Quan Guang Zhang

The neuroprotection of insulin on ischemic brain injury in rat hippocampus through negative regulation of JNK signaling pathway by PI3K/Akt activation.

Current studies demonstrated that cell survival is determined by a balance among signaling cascades, including those that recruit the Akt and JNK pathways. In our present work, the relationship between Akt1 and JNK1/2 was evaluated after cerebral ischemia-reperfusion in the hippocampus in a four-vessel occlusion model of Sprague-Dawley rats. This paper was based on our present and previous studies. Firstly, Akt1 had one active peak during reperfusion following 15 min ischemia. Secondly, two peaks of JNK1/2 activation occurred during reperfusion, respectively. Thirdly, the phosphorylation of JNK substrates c-Jun and Bcl-2, and the activation of a key protease of caspase-3 were detected. They only had one active peak, respectively, during reperfusion. To clarify the mechanism of Akt1 activation and further define whether JNK1/2 activation could be regulated by Akt1 through PI3K pathway, LY294002 and insulin were, respectively, administrated to the rats prior to ischemia. Our research indicated that LY294002, a PI3K inhibitor, significantly suppressed Akt1 activation. Furthermore, LY294002 significantly strengthened both peaks of JNK1/2 activation, c-Jun activation, Bcl-2 phosphorylation, and the activation of caspase-3 during reperfusion. In contrast, insulin, a PI3K agonist, not only obviously activated Akt1 during early and later reperfusion, but also inhibited phosphorylation of JNK1/2, c-Jun, and Bcl-2 and attenuated the activation of caspase-3. In addition, pretreatment of insulin significantly increased the number of the surviving CA1 pyramidal cells at 5 days of reperfusion. Consequently, our results indicated that the cross-talk between Akt1 and JNK1/2 could be mediated by insulin receptor through PI3K in rat hippocampus during reperfusion. This signaling pathway might play a neuroprotective role against ischemic insults via inhibition of the JNK pathway, involving the death effector of caspase-3.

Critical role of PTEN in the coupling between PI3K/Akt and JNK1/2 signaling in ischemic brain injury.

JNK pathway is an important pro‐apoptotic kinase cascade mediating cell death in response to a variety of extracellular stimuli including excitotoxicity, which results in selective and delayed neuronal death in the hippocampal CA1. On the contrary, activation of the protein kinase Akt, which is controlled by the opposing actions of PI3K and PTEN, contributes to enhanced resistance to apoptosis through multiple mechanisms. We here demonstrate that the temporal pattern of Akt activation reversely correlates with JNK1/2 activation following various time points of ischemic reperfusion. However, the activation of JNK1/2 could be decreased by the elevation of Akt activation via increasing the tyrosine phosphorylation of PTEN by bpv(pic), a potent PTPases inhibitor for PTEN, or by intracerebroventricular infusion of PTEN antisense oligodeoxynucleotides (AS‐ODNs). In contrast, JNK1/2 activation was significantly increased by preventing PTEN degradation after pretreatment with proteasome inhibitor. The neuroprotective effects of bpv(pic) and PTEN AS‐ODNs were significant in the CA1 subfield after transient global ischemia. In conclusion, the present results clearly show that PTEN plays a key regulatory role in the cross‐talk between cell survival PI3K/Akt pathway and pro‐death JNK pathway, and raise a new possibility that agents targeting phosphatase PTEN may offer a great promise to expand the therapeutic options in protecting neurons form ischemic brain damage.

Inhibition of MLK3-MKK4/7-JNK1/2 pathway by Akt1 in exogenous estrogen-induced neuroprotection against transient global cerebral ischemia by a non-genomic mechanism in male rats.

