Tyrosine Phosphorylation of Microtubule-Associated Protein Kinase After Transient Ischemia in the Gerbil Brain

The tyrosine phosphorylation of microtubule-associated protein (MAP) kinase was examined in the gerbil brain after transient ischemia and reperfusion. Phosphorylation of MAP kinase was maximal within 1 min of reperfusion following 5 min of ischemia and returned to control levels as early as 5 min postischemia. The greatest increase in MAP kinase phosphorylation was detected in the hippocampus, with minor increases in other ischemic regions of the brain. Several tyrosine-phosphorylated proteins were detected in the gerbil hippocampus; however, the ischemia and reperfusion injury only increased tyrosine phosphorylation of MAP kinase. The increase in tyrosine phosphorylation was prevented by the N-methyl-D-aspartate (NMDA) receptor blocker (+)-MK-801, whereas a non-NMDA receptor blocker, 6-cyano-7-nitroquinoxaline-2,3-dione, was ineffective. Pretreatment of gerbils with calcium channel blockers also prevented the tyrosine phosphorylation of MAP kinase in the ischemic brain. Altogether, these results imply an involvement of glutamate receptors and calcium during the tyrosine phosphorylation of MAP kinase. Tyrosine phosphorylation was also prevented when ischemia and reperfusion were conducted under hypothermic conditions, which protect against neurodegenerative damage. These findings implicate a role for MAP kinase in neuronal damage resulting from ischemia and reperfusion.     

Casein Kinase II Activity in the Postischemic Rat Brain Increases in Brain Regions Resistant to Ischemia and Decreases in Vulnerable Areas

Abstract: Casein kinase II (CKII) is a protein kinase acting in the intracellular cascade of reactions activated by growth factor receptors, and that has a profound influence on cell proliferation and survival. In this investigation, we studied the changes in the activity and levels of CKII in the rat brain exposed to 10. 15 and 20 min of transient forebrain ischemia followed by variable periods of reperfusion. The cytosolic CKII activity decreased during reperfusion by ∼ 30 and ∼ 50% in the selectively vulnerable areas, striatum and the CA1 region of the hippocampus, respectively. In the resistant CA3 region of hippocampus and neocortex, the activity increased by ∼ 20 and ∼ 60%, respectively. The postischemic changes in CKII activity were dependent on the duration of the ischemic insult. The levels of CKII did not change after ischemia, suggesting that the enzyme is modulated by covalent modification or is interacting with an endogenous inhibitor/activator. Treatment of the cytosolic fraction from cortex of rats exposed to ischemia and 1 h of reperfusion with agarose-bound phosphatase decreased the activity of CKII to control levels, suggesting that CKII activation after ischemia involves a phosphorylation of the enzyme. The correlation between postischemic CKII activity and neuronal survival implies that preservation or activation of CKII activity may be important for neuronal survival after cerebral ischemia.     

Block of P2X7 receptors could partly reverse the delayed neuronal death in area CA1 of the hippocampus after transient global cerebral ischemia

Transient global ischemia (which closely resembles clinical situations such as cardiac arrest, near drowning or severe systemic hypotension during surgical procedures), often induces delayed neuronal death in the brain, especially in the hippocampal CA1 region. The mechanism of ischemia/reperfusion (I/R) injury is not fully understood. In this study, we have shown that the P2X7 receptor antagonist, BBG, reduced delayed neuronal death in the hippocampal CA1 region after I/R injury; P2X7 receptor expression levels increased before delayed neuronal death after I/R injury; inhibition of the P2X7 receptor reduced I/R-induced microglial microvesicle-like components, IL-1β expression, P38 phosphorylation, and glial activation in hippocampal CA1 region after I/R injury. These results indicate that antagonism of the P2X7 receptor and signaling pathways of microglial MV shedding, such as src-protein tyrosine kinase, P38 MAP kinase and A-SMase, might be a promising therapeutic strategy for clinical treatment of transient global cerebral I/R injury.     

