Zhengui Xia

Opposing Effects of ERK and JNK-p38 MAP Kinases on Apoptosis

Apoptosis plays an important role during neuronal development, and defects in apoptosis may underlie various neurodegenerative disorders. To characterize molecular mechanisms that regulate neuronal apoptosis, the contributions to cell death of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38, were examined after withdrawal of nerve growth factor (NGF) from rat PC-12 pheochromocytoma cells. NGF withdrawal led to sustained activation of the JNK and p38 enzymes and inhibition of ERKs. The effects of dominant-interfering or constitutively activated forms of various components of the JNK-p38 and ERK signaling pathways demonstrated that activation of JNK and p38 and concurrent inhibition of ERK are critical for induction of apoptosis in these cells. Therefore, the dynamic balance between growth factor-activated ERK and stress-activated JNK-p38 pathways may be important in determining whether a cell survives or undergoes apoptosis.

Neuroprotection by brain-derived neurotrophic factor is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase.

Apoptosis is a form of programmed cell death that plays a pivotal role during development and in the homeostasis of the adult nervous systems. However, mechanisms that regulate neuronal apoptosis are not well defined. Here, we report that brain-derived neurotrophic factor (BDNF) protects cortical neurons against apoptosis induced by camptothecin or serum deprivation and activates the extracellular-signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Using pharmacological agents and transient transfection with dominant interfering or constitutive active components of the ERK or the PI 3-kinase pathway, we demonstrate that the ERK pathway plays a major role in BDNF neuroprotection against camptothecin. Furthermore, ERK is activated in cortical neurons during camptothecin-induced apoptosis, and inhibition of ERK increases apoptosis. In contrast, the PI 3-kinase pathway is the dominant survival mechanism for serum-dependent survival under normal culture conditions and for BDNF protection against serum withdrawal. These results suggest that the ERK pathway is one of several neuroprotective mechanisms that are activated by stress to counteract death signals in central nervous system neurons. Furthermore, the relative contribution of the ERK and PI 3-kinase pathways to neuronal survival may depend on the type of cellular injury.

Nerve Growth Factor Activates Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Pathways To Stimulate CREB Serine 133 Phosphorylation

ABSTRACT The mechanisms by which growth factor-induced signals are propagated to the nucleus, leading to the activation of the transcription factor CREB, have been characterized. Nerve growth factor (NGF) was found to activate multiple signaling pathways that mediate the phosphorylation of CREB at the critical regulatory site, serine 133 (Ser-133). NGF activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs), which in turn activate the pp90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases, all three members of which were found to catalyze CREB Ser-133 phosphorylation in vitro and in vivo. In addition to the ERK/RSK pathway, we found that NGF activated the p38 MAPK and its downstream effector, MAPK-activated protein kinase 2 (MAPKAP kinase 2), resulting in phosphorylation of CREB at Ser-133. Inhibition of either the ERK/RSK or the p38/MAPKAP kinase 2 pathway only partially blocked NGF-induced CREB Ser-133 phosphorylation, suggesting that either pathway alone is sufficient for coupling the NGF signal to CREB activation. However, inhibition of both the ERK/RSK and the p38/MAPKAP kinase 2 pathways completely abolished NGF-induced CREB Ser-133 phosphorylation. These findings indicate that NGF activates two distinct MAPK pathways, both of which contribute to the phosphorylation of the transcription factor CREB and the activation of immediate-early genes.

Disruption of the Type III Adenylyl Cyclase Gene Leads to Peripheral and Behavioral Anosmia in Transgenic Mice

Cyclic nucleotide-gated ion channels in olfactory sensory neurons (OSNs) are hypothesized to play a critical role in olfaction. However, it has not been demonstrated that the cAMP signaling is required for olfactory-based behavioral responses, and the contributions of specific adenylyl cyclases to olfaction have not been defined. Here, we report the presence of adenylyl cyclases 2, 3, and 4 in olfactory cilia. To evaluate the role of AC3 in olfactory responses, we disrupted the gene for AC3 in mice. Interestingly, electroolfactogram (EOG) responses stimulated by either cAMP- or inositol 1,4,5-triphosphate- (IP3-) inducing odorants were completely ablated in AC3 mutants, despite the presence of AC2 and AC4 in olfactory cilia. Furthermore, AC3 mutants failed several olfaction-based behavioral tests, indicating that AC3 and cAMP signaling are critical for olfactory-dependent behavior.

The role of calmodulin as a signal integrator for synaptic plasticity

Excitatory synapses in the brain show several forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD), which are initiated by increases in intracellular Ca2+ that are generated through NMDA (N-methyl-D-aspartate) receptors or voltage-sensitive Ca2+ channels. LTP depends on the coordinated regulation of an ensemble of enzymes, including Ca2+/calmodulin-dependent protein kinase II, adenylyl cyclase 1 and 8, and calcineurin, all of which are stimulated by calmodulin, a Ca2+-binding protein. In this review, we discuss the hypothesis that calmodulin is a central integrator of synaptic plasticity and that its unique regulatory properties allow the integration of several forms of signal transduction that are required for LTP and LTD.

