Comparative status of activated ERK1/2 and PARP cleavage in human gliomas

Gliomas are the most common form of cerebral tumors. Understanding molecular features of glioma will eventually allow for targeted intervention and more promising approaches for treating gliomas. The present study is therefore carried out to check the levels of activated ERK1/2 with respect to phospho‐tyrosine and cleavage of poly ADP‐ribose polymerase (PARP). Recent experiments support that extracellular signal regulated kinase (ERK), a mitogen activated protein (MAP) kinase might have a critical role in cell proliferation. PARP is a DNA‐repair enzyme activated by DNA strand breaks. Overactivation of PARP after cellular insult lead to cell death caused by rapid depletion of cellular ATP. Three glioblastoma multiforme (GBM) and two astrocytoma biopsies (core tumor) and peripheral tissues were analyzed for the expression of p‐ERK1/2 and PARP. Results indicate higher p‐ERK1/2 in GBM. Cleaved fragments of PARP (89 kDa) were found to be more in core tumor tissue samples as compared to peripheral tumor tissues of both astrocytoma and GBM.     

Protective effects of sesamin and sesamolin on hypoxic neuronal and PC12 cells

Reactive oxygen species (ROS) are important mediators of a variety of pathological processes, including inflammation and ischemic injury. The neuroprotective effects of sesame antioxidants, sesamin and sesamolin, against hypoxia or H2O2-induced cell injury were evaluated by cell viability or lactate dehydrogenase (LDH) activity. Sesamin and sesamolin reduced LDH release of PC12 cells under hypoxia or H2O2-stress in a dose-dependent manner. Dichlorofluorescein (DCF)-sensitive ROS production was induced in PC12 cells by hypoxia or H2O2-stress but was diminished in the presence of sesamin and sesamolin. We evaluated further the role of mitogen-activated protein kinases (MAPKs) and caspase-3 in hypoxia-induced PC12 cell death. Extracellular signal-regulated protein kinase (ERK) 1, c-jun N-terminal kinase (JNK), and p38 MAPKs of signaling pathways were activated during hypoxia. We found that the inhibition of MAPKs and caspase-3 by sesamin and sesamolin correlated well with the reduction in LDH release under hypoxia. Furthermore, the hypoxia-induced apoptotic-like cell death in cultured cortical cells as detected by a fluorescent DNA binding dye was reduced significantly by sesamin and sesamolin. Taken together, these results suggest that the protective effect of sesamin and sesamolin on hypoxic neuronal and PC12 cells might be related to suppression of ROS generation and MAPK activation.     

Activated glia induce neuron death via MAP kinase signaling pathways involving JNK and p38

Chronic glial activation in neurodegenerative diseases contributes to neuronal dysfunction and neuron loss through production of neuroinflammatory molecules. However, the molecular mechanisms, particularly the signal transduction pathways involved in glia-dependent neuron death, are poorly understood. As a first step to address this question, we used a neuron-glia co-culture system that allows diffusion of soluble molecules between glia and neurons to test the potential importance of mitogen-activated protein kinase (MAPK) signaling pathways in the glia-induced neuron death. Activation of glia in co-culture by lipopolysaccharide (LPS) induced apoptotic-like neuron death. The MAPKs tested (p38, JNK, ERK1/2) were activated in both glia and neurons following LPS treatment, suggesting their involvement in both glial activation and neuronal response to diffusible, glia-derived neurotoxic molecules. Inhibitors of p38 and JNK partially blocked neuron death in the LPS-treated co-culture, whereas an ERK1/2 pathway inhibitor did not protect neurons. These results show that p38 and JNK MAPKs, but not ERK1/2 MAPK, are important signal transduction pathways contributing to glia-induced neuron death.     