Akt inhibits MLK3/JNK3 signaling by inactivating Rac1: a protective mechanism against ischemic brain injury

The overall goal of this study was to determine the molecular basis by which mixed-lineage kinase 3 (MLK3) kinase and its signaling pathways are negatively regulated by the pro-survival Akt pathway in cerebral ischemia. We demonstrated that tyrosine phosphorylation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) underlies the increased Akt-Ser473 phosphorylation by orthovanadate. Co-immunoprecipitation analysis revealed that endogenous Akt physically interacts with Rac1 in the hippocampal CA1 region, and this interaction is promoted on tyrosine phosphatase inhibition. The elevated Akt activation can deactivate MLK3 by phosphorylation at the Ser71 residue of Rac1, a small Rho family of guanidine triphosphatases required for MLK3 autophosphorylation. Subsequently, inhibition of c-Jun N-terminal kinase 3 (JNK3) results in decreased serine phosphorylation of 14-3-3, a cytoplasmic anchor of Bax, and prevents ischemia-induced mitochondrial translocation of Bax, release of cytochrome c and activation of caspase 3. At the same time, the expression of Fas-ligand decreases in the CA1 region after inhibition of c-Jun activation. The neuroprotective effect of Akt activation is significant in the CA1 region after global cerebral ischemia. Our results suggest that the activation of the pro-apoptotic MLK3/JNK3 cascade induced by ischemic stress can be suppressed through activation of the anti-apoptotic phosphatidylinositol 3-kinase/Akt pathway, which provides a direct link between Akt and the family of stress-activated kinases.     

GDNF Selectively Induces Microglial Activation and Neuronal Survival in CA1/CA3 Hippocampal Regions Exposed to NMDA Insult through Ret/ERK Signalling

The glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for several neuronal populations in different brain regions, including the hippocampus. However, no information is available on the: (1) hippocampal subregions involved in the GDNF-neuroprotective actions upon excitotoxicity, (2) identity of GDNF-responsive hippocampal cells, (3) transduction pathways involved in the GDNF-mediated neuroprotection in the hippocampus. We addressed these questions in organotypic hippocampal slices exposed to GDNF in presence of N-methyl-D-aspartate (NMDA) by immunoblotting, immunohistochemistry, and confocal analysis. In hippocampal slices GDNF acts through the activation of the tyrosine kinase receptor, Ret, without involving the NCAM-mediated pathway. Both Ret and ERK phosphorylation mainly occurred in the CA3 region where the two activated proteins co-localized. GDNF protected in a greater extent CA3 rather than CA1 following NMDA exposure. This neuroprotective effect targeted preferentially neurons, as assessed by NeuN staining. GDNF neuroprotection was associated with a significant increase of Ret phosphorylation in both CA3 and CA1. Interestingly, confocal images revealed that upon NMDA exposure, Ret activation occurred in microglial cells in the CA3 and CA1 following GDNF exposure. Collectively, this study shows that CA3 and CA1 hippocampal regions are highly responsive to GDNF-induced Ret activation and neuroprotection, and suggest that, upon excitotoxicity, such neuroprotection involves a GDNF modulation of microglial cell activity.     

Hyperphosphorylation at serine 199/202 of tau factor in the gerbil hippocampus after transient forebrain ischemia

We examined the phosphorylation state of tau factor in hippocampal delayed neuronal death (DND) after transient forebrain ischemia. A transient phosphorylation increase at serine 199/202 but not serine 396 of tau factor after transient ischemia was clearly observed. Intraventricular injections of olomoucine and U-0126 (CDK5 and MAP kinase inhibitors, respectively) inhibited hyperphosphorylation. In contrast, wortmannin (PI3 kinase inhibitor) increased phosphorylation at serine 199/202 and corresponded with an increase in GSK3 phosphorylation. Our findings suggest that CDK5, MAP kinase, and GSK3 phosphorylate these sites after ischemia. We prepared recombinant normal human tau (N-Tau40) with TAT-HA protein and dephosphorylated-form human Tau-40 (D-tau40) in which 199/202 serines were changed to alanine by site-directed mutagenesis. Intraventricularly injected D-tau40 protected somewhat against DND while N-Tau40 did not. These data suggest that hyperphosphorylation at serine 199/202 of tau factor is induced by MAP kinase, CDK5, and GSK3, and contributes to ischemic neuronal injury.     