CREB Transcriptional Activity in Neurons Is Regulated by Multiple, Calcium-Specific Phosphorylation Events

The transcription factor CREB mediates diverse responses in the nervous system. It is not known how CREB induces specific patterns of gene expression in response to different extracellular stimuli. We find that Ca(2+) influx into neurons induces CREB phosphorylation at Ser133 and two additional sites, Ser142 and Ser143. While CREB Ser133 phosphorylation is induced by many stimuli, phosphorylation at Ser142 and Ser143 is selectively activated by Ca(2+) influx. The triple phosphorylation of CREB is required for effective Ca(2+) stimulation of CREB-dependent transcription, but the phosphorylation of Ser142 and Ser143, in addition to Ser133, disrupts the interaction of CREB with its cofactor CBP. These results suggest that Ca(2+) influx triggers a specific program of gene expression in neurons by selectively regulating CREB phosphorylation.

Distribution of mRNA for the calmodulin-sensitive adenylate cyclase in rat brain: Expression in areas associated with learning and memory

The Drosophila learning mutant, rutabaga, is deficient in the calmodulin-sensitive adenylate cyclase, and studies of associative learning in Aplysia have implicated this enzyme in neuroplasticity. Therefore, the distribution of mRNA encoding the calmodulin-sensitive adenylate cyclase in rat brain was examined by in situ hybridization. mRNA for this enzyme is expressed in specific areas of brain that have been implicated in learning and memory, including the neocortex, the hippocampus, and the olfactory system. The presence of mRNA for this enzyme in the pyramidal and granule cells of the hippocampal formation provides evidence that it is found in neurons. These data are consistent with the proposal that the calmodulin-sensitive adenylate cyclase plays an important role in learning and memory.

Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP, or paraquat

Inhibition of mitochondrial complex I is one of the leading hypotheses for dopaminergic neuron death associated with Parkinsons disease (PD). To test this hypothesis genetically, we used a mouse strain lacking functional Ndufs4, a gene encoding a subunit required for complete assembly and function of complex I. Deletion of the Ndufs4 gene abolished complex I activity in midbrain mesencephalic neurons cultured from embryonic day (E) 14 mice, but did not affect the survival of dopaminergic neurons in culture. Although dopaminergic neurons were more sensitive than other neurons in these cultures to cell death induced by rotenone, MPP+, or paraquat treatments, the absence of complex I activity did not protect the dopaminergic neurons, as would be expected if these compounds act by inhibiting complex 1. In fact, the dopaminergic neurons were more sensitive to rotenone. These data suggest that dopaminergic neuron death induced by treatment with rotenone, MPP+, or paraquat is independent of complex I inhibition.

Type I calmodulin-sensitive adenylyl cyclase is neural specific.

Abstract: The distribution of type I calmodulin‐sensitive adenylyl cyclase in bovine and rat tissues was examined by northern blot analysis and in situ hybridization. Northern blot analysis using poly(A)+‐selected RNA from various bovine tissues indicated that mRNA for type I adenylyl cyclase was found only in brain, retina, and adrenal medulla, suggesting that this enzyme is neural specific. In situ hybridization studies using bovine, rabbit, and rat retina indicated that mRNA for type I adenylyl cyclase is found in all three nuclear layers of the neural retina and is particularly abundant in the inner segment of the photoreceptor cells. The neural‐specific distribution of type I adenylyl cyclase mRNA and its restricted expression in areas of brain implicated in neuroplasticity are consistent with the proposal that this enzyme plays an important role in various neuronal functions including learning and memory.

p38 MAP Kinase Mediates Apoptosis through Phosphorylation of BimEL at Ser-65

The stress-activated c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein (MAP) kinase (p38) regulate apoptosis induced by several forms of cellular insults. Potential targets for these kinases include members of the Bcl-2 family proteins, which mediate apoptosis generated through the mitochondria-initiated, intrinsic cell death pathway. Indeed, the activities of several Bcl-2 family proteins, both pro- and anti-apoptotic, are controlled by JNK phosphorylation. For example, the pro-apoptotic activity of BimEL, a member of the Bcl-2 family, is stimulated by JNK phosphorylation at Ser-65. In contrast, there is no reported evidence that p38-induced apoptosis is due to direct phosphorylation of Bcl-2 family proteins. Here we report evidence that sodium arsenite-induced apoptosis in PC12 cells may be due to direct phosphorylation of BimEL at Ser-65 by p38. This conclusion is supported by data showing that ectopic expression of a wild type, but not a non-phosphorylatable S65A mutant of BimEL, potentiates sodium arsenite-induced apoptosis and by experiments showing direct phosphorylation of BimEL at Ser-65 by p38 in vitro. Furthermore, sodium arsenite induced BimEL phosphorylation at Ser-65, which was blocked by p38 inhibition. This study provides the first example whereby p38 induces apoptosis by phosphorylating a member of the Bcl-2 family and illustrates that phosphorylation of BimEL on Ser-65 may be a common regulatory point for cell death induced by both JNK and p38 pathways.