Numerous studies have demonstrated the neuroprotective effects of estrogen in experimental cerebral ischemia. To investigate molecular mechanisms of estrogen neuroprotection in global ischemia, immunoblotting, immunohistochemistry and Nissel‐staining analysis were used. Our results showed that chronic pretreatment with β‐estradiol 3‐benzoate (E2) enhanced Akt1 activation and reduced the activation of mixed‐lineage kinase 3 (MLK3), mitogen‐activated protein kinase kinase 4/7 (MKK4/7), and c‐Jun N‐terminal kinase 1/2 (JNK1/2) in the hippocampal CA1 subfield during reperfusion after 15 min of global ischemia. In addition, E2 reduced downstream JNK nuclear and non‐nuclear components, c‐Jun and Bcl‐2 phosphorylation and Fas ligand protein expression induced by ischemia/reperfusion. Administration of phosphoinositide 3‐kinase (PI3K) inhibitor LY 294,002 prevented both activation of Akt1 and inhibition of MLK3, MKK4/7 and JNK1/2. The interaction between ERα and the p85 subunit of PI3K was also examined. E2 and antiestrogen ICI 182,780 promoted and prevented this interaction, respectively. Furthermore, ICI 182,780 blocked both the activation of Akt1 and the inhibition of MLK3, MKK4/7 and JNK1/2. Photomicrographs of cresyl violet‐stained brain sections showed that E2 reduced CA1 neuron loss after 5 days of reperfusion, which was abolished by ICI 182,780 and LY 294,002. Our data indicate that in response to estrogen, ERα interacts with PI3K to activate Akt1, which may inhibit the MLK3‐MKK4/7‐JNK1/2 pathway to protect hippocampal CA1 neurons against global cerebral ischemia in male rats.

Neuroprotective effects of preconditioning ischaemia on ischaemic brain injury through inhibition of mixed-lineage kinase 3 via NMDA receptor-mediated Akt1 activation

A number of works show that the mitogen‐activated protein kinase (MAPK) signalling pathway responds actively in cerebral ischaemia and reperfusion. We undertook our present studies to clarify the role of mixed‐lineage kinase 3 (MLK3), a MAPK kinase kinase (MAPKKK) in MAPK cascades, in global ischaemia and ischaemic tolerance. The mechanism concerning NMDA receptor‐mediated Akt1 activation underlying ischaemic tolerance, was also investigated. Sprague–Dawley rats were subjected to 6 min of ischaemia and differing times of reperfusion. Our results showed MLK3 was activated in the hippocampal CA1 region with two peaks occurring at 30 min and 6 h, respectively. This activation returned to base level 3 days later. Both preconditioning with 3 min of sublethal ischaemia and NMDA pretreatment inhibited the 6‐h peak of activation. However, pretreatment of ketamine before preconditioning reversed the inhibiting effect of preconditioning on MLK3 activation at 6 h of reperfusion. In the case of Akt1, however, preconditioning and NMDA pretreatment enhanced Akt1 activation at 10 min of reperfusion. Furthermore, ketamine pretreatment reversed preconditioning‐induced increase of Akt1 activation. We also noted that pretreatment of LY294002 before preconditioning reversed both the inhibition of MLK3 activation at 6 h of reperfusion and the increase in Akt1 activation at 10 min of reperfusion. The above‐mentioned results lead us to conclude that, in the hippocampal CA1 region, preconditioning inhibits MLK3 activation after lethal ischaemia and reperfusion and, furthermore, this effect is mediated by Akt1 activation through NMDA receptor stimulation.

Activation of c-Jun NH2-terminal kinase 3 is mediated by the GluR6.PSD-95.MLK3 signaling module following cerebral ischemia in rat hippocampus.