Identification of the mitogen-activated protein kinase phosphorylation sites on human Sos1 that regulate interaction with Grb2.

The Son of sevenless proteins (Sos) are guanine nucleotide exchange factors involved in the activation of Ras by cytoplasmic and receptor tyrosine kinases. Growth factor stimulation rapidly induces the phosphorylation of Sos on multiple serine and threonine sites. Previous studies have demonstrated that growth factor-induced Sos phosphorylation occurs at the C-terminal region of the protein and is mediated, in part, by mitogen-activated protein (MAP) kinase. In this report, we describe the identification of five MAP kinase sites (S-1137, S-1167, S-1178, S-1193, and S-1197) on hSos1. We demonstrate that four of these sites, S-1132, S-1167, S-1178, and S-1193, become phosphorylated following growth factor stimulation. The MAP kinase phosphorylation sites are clustered within a region encompassing three proline-rich SH3-binding sites in the C-terminal domain of hSos1. Replacing the MAP kinase phosphorylation sites with alanine residues results in an increase in the binding affinity of Grb2 to hSos1. Interestingly, hSos2 contains only one MAP kinase phosphorylation site and, as demonstrated previously, has an increased affinity toward Grb2 compared with hSos1. These results suggest a role for MAP kinase in the regulation of Grb2-Sos interactions. Since the binding of Grb2 is important for Sos function, the phosphorylation-dependent modulation of Grb2-Sos association may provide a means of controlling Ras activation.     

High expression of GLT-1 in hippocampal CA3 and dentate gyrus subfields contributes to their inherent resistance to ischemia in rats

It is well known that neurons in the CA3 and dentate gyrus (DG) subfields of the hippocampus are resistant to short period of ischemia which is usually lethal to pyramidal neurons in hippocampal CA1 subfield. The present study was undertaken to clarify whether the inherent higher resistance of neurons in CA3 and DG to ischemia is associated with glial glutamate transporter-1 (GLT-1) in rats. Western blot analysis and immunohistochemistry assay showed that the basal expressions of GLT-1 in both CA3 and DG were much higher than that in CA1 subfield. Mild global brain ischemia for 8 min induced delayed death of almost all CA1 pyramidal neurons and marked GLT-1 down-regulation in the CA1 subfield, but it was not lethal to the neurons in either CA3 or DG and induced GLT-1 up-regulation and astrocyte activation showed normal soma and aplenty slender processes in the both areas. When the global brain ischemia was prolonged to 25 min, neuronal death was clearly observed in CA3 and DG accompanied with down-regulation of GLT-1 expression and abnormal astrocytes represented with hypertrophic somas, but shortened processes. After down-regulating of GLT-1 expression and function by its antisense oligodeoxynucleotides or inhibiting GLT-1 function by dihydrokainate, an inhibitor of GLT-1, the mild global brain ischemia for 8 min, which usually was not lethal to CA3 and DG neurons, induced the neuronal death in CA3 and DG subfields. Taken together, the higher expression of GLT-1 in the CA3 and DG contributes to their inherent resistance to ischemia.     