Tumor necrosis factor‐alpha mediates activation of NF‐κB and JNK signaling cascades in retinal ganglion cells and astrocytes in opposite ways

Tumor necrosis factor‐alpha (TNF) is an important mediator of the innate immune response in the retina. TNF can activate various signaling cascades, including NF‐κB, nuclear factor kappa B (NF‐κB) and c‐Jun N‐terminal kinase (JNK) pathways. The harmful role of these pathways, as well as of TNF, has previously been shown in several retinal neurodegenerative conditions including glaucoma and retinal ischemia. However, TNF and TNF‐regulated signaling cascades are capable not only of mediating neurotoxicity, but of being protective. We performed this study to delineate the beneficial and detrimental effects of TNF signaling in the retina. To this end, we used TNF‐treated primary retinal ganglion cell (RGC) and astrocyte cultures. Levels of expression of NF‐κB subunits in RGCs and astrocytes were evaluated by quantitative RT‐PCR (qRT‐PCR) and Western blot (WB) analysis. NF‐κB and JNK activity in TNF‐treated cells was determined in a time‐dependent manner using ELISA and WB. Gene expression in TNF‐treated astrocytes was measured by qRT‐PCR. We found that NF‐κB family members were present in RGCs and astrocytes at the mRNA and protein levels. RGCs failed to activate NF‐κB in the presence of TNF, a phenomenon that was associated with sustained JNK activation and RGC death. However, TNF initiated the activation of NF‐κB and mediated transient JNK activation in astrocytes. These events were associated with glial survival and increased expression of neurotoxic pro‐inflammatory factors. Our findings suggest that, in the presence of TNF, NF‐κB and JNK signaling cascades are activated in opposite ways in RGCs and astrocytes. These events can directly and indirectly facilitate RGC death.     

Expression of pERK and pAKT in pediatric high grade astrocytomas: Correlation with YKL40 and prognostic significance

The Ras signaling pathway, consisting of mitogen‐activated protein kinase (MAPK) and PI3K/AKT signaling, is a prominent oncogenic pathways in adult diffuse gliomas, but few studies have evaluated such pathways in pediatric malignant gliomas. We investigated by immunohistochemistry MAPK and AKT signaling in a series of 28 pediatric high‐grade gliomas (WHO grade III and IV). We sought a possible association of phospho‐ERK (p‐ERK) and phospho‐AKT (p‐AKT) with expression of other proteins involved in the Ras pathway, that is, YKL40, epidermal growth factor receptor (EGFR), EGFR vIII and c‐Met. Moreover we correlated the expression of p‐ERK and p‐AKT with prognosis. No cases showed expression for c‐Met and EGFR, and only one case was positive for EGFR vIII. YKL‐40 protein was expressed in 43% of cases. We detected expression of p‐ERK and p‐AKT in 61% and 57%, respectively, of pediatric high grade gliomas. Statistical analysis comparing the two groups in term of high and low p‐ERK and p‐AKT expression showed a trend toward worse overall survival in patients with high expression of p‐AKT. The activation of ERK and AKT suggest a possible role of this protein in inducing activation of the Ras signaling pathway in pediatric high‐grade gliomas. Moreover high levels of p‐AKT are associated with worse overall survival.     

Activation of STAT3, MAPK, and AKT in malignant astrocytic gliomas: correlation with EGFR status, tumor grade, and survival

Diffuse astrocytic gliomas are the most common human glial tumors with glioblastoma being the most malignant form. Epidermal growth factor receptor (EGFR) gene amplification is one of the most common genetic changes in glioblastoma and can lead to the activation of various downstream signaling molecules, including STAT3, MAPK, and AKT. In this study, we investigated the activation status of these 3 signaling molecules as well as wild-type (EGFRwt) and mutant (EGFRvIII) EGFR in 82 malignant astrocytic gliomas (55 glioblastomas and 27 anaplastic astrocytomas) using immunohistochemistry. The presence of EGFRwt, but not EGFRvIII, immunopositivity correlated significantly with prevalent EGFR gene amplification in glioblastomas. STAT3 and AKT activation correlated significantly with EGFR status, although the correlation for p-STAT3 was attributed exclusively to EGFRvIII. The distribution of these 3 activated molecules varied significantly with tumor grade; although activation of STAT3 was essentially identical between anaplastic astrocytomas and glioblastomas, an increase in the activation of MAPK and AKT appeared to correlate with the progression of anaplastic astrocytoma to glioblastoma. Finally, activated STAT3 and AKT were marginally predictive of improved and worse prognosis, respectively. Taken together, these findings begin to elucidate the interrelationship between these signaling pathways in astrocytic gliomas in vivo.     