Kainate receptor glutamate receptor 6 (GluR6) binds to the postsynaptic density protein 95 (PSD-95), which in turn anchors mixed lineage kinase 3 (MLK3) via SH3 domain in rat brain tissue. MLK3 subsequently activates c-Jun NH(2)-terminal kinase (JNK) via MAP kinase kinases (MKKs). We investigated the association of PSD-95 with GluR6 and MLK3, MLK3 autophosphorylation, the interaction of MLK3 with JNK3, and JNK3 phosphorylation following cerebral ischemia in rat hippocampus. Our results indicate that the GluR6.PSD-95.MLK3 complex peaked at 6 h of reperfusion. Furthermore, MLK3 autophosphorylation and the interaction of MLK3 with JNK3 occurred with the alteration of GluR6.PSD-95.MLK3 signaling module. To further prove whether JNK3 activation in ischemic hippocampus is mediated by GluR6.PSD-95.MLK3 signaling pathway, the AMPA/KA receptor antagonist 6,7-dinitroquinoxaline-2, (1H, 4H)-dione (DNQX), the GluR6 antagonist 6,7,8,9-Tetrahydro-5-nitro-1H-benz[g]indole-2,3-dione-3-oxime (NS102), the AMPA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzo diazepine (GYKI52466), and the NMDA receptor antagonist ketamine were given to the rats 20 min prior to ischemia. Our findings indicate that both DNQX and NS102 significantly attenuated the association of PSD-95 with GluR6 and MLK3, MLK3 autophosphorylation, interaction of MLK3 with JNK3, and JNK3 phosphorylation, while GYKI52466 and ketamine had no effect. Moreover, administration of NS102 before cerebral ischemia significantly increased the number of the surviving hippocampal CA1 pyramidal cells at 5 days of reperfusion. Consequently, GluR6, one subunit of kainate receptor, plays a critical role in inducing JNK3 activation after ischemic injury.

Biphasic activation of apoptosis signal-regulating kinase 1-stress-activated protein kinase 1-c-Jun N-terminal protein kinase pathway is selectively mediated by Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors involving oxidative stress following brain ischemia in rat hippocampus

Stress-activated protein kinase/extracellular signal-regulated kinase-1 (SEK1/MKK4) was examined in a rat model of global brain ischemia. Western blot assay showed that SEK1 activation was biphasic in CA1 but not CA3/dentate gyrus. The second activation peak (3 days after ischemia) was prevented by pretreatment with l-naphthyl acetyl spermine (Naspm), a channel blocker of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, or N-acetylcysteine (NAC), a free radical scavenger. Concomitantly, the late activation of apoptosis signal-regulating kinase 1 (ASK1) and c-Jun N-terminal protein kinase (JNK) was also prevented by Naspm or NAC. Moreover, phospho-SEK1 and phospho-JNK co-immunoprecipitated with ASK1 and the bindings peaked at 3 days of reperfusion. Together with previous results, these findings indicate that Ca(2+)-permeable AMPA receptors are important routes to mediate the late activation of ASK1-SEK1-JNK pathway involving oxidative stress in hippocampal CA1 region after ischemia.

Antioxidant N-acetylcysteine and AMPA/KA receptor antagonist DNQX inhibited mixed lineage kinase-3 activation following cerebral ischemia in rat hippocampus

We measured the MLK3 expression, activity and backphosphorylation following cerebral ischemia. Our data showed that MLK3 protein levels were unalterable during ischemia and reperfusion. However, during ischemia MLK3 activity gradually increased and reached its peak at 30 min of ischemia. While its backphosphorylation reduced from 5 min of ischemia to 30 min of ischemia. In addition, we also detected MLK3 alteration at various time points of reperfusion after 15 min of ischemia, which showed that MLK3 activity increased twice, whereas MLK3 backphosphorylation was similarly consistent with its activity during reperfusion. To further analyze the reason of MLK3 activation, antioxidant N-acetylcysteine (NAC) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA)/kainate (KA) receptor antagonist 6,7-dinitroquinoxaline-2,3(1H, 4H)-dione (DNQX) were given to the rats 20 min prior to ischemia. The results illustrated that NAC preferably inhibited the MLK3 activation during the ischemia and the early reperfusion, whereas DNQX effectively attenuated the MLK3 activation of the late reperfusion. We think that MLK3 activation is certainly associated with reactive oxygen species (ROS) and AMPA/KA receptor in response to ischemic insult.

