Neuroprotection of GluK1 kainate receptor agonist ATPA against ischemic neuronal injury through inhibiting GluK2 kainate receptor-JNK3 pathway via GABA(A) receptors.

It is well known that GluK2-containing kainate receptors play essential roles in seizure and cerebral ischemia-induced neuronal death, while GluK1-containing kainate receptors could increase tonic inhibition of post-synaptic pyramidal neurons. This research investigated whether GluK1 could inhibit activation of c-Jun N-terminal kinase 3 (JNK3) signaling pathway mediated by the GluK2 in cerebral ischemia-reperfusion. The results show that GluK1 activation by (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) at 1nmol per rat could inhibit the assembly of GluK2·Postsynaptic density 95·mixed lineage kinase 3 signaling module, activation of JNK3 and its downstream signal molecules. However, the inhibition of ATPA could be prevented by GluK1 antagonist NS3763, GluK1 antisense, and GABA(A) receptor antagonist bicuculline. In addition, ATPA played a neuroprotective role against cerebral ischemia. In sum, the findings indicate that activation of GluK1 by ATPA at specific dosages may promote GABA release, which then suppresses post-synaptic GluK2-JNK3 signaling-mediated cerebral ischemic injury via GABA(A)R.

S-nitrosylation of c-Src via NMDAR-nNOS module promotes c-Src activation and NR2A phosphorylation in cerebral ischemia/reperfusion.

Previous studies suggested that activated c-Src promote the tyrosine phosphorylation of NMDA receptor subunit NR2A, and thus aggravate the injury induced by transient cerebral ischemia/reperfusion (I/R) in rat hippocampus CA1 region. In this study, we examined the effect of nitric oxide (NO) on the activation of c-Src and the tyrosine phosphorylation of NMDA receptor NR2A subunit. The results show that S-nitrosylation and the phosphorylation of c-Src were induced after cerebral I/R in rats, and administration of nNOS inhibitor 7-NI, nNOS antisense oligonucleotides and exogenous NO donor sodium nitroprusside diminished the increased S-nitrosylation and phosphorylation of c-Src during cerebral I/R. The cysteine residues of c-Src modified by S-nitrosylation are Cys489, Cys498, and Cys500. On the other hand, NMDAR antagonist MK-801 could attenuate the S-nitrosylation and activation of c-Src. Taken together, the S-nitrosylation of c-Src is provoked by NO derived from endogenous nNOS, which is activated by Ca(2+) influx from NMDA receptors, and promotes the auto-phosphorylation at tyrosines and further phosphorylates NR2A. The molecular mechanism we outlined here is a novel postsynaptic NMDAR-nNOS/c-Src-mediated signaling amplification, the 'NMDAR-nNOS â†’ NO â†’ SNO-c-Src â†’ p-c-Src â†’ NMDAR-nNOS' cycle, which presents the possibility as a potential therapeutic target for stroke treatment.

Knock-down of postsynaptic density protein 95 expression by antisense oligonucleotides protects against apoptosis-like cell death induced by oxygen-glucose deprivation in vitro.

OBJECTIVE: Postsynaptic density protein 95 (PSD-95) plays important roles in the regulation of glutamate signaling, such as that of N-methyl-D-aspartate receptors (NMDARs). In this study, the functional roles of PSD-95 in tyrosine phosphorylation of NMDAR subunit 2A (NR2A) and in apoptosis-like cell death induced by oxygen-glucose deprivation (OGD) in cultured rat cortical neurons were investigated. METHODS: We used immunoprecipitation and immunoblotting to detect PSD-95 protein level, tyrosine phosphorylation level of NR2A, and the interaction between PSD-95 and NR2A or Src. Apoptosis-like cells were observed by 4,6-diamidino-2-phenylindole staining. RESULTS: Tyrosine phosphorylation of NR2A and apoptosis-like cell death were increased after recovery following 60-min OGD. The increases were attenuated by pretreatment with antisense oligonucleotides against PSD-95 before OGD, but not by missense oligonucleotides or vehicle. PSD-95 antisense oligonucleotides also inhibited the increased interaction between PSD-95 and NR2A or Src, while NR2A expression did not change under this condition. CONCLUSION: PSD-95 may be involved in regulating NR2A tyrosine phosphorylation by Src kinase. Inhibition of PSD-95 expression can be neuroprotective against apoptosis-like cell death after recovery from OGD.