Induction of apoptosis signal regulating kinase 1 (ASK1) after spinal cord injury in rats: possible involvement of ASK1-JNK and -p38 pathways in neuronal apoptosis.

The aims of this study were to clarify the mechanism of cell death by apoptosis in the spinal cord after traumatic injury, and to examine the role of the mitogen-activated protein kinase (MAPK) pathways in the transmission of apoptosis signals. The rat spinal cord, experimentally injured by extradural static weight-compression, was studied by hematoxylin and eosin staining, Nissl-staining, terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) staining, and immunostaining using polyclonal antibodies against Apoptosis Signal-regulating Kinase 1 (ASK1), c-Jun N-terminal kinase (JNK), and p38 MAPK. TUNEL-positive cells were present at all stages studied until 7 days after injury, and percentage positivity for these cells was maximal at 3 days after injury. Electron microscopic analysis revealed the occurrence of apoptosis in both neuronal cells and glial cells. TUNEL-positive glial cells were stained by oligodendrocyte-specific maker. Expression of ASK1 was maximal at 24 h after injury in the gray matter and at 3 days after injury in the white matter. Following the expression of ASK1, activated forms of JNK and p38 were observed in apoptotic cells detected by the TUNEL method. Colocalization of ASK1 and activated JNK or activated p38 was observed in the same cell. These findings suggest the involvement of the stress-activated MAPK pathways including ASK1 in the transmission of apoptosis signals after spinal cord injury.     

Brain-derived growth factor and glial cell line-derived growth factor use distinct intracellular signaling pathways to protect PD cybrids from H2O2-induced neuronal death

The cause of idiopathic PD is obscure, and most cases are sporadic. Oxidative stress and deficiency of various neurotrophic factors (NTFs) could be factors triggering neurodegeneration in the substantia nigra (SN). Cytoplasmic hybrid cells (cybrids) made from mitochondrial DNA of idiopathic PD subjects have reduced glutathione (GSH) levels and increased vulnerability to H2O2. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) rescue PD cybrids from H2O2-induced cell death. GDNF mediated effects require Src kinase and phosphatidylinositol 3-kinase (PI3K)/Akt activation. Inhibiting either PI3K/Akt or ERK pathways blocks the effects of BDNF. Inhibiting p38MAPK and c-Jun N-terminal kinase (JNK) pathways enhances the neuroprotective effects of both NTFs. These results demonstrate that expression of PD mitochondrial genes in cybrids increases vulnerability to oxidative stress that is ameliorated by both BDNF and GDNF, which utilize distinct signaling cascades to increase intracellular GSH and enhance survival-promoting cell signaling.     

Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

The pathological basis of neonatal hypoxia–ischemia (HI) brain damage is characterized by neuronal cell loss. Oxidative stress is thought to be one of the main causes of HI‐induced neuronal cell death. The p38 mitogen‐activated protein kinase (MAPK) is activated under conditions of cell stress. However, its pathogenic role in regulating the oxidative stress associated with HI injury in the brain is not well understood. Thus, this study was conducted to examine the role of p38 MAPK signaling in neonatal HI brain injury using neonatal rat hippocampal slice cultures exposed to oxygen/glucose deprivation (OGD). Our results indicate that OGD led to a transient increase in p38 MAPK activation that preceded increases in superoxide generation and neuronal death. This increase in neuronal cell death correlated with an increase in the activation of caspase‐3 and the appearance of apoptotic neuronal cells. Pre‐treatment of slice cultures with the p38 MAPK inhibitor, SB203580, or the expression of an antisense p38 MAPK construct only in neuronal cells, through a Synapsin I‐1‐driven adeno‐associated virus vector, inhibited p38 MAPK activity and exerted a neuroprotective effect as demonstrated by decreases in OGD‐mediated oxidative stress, caspase activation and neuronal cell death. Thus, we conclude that the activation of p38 MAPK in neuronal cells plays a key role in the oxidative stress and neuronal cell death associated with OGD.     