Autophosphorylated calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) reversibly targets to and phosphorylates N-methyl-d-aspartate receptor subunit 2B (NR2B) in cerebral ischemia and reperfusion in hippocampus of rats

It has been reported that cerebral ischemia induces Thr286 autophosphorylation and translocation of CaMKIIalpha which targets to and phosphorylates NR2B in hippocampus of rats [Neuroscience 96 (2000) 665; J. Biol. Chem. 275 (2000) 23798]. To further illustrate the mechanisms underlying these processes, we examined the effects of ketamine (a selective antagonist of NMDA receptor), KN-62 (1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, a selective inhibitor of CaMKII) and reperfusion on CaMKII and NMDA receptors and the interactions between these signal proteins. Firstly, our results showed that ketamine decreased the ischemia-induced autophosphorylation, translocation and the targeting of CaMKIIalpha to NR2B and the serine-phosphorylation of NR2B. Secondly, KN-62 also inhibited the autophosphorylation of CaMKIIalpha, NR2B serine-phosphorylation and the binding of CaMKIIalpha to NR2B but had no effect on the translocation of CaMKII. These data strongly suggest that NMDA receptor channels mediated the Ca(2+)-dependent activation of CaMKII and NMDA receptors surely were the substrates on membranes of active CaMKII. Thirdly, our results indicated the concomitant phosphorylation and dephosphorylation of CaMKII and NR2B following ischemia or longer reperfusion. Moreover, the dissociation of CaMKII from NR2B had the same trend as that of the return of CaMKII to cytosol. All these data imply the close relationships between CaMKII and NR2B during ischemia and reperfusion, namely, CaMKII might act as an amplifier of detrimental cellular calcium signal regulated by NMDA receptors when becoming autophosphorylated and targeting to NR2B; conversely, autophosphorylated CaMKII could modulate NMDA receptor channel properties by phosphorylating NR2B.

Co-activation of GABA receptors inhibits the JNK3 apoptotic pathway via the disassembly of the GluR6-PSD95-MLK3 signaling module in cerebral ischemic-reperfusion

In this study, we investigated whether the increase of inhibitory γ‐amino butyric acid (GABA) signal suppresses the excitatory glutamate signal induced by cerebral ischemia and the underlying mechanisms. In global cerebral ischemia, focal cerebral ischemia and oxygen‐glucose deprivation, application of muscimol and baclofen, agonists of GABA(A) receptor and GABA(B) receptor, exerted neuroprotection. The agonists inhibited the increased assembly of the GluR6‐PSD‐95‐MLK3 module induced by cerebral ischemia and the activation of the MLK3‐MKK4/7‐JNK3 cascade. Our results suggest that stimulation of the inhibitory GABA receptors can attenuate the excitatory JNK3 apoptotic signaling pathway via inhibiting the increased assembly of the GluR6‐PSD‐95‐MLK3 signaling module in cerebral ischemia.

Free radicals are involved in continuous activation of nonreceptor tyrosine protein kinase c-Src after ischemia/reperfusion in rat hippocampus

NMDA receptor-mediated calcium over-loading and the following free radical generation play essential roles in cerebral ischemia lesion, leading to neuronal cell apoptosis or necrosis through complicated intracellular signaling cascades. Here we evaluated the effects of NMDA receptor antagonist (ketamine) and antioxidant (N-acetylcysteine) on ischemia- and reperfusion-induced activation of tyrosine kinase c-Src. The in vitro kinase assay showed that the ischemia-induced rapid activation of c-Src reached its peak at 5 min, and the reperfusion-induced continuous activation of c-Src reached another peak at 6 h reperfusion after 15 min ischemia (4.2 and 3.0 fold vs. sham control, respectively). Ketamine might suppress both peaks described above, but N-acetylcysteine, a free radical scavenger, was only able to partly reduce the peak activation elicited by 6 h reperfusion. These results suggest that free radical production is involved in NMDA receptor-mediated continuous activation of c-Src during ischemia/reperfusion but not that during ischemia.