S-nitrosylation of mixed lineage kinase 3 contributes to its activation after cerebral ischemia.

Previous studies in our laboratory have shown that mixed lineage kinase 3 (MLK3) can be activated following global ischemia. In addition, other laboratories have reported that the activation of MLK3 may be linked to the accumulation of free radicals. However, the mechanism of MLK3 activation remains incompletely understood. We report here that MLK3, overexpressed in HEK293 cells, is S-nitrosylated (forming SNO-MLK3) via a reaction with S-nitrosoglutathione, an exogenous nitric oxide (NO) donor, at one critical cysteine residue (Cys-688). We further show that the S-nitrosylation of MLK3 contributes to its dimerization and activation. We also investigated whether the activation of MLK3 is associated with S-nitrosylation following rat brain ischemia/reperfusion. Our results show that the administration of 7-nitroindazole, an inhibitor of neuronal NO synthase (nNOS), or nNOS antisense oligodeoxynucleotides diminished the S-nitrosylation of MLK3 and inhibited its activation induced by cerebral ischemia/reperfusion. In contrast, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (an inhibitor of inducible NO synthase) or nNOS missense oligodeoxynucleotides did not affect the S-nitrosylation of MLK3. In addition, treatment with sodium nitroprusside (an exogenous NO donor) and S-nitrosoglutathione or MK801, an antagonist of the N-methyl-D-aspartate receptor, also diminished the S-nitrosylation and activation of MLK3 induced by cerebral ischemia/reperfusion. The activation of MLK3 facilitated its downstream protein kinase kinase 4/7 (MKK4/7)-JNK signaling module and both nuclear and non-nuclear apoptosis pathways. These data suggest that the activation of MLK3 during the early stages of ischemia/reperfusion is modulated by S-nitrosylation and provides a potential new approach for stroke therapy whereby the post-translational modification machinery is targeted.

N-methyl-D-aspartate receptor-dependent denitrosylation of neuronal nitric oxide synthase increase the enzyme activity.

Our laboratory once reported that neuronal nitric oxide synthase (nNOS) S-nitrosylation was decreased in rat hippocampus during cerebral ischemia-reperfusion, but the underlying mechanism was unclear. In this study, we show that nNOS activity is dynamically regulated by S-nitrosylation. We found that overexpressed nNOS in HEK293 (human embryonic kidney) cells could be S-nitrosylated by exogenous NO donor GSNO and which is associated with the enzyme activity decrease. Cys(331), one of the zinc-tetrathiolate cysteines, was identified as the key site of nNOS S-nitrosylation. In addition, we also found that nNOS is highly S-nitrosylated in resting rat hippocampal neurons and the enzyme undergos denitrosylation during the process of rat brain ischemia/reperfusion. Intrestingly, the process of nNOS denitrosylation is coupling with the decrease of nNOS phosphorylation at Ser(847), a site associated with nNOS activation. Further more, we document that nNOS denitrosylation could be suppressed by pretreatment of neurons with MK801, an antagonist of NMDAR, GSNO, EGTA, BAPTA, W-7, an inhibitor of calmodulin as well as TrxR1 antisense oligonucleotide (AS-ODN) respectively. Taken together, our data demonstrate that the denitrosylation of nNOS induced by calcium ion influx is a NMDAR-dependent process during the early stage of ischemia/reperfusion, which is majorly mediated by thioredoxin-1 (Trx1) system. nNOS dephosphorylation may be induced by the enzyme denitrosylation, which suggest that S-nitrosylation/denitrosylation of nNOS may be an important mechanism in regulating the enzyme activity.