Time-Resolved Signaling Pathways of Nerve Growth Factor Diverge Downstream of the p140trk Receptor Activation Between Chick Sympathetic and Dorsal Root Ganglion Sensory Neurons

Abstract: We have recently shown that the small GTP binding protein p21 ras is essential for nerve growth factor (NGF)-mediated survival of peripheral embryonic chick dorsal root ganglia (DRG) sensory but not sympathetic neurons. To investigate at which level of the signaling cascade the pathways diverge, we have studied the time-resolved pattern of NGF-stimulated tyrosine phosphorylation of proteins within 4 h after addition of the neurotrophin. In both chick sympathetic neurons [embryonic day (E) 12] and DRG sensory neurons (E9) NGF induces within 1 min the autophosphorylation of the receptor tyrosine kinase p140trk. However, the pattern of substrate protein tyrosine phosphorylation downstream of p140trk is distinctly different in both neuronal subtypes. In sympathetic neurons, we observe within 1 min the tyrosine phosphorylation of a new substrate protein, p105, reaching maximal levels at 3 min. Tyrosine phosphorylation of p105 remains elevated for up to 4 h. Subsequent to p105, NGF induces the tyrosine phosphorylation of p42, a protein belonging to the family of mitogen-activated protein (MAP) kinases. This stimulation is transient, reaching maximal levels at 10 min and returning to very low levels already after 2 h. In DRG sensory neurons, tyrosine phosphorylation of p105 is weak and very short lived, disappearing already after treatment with NGF for 10 min. In contrast, activation of MAP kinase p42 in DRG sensory neurons is more stable than in sympathetic neurons. All NGF-stimulated tyrosine phosphorylation events were inhibited by preincubation of neurons with the tropomyosin-related kinase (trk) inhibitor K252a. We suggest the working hypothesis that persistent tyrosine phosphorylation of p105 may play a role in the p21ras-independent NGF survival pathway of chick sympathetic neurons.     

利多卡因和硫喷妥钠对培养的大鼠海马脑片神经元损伤的影响

目的 :观察利多卡因和硫喷妥钠对生后 2 2d大鼠培养海马脑片的实验型缺血后神经元损伤的影响。方法 :将培养的SD大鼠海马脑片实验型缺血 (缺氧缺糖 ) 1 0min ,给药组在缺血前 1 0min给予 1 0nmol/L、1 0 0nmol/L的利多卡因或 2 50nmol/L、60 0nmol/L的硫喷妥钠 ,缺血后换用正常培养基继续培养 7d ,并用荧光染料PropidiumIo dide(PI)连续观察海马CA1区和齿状回神经元的损伤。结果 :缺血后第 1d缺血组即出现神经元损伤高峰 ,CA1区和齿状回的PI指数显著增加 (P <0 .0 1 ) ;直至缺血后第 7d其损伤指数仍显著高于缺血前水平 (P <0 .0 1 )。两浓度的利多卡因和硫喷妥钠均可降低缺血后CA1区和齿状回神经元损伤的程度 (P <0 .0 1 ) ,并可将CA1区和齿状回的神经元损伤高峰推迟至缺血后第 3d。结论 :利多卡因和硫喷妥可减轻缺血后海马CA1区和齿状回的神经元损伤 ,推迟神经元的损伤高峰。     

Transient forebrain ischemia effects FAK-coupled signaling in gerbil hippocampus

Focal adhesion kinase (FAK) is thought to play a major role in transducing extracellular matrix (ECM)-derived survival signals into cells. The function of FAK is linked to its autophosphorylation at Tyr-397 and then recruitment of several effector molecules. Thus, modulation of FAK activity may affect several intracellular signaling pathways and may participate in a variety of pathological settings. In the present study, we investigated the effect of short-term 5 min forebrain ischemia on levels and Tyr-397 phosphorylation of focal adhesion kinase and the interaction of this enzyme with Src protein tyrosine kinase and adapter protein p130Cas, involved in FAK-mediated signaling pathway in gerbil hippocampus. The total amount of focal adhesion kinase as well as its Tyr-397 phosphorylation declined substantially between 24 and 48 h after the insult, particularly in CA1 region of hippocampus. Concomitantly, a decreased amount of FAK/Src kinase complex has been observed. These data indicate that inhibition of FAK/Src-coupled signaling pathway may participate in the ischemia-induced neuronal degeneration in gerbil hippocampus. The temporal profile of FAK down-regulation in CA1 area coincides with metalloproteinases (MMPs) activation. These results suggest that extracellular proteolysis might belong to the mechanisms which govern the FAK-coupled pathway in ischemic hippocampus.     

Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival.

Adhesion of human primary skin fibroblasts and ECV304 endothelial cells to immobilized matrix proteins, beta1 or alphav integrin antibodies stimulates tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. This tyrosine phosphorylation is transiently induced, reaching maximal levels 30 min after adhesion, and it occurs in the absence of receptor ligands. Similar results were observed with EGF receptor-transfected NIH-3T3 cells. Use of a kinase-negative EGF receptor mutant demonstrates that the integrin-stimulated tyrosine phosphorylation is due to activation of the receptor's intrinsic kinase activity. Integrin-mediated EGF receptor activation leads to Erk-1/MAP kinase induction, as shown by treatment with the specific inhibitor tyrphostin AG1478 and by expression of a dominant-negative EGF receptor mutant. EGF receptor and Erk-1/MAP kinase activation by integrins does not lead per se to cell proliferation, but is important for entry into S phase in response to EGF or serum. EGF receptor activation is also required for extracellular matrix-mediated cell survival. Adhesion-dependent MAP kinase activation and survival are regulated through EGF receptor activation in cells expressing this molecule above a threshold level (5x10(3) receptors per cell). These results demonstrate that integrin-dependent EGF receptor activation is a novel signaling mechanism involved in cell survival and proliferation in response to extracellular matrix.     

Preconditioning-induced activation of ERK5 is dependent on moderate Ca2+ influx via NMDA receptors and contributes to ischemic tolerance in the hippocampal CA1 region of rats

Accumulating evidence implicates activation (phosphorylation) of mitogen-activated protein kinases (MAPK) during nonlethal ischemic preconditioning in the protection of hippocampal CA1 neuron against subsequent ischemic events. In this paper, we undertook to identify the role of extracellular signal regulated kinase (ERK) 5 in cerebral ischemic preconditioning (CIP). Three minutes of ischemia was induced as preconditioning stimulus. Three days later, 6 min of ischemia was induced. The levels of ERK5 protein expression and its activation were detected with or without the CIP in hippocampal CA1 and the dentate gyrus (DG) regions. Our results showed that ERK5 was activated selectively in hippocampal CA1 region with, but not without, the ischemic preconditioning. Notably, during the later phase of reperfusion, the rise in ERK5 activation was strong and persistent with a peak occurring at the third day. The activation peak was effectively prevented and ERK5 protein expression was significantly decreased by intracerebroventricular infusion of ERK5 antisense oligonucleotide (every 24 h for 3 days before the preconditioning), but not by sense oligonucleotide or vehicle. Subsequently, the CA1 neuronal loss was largely elevated. Moreover, both MK801 (10 microM), an antagonist of NMDA receptor, and EGTA (100 mM, but neither 50 nor 150 mM), an extracellular Ca2+ chelator, not only effectively inhibited the ERK5 activation but also markedly abolished CIP-induced survival of the CA1 neurons. These results suggested that activation of the ERK5 pathway by CIP was at least partly dependent on moderate Ca2+ influx via NMDA receptor, which might contribute to ischemic tolerance in hippocampal CA1 region of rats.     