Mevastatin‐induced neurite outgrowth of neuroblastoma cells via activation of EGFR

Neuroblastoma cell lines are commonly used as models to study neuronal differentiation, as they retain the capacity to differentiate into a neuronal‐like phenotype. Receptor tyrosine kinase (RTK) signaling is essential for neuronal differentiation during development, and cholesterol‐containing lipid‐rafts are important for RTK signaling. Hydroxymethylglutaryl–coenzyme A reductase inhibitors of the statin family impair cholesterol biosynthesis and are in widespread clinical use for the treatment of cardiovascular diseases. It is of great clinical interest that statin treatment also correlates with a lower incidence of malignancies. We found that mevastatin triggered neurite outgrowth of neuroblastoma cells and examined the responsible signaling pathways. Treatment of Neuro2a cells with mevastatin for 24 hr induced neurite outgrowth associated with up‐regulation of the neuronal marker protein NeuN. Interestingly, we found that mevastatin triggered phosphorylation of the key kinases epidermal growth factor receptor (EGFR), ERK1/2, and Akt/protein kinase B. Inhibition of EGFR, PI3K, and the mitogen‐activated protein kinase cascade blocked mevastatin‐induced neurite outgrowth. Moreover, add‐back experiments of cell‐permeable cholesterol precursors indicated that farnesylated and geranylgeranylated proteins play a major role in statin‐induced neurite outgrowth. Taken together, our results provide the first mechanistic insight into statin‐triggered signaling pathways that lead to neurite outgrowth in neuroblastoma cells. Surprisingly, we revealed that mevastatin triggered the phosphorylation of the EGFR and that this was because of the inhibition of farnesylated and geranylgeranylated proteins. We propose that members of the large family of farnesylated or geranylgeranylated small GTPases (such as Rabs or Rap1) regulating the trafficking and signaling of EGFR might be responsible for the statin‐induced effects on EGFR signaling. © 2009 Wiley‐Liss, Inc.     

c-Jun N-Terminal Kinase (JNK) and p38 Play Different Roles in Age-Related Purkinje Cell Death in Murine Organotypic Culture

Several studies have shown that Purkinje cells die by apoptosis in organotypic slice cultures from postnatal 3-day-old (P3) mice. This cell death is age-dependent and has been proposed as indirect evidence for the programmed Purkinje cell death occurring in in vivo cerebellum. Here, we studied whether c-jun N-terminal kinase (JNK) and p38 kinase pathways contribute to the Purkinje cell death observed in cerebellar slice cultures obtained from P3 mice. Slice culture treatment with D-JNKI1 or SB203580, respectively inhibitors of JNK and p38 MAP kinases, results in a better survival of Purkinje cells. Interestingly, the combined treatment with the two inhibitors potentiated single treatment effects. These results suggest that p38 and JNK pathways might be differently implicated in this Purkinje cell death. Time course experiments found p38 activation immediately post-slicing, whereas JNK activation was detected only 2 h after the culture. We hypothesize that p38 activation might be due to the “sliced condition,” and JNK activation might be more specific to P3 age-dependent cell death. The study of JNK and p38 activation in cerebellar lysates from P0 slice culture confirmed JNK activation being specific for the P3 explants, whereas p38 is activated both from P0 and P3 cerebellar slice culture lysates. These results suggest that p38 is activated by the slicing, whereas JNK activation is related to developmental Purkinje cell death.     