Depolarization induces NR2A tyrosine phosphorylation and neuronal apoptosis.

BACKGROUND: Cytosol Ca2+ overload plays a vital role in ischemic neuronal damage, which is largely contributed by the Ca2+ influx through L-type voltage-gated calcium channels (L-VGCCs) and N-methyl-D-aspartate (NMDA) type glutamate receptors. In this article, L-VGCCs were activated by depolarization to investigate the cross-talk between NMDA receptors and L-VGCCs. METHODS: Depolarization was induced by 20 minutes incubation of 75 mM KCl in cultured rat cortical neuron. Apoptosis-like neuronal death was detected by DAPI staining. Tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A), interactions of Src and NR2A were detected by immunoblot and immunoprecipitation. RESULTS: Depolarization induced cortical neuron apoptosis-like cell death after 24 hours of restoration. The apoptosis was partially inhibited by 5 mM EGTA, 100 µM Cd2+, 10 µM nimodipine, 100 µM genistein, 20 µM MK-801, 2 µM PP2 and combined treatment of nimodipine and MK-801. NR2A tyrosine phosphorylation increased after depolarization, and the increase was inhibited by the drugs listed above. Moreover, non-receptor tyrosine kinase Src bound with NR2A after depolarization and restoration. The binding was also inhibited by the drugs listed above. CONCLUSIONS: The results indicated that depolarization-induced neuronal death might be due to extracellular Ca2+ influx through L-VGCCs and subsequently Src activationmediated NR2A tyrosine phosphorylation.

Overexpression of the PDZ1 domain of PSD-95 diminishes ischemic brain injury via inhibition of the GluR6.PSD-95.MLK3 pathway.

Recent studies have shown that kainate (KA) receptors are involved in neuronal cell death induced by seizure, which is mediated by the GluR6.PSD-95.MLK3 signaling module and subsequent JNK activation. In our previous studies, we demonstrated the neuroprotective role of a GluR6 c-terminus containing peptide against KA or cerebral ischemia-induced excitotoxicity in vitro and in vivo. Here, we first report that overexpression of the PDZ1 domain of PSD-95 protein exerts a protective role against neuronal death induced by cerebral ischemia-reperfusion in vivo and can prevent neuronal cell death induced by oxygen-glucose deprivation. Further studies show that overexpression of PDZ1 can perturb the interaction of GluR6 with PSD-95 and suppress the assembly of the GluR6.PSD-95.MLK3 signaling module and therefore inhibit JNK activation. Thus, it not only inhibits phosphorylation of c-Jun and down-regulates Fas ligand expression but also inhibits phosphorylation of 14-3-3 and decreases Bax translocation to mitochondria, decreases the release of cytochrome c, and decreases caspase-3 activation. Overall, the essential role of the PDZ1 domain of PSD-95 in apoptotic cell death in neurons provides an experimental foundation for gene therapy of neurodegenerative diseases with overexpression of the PDZ1 domain.

Overexpression of PDZ1 domain prevents apoptosis of rat hippocampal neurons induced by kainic acid.

In our previous studies, Tat-GluR6-9c (a glutamate receptor 6 C-terminus peptide fused the TAT protein transduction sequence) not only inhibited the activation of MLK3 (mixed lineage kinase 3) and JNK (c-Jun N-terminal kinase) via the GluR6.PSD-95 (postsynaptic density protein 95).MLK3 signaling module but also diminished neuronal death induced by kainic acid or transient cerebral ischemia in rat hippocampus. Here, we investigate whether overexpression of the PDZ1 domain of PSD-95 protein could suppress the binding of GluR6 with PSD-95 and the activation of MLK3, MKK7 (mitogen-activated kinase kinase 7) and JNK1/2, and rescused neuronal cell death induced by kainic acid. Our results showed that overexpression of the PDZ1 domain of PSD-95 protein could prevent nuclear accumulation and abrogate neuronal cell death in SD (Sprague-Dawley) rat hippocampal neuronal cells. Further studies indicated that overexpression of PDZ1 could inhibit the enhancement of binding of GluR6 to PSD-95 and prevent the activation of MLK3, MKK7 and JNK1/2 induced by kainic acid. Taken together, the essential role of the PDZ1 domain of PSD-95 in apoptotic cell death in neurons provides an experimental foundation for gene therapy of neurodegenerative diseases with overexpression of the PDZ1 domain.

