family of proteins is an abundant group of cytoskeletal components which are predominantly expressed in neurons and serve as substrates for most of protein kinases and phosphatases present in neurons. MAP2 phosphorylation seems to control its association with the cytoskeleton and it is developmentally regulated. Moreover, MAP2 may perform many functions including the nucleation and stabilization of microtubules (and maybe microfilaments), the regulation of organelle transport within axons and dendrites, as well as the anchorage of regulatory proteins such as protein kinases which may be important for signal transduction. These putative functions of MAP2 have also been proposed to play important roles in the outgrowth of neuronal processes, synaptic plasticity and neuronal cell death. Thus, MAP2 constitutes an interesting case to understand the regulation of neuronal function by the alteration of the phosphorylation state of cytoskeletal proteins in response to different extracellular signals. Here we will review the current knowledge about the regulation of MAP2 function through phosphorylation/dephosphorylation and its relevance in the broader context of neuronal functions.     

Insulin-like growth factor-1 activates Akt and Jun N-terminal kinases (JNKs) in promoting the survival of T lymphocytes

Insulin-like growth factor 1 receptor (IGF-1R) expression is augmented on T cells upon ligation of CD28, and this promotes IGF-1-mediated protection from Fas-induced cell death for up to 6 days. To determine the mechanism of action of IGF-1R in T-cell expansion, we investigated the signalling pathways activated by IGF-1 in T cells and in Jurkat cells. We found that IGF-1 transiently induces Akt, jun N-terminal kinases (JNK), and c-Jun phosphorylation in activated T cells, with JNK and c-Jun phosphorylation occurring faster than Akt phosphorylation. To mimic IGF-1R expression levels in CD28-stimulated Jurkat cells these cells were stably transfected to over-express the IGF-1R. Jurkat/IGF-1R cells exhibited enhanced constitutive Akt phosphorylation compared with mock-transfected controls, but IGF-1 induced transient phosphorylation of MKK4, JNKs, and c-Jun. Inhibition of PI-3 kinase activity and Akt phosphorylation with LY294002 totally suppressed IGF-1-mediated protection from Fas killing in activated T cells, but only partially suppressed IGF-1-mediated protection in Jurkat/IGF-1R cells. However, either dicumarol in T cells or a dominant negative JNK1 (APF) in Jurkat/IGF-1R cells greatly suppressed IGF-1-mediated protection from Fas killing. Together, these data demonstrate that IGF-1-mediated activation of JNKs and PI-3 kinase contributes to normal T-cell survival, whereas the JNK pathway may be more important in Jurkat leukaemia cells.     

Subunit- and site-specific pharmacology of the NMDA receptor channel.

N-Methyl-D-aspartate (NMDA) receptor channels play important roles in various physiological functions such as synaptic plasticity and synapse formation underlying memory, learning and formation of neural networks during development. They are also important for a variety of pathological states including acute and chronic neurological disorders, psychiatric disorders, and neuropathic pain syndromes. cDNA cloning has revealed the molecular diversity of NMDA receptor channels. The identification of multiple subunits with distinct distributions, properties and regulation, implies that NMDA receptor channels are heterogeneous in their pharmacological properties, depending on the brain region and the developmental stage. Furthermore, mutation studies have revealed a critical role for specific amino acid residues in certain subunits in determining the pharmacological properties of NMDA receptor channels. The molecular heterogeneity of NMDA receptor channels as well as their dual role in physiological and pathological functions makes it necessary to develop subunit- and site-specific drugs for precise and selective therapeutic intervention. This review summarizes from a molecular perspective the recent advances in our understanding of the pharmacological properties of NMDA receptor channels with specific references to agonists binding sites, channel pore regions, allosteric modulation sites for protons, polyamines, redox agents, Zn2+ and protein kinases, phosphatases.