Pyrrolidine dithiocarbamate-induced neuronal cell death is mediated by Akt,casein kinase 2, c-Jun N-terminal kinase,and IkappaB kinase in embryonic hippocampal progenitor cells

Pyrrolidine dithiocarbamate (PDTC) is known to induce cell death by the stimulation of intracellular zinc transport and subsequent modulation of nuclear factor-kappaB (NF-kappaB) activity. Zinc is a signaling messenger that is released by neuronal activity at many central excitatory synapses. Excessive synaptic release of zinc followed by entry into vulnerable neurons contributes to severe neuronal cell death. In the present study, we explored how PDTC modulates intracellular signal transduction pathways, leading to neuronal cell death. The exposure of immortalized embryonic hippocampal cells (H19-7) to PDTC within the range of 1-100 microM caused cell death in a dose-dependent manner. During the cell death, NF-kappaB activity increased in response to PDTC, and this activity corresponded well with the increase of intracellular free zinc levels, implying that the activation of NF-kappaB transmits the cell death signals of PDTC. Furthermore, PDTC caused the activation of IkappaB kinase (IKK), casein kinase 2 (CK2), phosphatidylinositol 3-kinase (PI-3K), and Akt, as well as mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but not p38 kinase. The blockade of PI-3K, JNK, and CK2 pathways resulted in a remarkable suppression of PDTC-induced cell death and also the activation of IKK, which subsequently led to a decrease of IkappaB phosphorylation. Although the overexpression of dominant-negative SEK in a transient manner did not inhibit the activation of Akt by PDTC, the transfection of kinase-inactive Akt mutants did cause a remarkable blockade of JNK activation, implying that Akt is present upstream of JNK in the PDTC-signaling pathways. Moreover, whereas selective CK2 inhibitors suppressed PDTC-induced JNK activation, the inhibition of JNK did not affect CK2 activity, suggesting that CK2 is directly related to the regulation of cell viability by PDTC and that the CK2-JNK pathway could be a downstream target of PDTC. Taken together, our results suggest that PDTC-mediated accumulation of intracellular zinc ions may affect cell viability by modulating several intracellular signaling pathways in neuronal hippocampal progenitor cells.     

Mechanisms of minocycline-induced suppression of simian immunodeficiency virus encephalitis: inhibition of apoptosis signal-regulating kinase 1

Human immunodeficiency virus (HIV) infection of the central nervous system (CNS) can lead to cognitive dysfunction, even in individuals treated with highly active antiretroviral therapy. Using an established simian immunodeficiency virus (SIV)/macaque model of HIV CNS disease, we previously reported that infection shifts the balance of activation of mitogen-activated protein kinase (MAPK) signaling pathways in the brain, resulting in increased activation of the neurodegenerative MAPKs p38 and JNK. Minocycline treatment of SIV-infected macaques reduced the incidence and severity of SIV encephalitis in this model, and suppressed the activation of p38 in the brain. The purpose of this study was to further examine the effects of minocycline on neurodegenerative MAPK signaling. We first demonstrated that minocycline also decreases JNK activation in the brain and levels of the inflammatory mediator nitric oxide (NO). We next used NO to activate these MAPK pathways in vitro, and demonstrated that minocycline suppresses p38 and c-Jun N-terminal kinase (JNK) activation by reducing intracellular levels, and hence, activation of apoptosis signal-regulating kinase 1 (ASK1), a MAPK kinase capable of selectively activating both pathways. We then demonstrated that ASK1 activation in the brain during SIV infection is suppressed by minocycline. By suppressing p38 and JNK activation pathways, which are important for the production of and responses to inflammatory mediators, minocycline may interrupt the vicious cycle of inflammation that both results from, and promotes, virus replication in SIV and HIV CNS disease.