Neuroprotection against transient focal cerebral ischemia and oxygen-glucose deprivation by interference with GluR6-PSD95 protein interaction.

Previous studies have shown that KA receptor subunit GluR6 mediated c-Jun N-terminal protein kinase (JNK) signaling is involved in global ischemia injury. Our present study indicates that focal ischemic brain insult on rat middle cerebral artery occlusion (MACo) model enhances the assembly of the GluR6-PSD95-MLK3 module and facilitates the phosphorylation of JNK. Most importantly, a peptide containing the TAT protein transduction sequence, Tat-GluR6-9c, can perturb the assembly of the GluR6-PSD95-MLK3 signaling module and suppress the activation of MLK3, MKK7/4 and JNK. As result, the inhibition of JNK activation caused by Tat-GluR6-9c diminishes the phosphorylation of the transcription factor c-Jun, down-regulates FasL expression and attenuates bax translocation, release of cytochrome c and the activation of caspase-3. Furthermore, MCAo induced infract volume is reduced by intracerebroventricular injection of Tat-Glur6-9c. Oxygen-glucose-deprivation (OGD) cultured cortical neuronal cell also shows an improved cell viability by application of Tat-GluR6-9c. Taken together, our findings strongly suggest that GluR6-PSD95-MLK3 signaling module mediated activation of nuclear and non-nuclear pathways of JNK activation are involved in focal ischemia injury and OGD. Tat-GluR6-9c, the peptide we constructed, gives a new insight into the therapy for ischemic stroke.

Different protection of K252a and N-acetyl-L-cysteine against amyloid-beta peptide-induced cortical neuron apoptosis involving inhibition of MLK3-MKK7-JNK3 signal cascades.

Amyloid-beta peptide (Abeta) has been implicated in the etiopathogenesis of Alzheimer's disease (AD). However, the molecular mechanisms underlying Abeta neurotoxicity remain to be elucidated. This study showed that Abeta treatment resulted in the increased phosphorylation (activation) of MLK3, MKK7, and JNK3 in cultured cortical neurons, which characterized as biphasic activation (first peaked at 1 hr and second peaked at 12 hr after Abeta treatment). K252a blocked Abeta-induced neuronal apoptosis, both early and late phases of MLK3-MKK7-JNK3 activation, as well as downstream signal events involving p-JNKs nuclear translocation, c-Jun phosphorylation, and Bad translocation to the mitochondria. The neuroprotective effect of K252a on Abeta-induced apoptosis was partially dependent on Akt activation. In contrast, antioxidant N-acetyl-L-cysteine (NAC) reduced early, but not late, MLK3-MKK7-JNK3 activation by Abeta treatment and provided a weak neuroprotective ability in Abeta-induced apoptosis. Taken together, Abeta neurotoxicity is mainly due to MLK3-MKK7-JNK3 signal cascades. The late signal events of MLK3 activation after Abeta treatment may play an important role in AD neuronal loss and will be a promising pharmacological target for AD therapeutic intervention.

[Overexpression of PDZ1 domain of PSD-95 protein rescues hippocampal neurons from apoptosis induced by oxygen-glucose deprivation].