Nicotine-induced phosphorylation of Akt through epidermal growth factor receptor and Src in PC12h cells

Nicotine treatment triggers calcium influx into neuronal cells, which promotes cell survival in a number of neuronal cells. Phosphoinositide (PI) 3-kinase and downstream PI3-kinase target Akt have been reported to be important in the calcium-mediated promotion of survival in a wide variety of cells. We investigated the mechanisms of nicotine-induced phosphorylation of Akt in PC12h cells, in comparison with nicotine-induced ERK phosphorylation. Nicotine induced Akt phosphorylation in a dose-dependent manner. A nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitor had no significant effect on nicotine-induced Akt phosphorylation, while a non-selective nAChR antagonist inhibited the phosphorylation. L-type voltage-sensitive calcium channel (VSCC) antagonists, calmodulin antagonist, and Ca2+/calmudulin-dependent protein kinase (CaM kinase) inhibitor prevented the nicotine-induced Akt phosphorylation. Three epidermal growth factor receptor (EGFR) inhibitors prevented the nicotine-induced phosphorylation of both extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and Akt. In contrast, an inhibitor of the Src family tyrosine kinase prevented the nicotine-induced Akt phosphorylation but not ERK phosphorylation. These results suggested that nicotine induces the activation of both PI3-kinase/Akt and ERK pathways via common pathways including non-alpha7-nAChRs, L-type VSCC, CaM kinase II and EGFR in PC12h cells, but Src family tyrosine kinases only participate in the pathway to activate Akt.     

Differential response to ischemia in adjacent hippocampalsectors: neuronal death in CA1 versus neurogenesis in dentate gyrus

Two hippocampal sectors show distinct responses to transient ischemia: the cornu Ammonis (CA)1 sector undergoes a delayed neuronal death followed by a lack of neuronal generation, while the dentate gyrus (DG) shows slight postischemic damage followed by an increased neurogenesis. Using the monkey experimental paradigm of transient whole brain global ischemia, the 'calpain-cathepsin hypothesis' was formulated in 1998. This hypothesis proposes that following ischemia calpain compromises the integrity of lysosomal membrane, causing a leakage of degrading hydrolytic enzymes--cathepsins--into the cytoplasm. Ischemia induces Ca(2+) mobilization, calpain activation, lysosomal membrane disruption, and cathepsin release, which all occur specifically in the CA1 sector and cause neuronal death. In the postischemic DG, a vascular niche has been implicated in adult neurogenesis, in that adventitial cells of the DG microvascular environment provoke postischemic up-reguation of neurogenesis with the aid of brain-derived neurotrophic factor and polysialylated form of the neural cell adhesion molecule. In parallel, Down's syndrome cell adhesion molecule has recently been shown to be expressed specifically in the neural progenitor cells of DG. In this review, we focus on the monkey experimental paradigm to reveal the remarkable contrasts between CA1 and DG in response to the ischemic insult.     

Transient ischemia enhances tyrosine phosphorylation and binding of the NMDA receptor to the Src homology 2 domain of phosphatidylinositol 3-kinase in the rat hippocampus

Tyrosine phosphorylation of the NMDA receptor has been implicated in the regulation of the receptor channel. We investigated the effects of transient (15 min) global ischemia on tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B, and the interaction of NR2 subunits with the SH2 domain of phosphatidylinositol 3-kinase (PI3-kinase) in vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. Transient ischemia induced a marked increase in the tyrosine phosphorylation of NR2A in both regions. The tyrosine phosphorylation of NR2B in CA3/dentate gyrus after transient ischemia was sustained and greater than that in CA1. PI3-kinase p85 was co-precipitated with NR2B after transient global ischemia. The SH2 domain of the p85 subunit of PI3-kinase bound to NR2B, but not to NR2A. Binding to NR2B was increased following ischemia and the increase in binding in CA3/dentate gyrus (4.5-fold relative to sham) was greater than in CA1 (1.7-fold relative to sham) at 10 min of reperfusion. Prior incubation of proteins with an exogenous protein tyrosine phosphatase or with a phosphorylated peptide (pYAHM) prevented binding. The results suggest that sustained increases in tyrosine phosphorylation and increased interaction of NR2B with the SH2 domain of PI3-kinase may contribute to altered signal transduction in the CA3/dentate gyrus after transient ischemia.     

GluR2 protein synthesis and metabolism in rat hippocampus following transient ischemia and ischemic tolerance induction

In this study we have determined the metabolic half-life, protein synthesis and expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR2 in the hippocampus of the living rat. Synthesized proteins were pulse labeled in vivo using intracarotid infusion or intrahippocampal injection of L-[(35)S] labeled amino acids, and the GluR2 protein immunoprecipitated in order to measure the tracer incorporation at different survival time-points. A limited time course study suggested a metabolic half-life of 144 and 108 h in the CA1 region in control animals following carotid artery infusion and intrahippocampal injection, respectively. Twenty-four hours following a moderate ischemic insult, GluR2 protein synthesis was decreased significantly in both the CA1 and DG/CA3 region, whereas the total protein synthesis was decreased significantly only in the CA1 region. Twenty-four hours following ischemic tolerance induction, a significant increase in GluR2 expression was found in the CA1 region using quantitative Western blotting, while no change was found in the dentate gyrus (DG)/CA3 or in expression of GluR1 protein. Data from labeling experiments did not reveal the reason for the increased amount of GluR2 in the CA1 region of the tolerant animals. This study shows that following global ischemia the GluR2 synthesis is decreased both in the CA1 and DG/CA3, which, together with the found GluR2 metabolic half-life, contradict a selective loss of GluR2 protein as a triggering mechanism for the delayed CA1 pyramidal cell death. Twenty-four hours following tolerance induction, we found an increased GluR2 expression in the CA1 region, suggesting that GluR2 plays a role in the acquisition of ischemic tolerance. Our study suggests the ability of neurons to regulate the AMPA receptor subunit expression through changes in protein synthesis and stability.     

Selenazoles (selenium compounds) facilitate survival of cultured rat pheochromocytoma PC12 cells after serum-deprivation and stimulate their neuronal differentiation via activation of Akt and mitogen-activated protein kinase, respectively

The activation of extracellular receptor kinase (ERK) is one of the checkpoints to assess the activation of the classical Ras/mitogen-activated protein kinase (MAPK) cascade. Therefore, we tested more than 100 selenium-containing compounds for their ability to activate the MAPK signal pathway. Among them, we found that three selenazoles, 5-chloroacetyl-2-piperidino-1,3-selenazole (CS1), 5-chloroacetyl-2-morpholino-1,3-selenazole (CS2), and 5-chloroacetyl-2-dimethylamino-1,3-selenazole (CS3), induced the phosphorylation of ERK. These compounds also enhanced the phosphorylation of Akt, a signal transducing protein kinase for cell survival; and this phosphorylation was followed by suppression of cell death, thus suggesting that they had anti-apoptotic effects. Moreover, CSs 1-3 induced neurite outgrowth and facilitated the expression of neurofilament-M of PC12 cells, demonstrating that they induced neuronal differentiation of these cells. On the other hand, the CS-induced phosphorylation of MAPK was enhanced by buthionine sulfoximine (BSO), an activator of protein tyrosine phosphatases (PTPs), but inhibited by N-acetyl-l-cysteine (NAC), an inhibitor of receptor tyrosine kinase. These results imply that activation of some receptor tyrosine kinase(s) is involved in the mechanism of action of CSs 1-3. The activation of MAPK by CSs 1-3 was suppressed by U0126, a MEK inhibitor, but not by K252a, an inhibitor of TrkA; AG1478, an antagonist of epidermal growth factor receptor (EGFR); or by pertussis toxin. These results demonstrate that the CS-induced phosphorylation of Akt and MAP kinase (receptor tyrosine kinase(s)-MEK1/2-ERK1/2) cascades was responsible for suppression of apoptosis and facilitation of neuronal differentiation of PC12 cells, respectively. Our results suggest that CSs 1-3 are promising candidates as neuroprotective and/or neurotrophic agents for the treatment of various neurodegenerative neurological disorders.