To detect the effect of PDZ1, domain of postsynaptic density 95 (PSD-95), on apoptosis of hippocampal neurons induced by oxygen-glucose deprivation (OGD), Sprague-Dawley rat hippocampal neurons were infected with PDZ1-viruses after 21 days of plating. Twenty-four hours after infection, cells were treated with OGD for 1.5 h, then were incubated with DAPI and apoptosis-like cells were characterized, or were collected for co-immunoprecipitation and Western blot analyses. The results showed that: (1) PDZ1 overexpression was observed in hippocampal neurons; (2) Apoptosis induced by OGD was obviously decreased in neurons overexpressing PDZ1 (P<0.05); (3) Overexpression of PDZ1 prevented the binding of GluR6 to PSD-95; (4) Overexpression of PDZ1 inhibited MLK3 and JNK1/2 activation induced by OGD. These results indicate that overexpression of PDZ1 may prevent hippocampal neurons from apoptosis induced by OGD.

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 gamma-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.

Neuroprotection against ischaemic brain injury by a GluR6-9c peptide containing the TAT protein transduction sequence.

It is well documented that N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors play a pivotal role in ischaemic brain injury. Recent studies have shown that kainate (KA) receptors are involved in neuronal cell death induced by seizure, which is mediated by the GluR6*PSD-95*MLK3 signalling module and subsequent c-Jun N-terminal kinase (JNK) activation. Here we investigate whether GluR6 mediated JNK activation is correlated with ischaemic brain injury. Our results show that cerebral ischaemia followed by reperfusion can enhance the assembly of the GluR6*PSD-95*MLK3 signalling module and JNK activation. As a result, activated JNK can not only phosphorylate the transcription factor c-Jun and up-regulate Fas L expression but can also phosphorylate 14-3-3 and promote Bax translocation to mitochondria, increase the release of cytochrome c and increase caspase-3 activation. These results indicate that GluR6 mediated JNK activation induced by ischaemia/reperfusion ultimately results in neuronal cell death via nuclear and non-nuclear pathways. Furthermore, the peptides we constructed, Tat-GluR6-9c, show a protective role against neuronal death induced by cerebral ischaemia/reperfusion through inhibiting the GluR6 mediated signal pathway. In summary, our results indicate that the KA receptor subunit GluR6 mediated JNK activation is involved in ischaemic brain injury and provides a new approach for stroke therapy.

Activation and involvement of JNK1/2 in hydrogen peroxide-induced neurotoxicity in cultured rat cortical neurons.

AIM: To investigate the role of c-Jun N-terminal protein kinase 1 and 2 (JNK1/2) and the main signal pathway for its activation in hydrogen peroxide (H(2)O(2)) induced apoptotic-like cortical cell death. METHODS: Using the model of oxidative stress induced by H(2)O(2), the expression and diphosphorylation of JNK1/2 was examined by immunoblotting analysis, and neuronal apoptotic like cell death was determined by 4',6-diamidino-2-phenylindole (DAPI) staining. RESULTS: The elevation in diphosphorylation level of JNK1/2 (4.40-/5.61-fold vs sham control) was associated with the concentration of H(2)O(2) (0-100 micromol/L) and the development of apoptotic-like cell death (11.04 %-81.01 %). There was no alteration of JNK1/2 protein expression following H(2)O(2) treatment and recovery at different time points. Administration with JNK1/2 antisense oligonucleotides not only significantly decreased JNK1/2 protein expression and activation level, but also significantly reduced cortical cell death induced by H(2)O(2) exposure. Furthermore, both JNK1/2 diphosphorylation and apoptotic-like cell death were largely prevented by pretreatment with (5S,10R)-(-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) or omission of Ca(2+) in incubation medium with ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). CONCLUSION: JNK1/2 is activated and participates in H(2)O(2)-induced apoptotic-like death in cultured rat cortical neurons mainly via N-methyl-D-aspartate (NMDA) receptor-mediated influx of extracellular Ca(2+).

Suppression of postsynaptic density protein 95 expression attenuates increased tyrosine phosphorylation of NR2A subunits of N-methyl-D-aspartate receptors and interactions of Src and Fyn with NR2A after transient brain ischemia in rat hippocampus.

The effects of suppression of postsynaptic density protein 95 (PSD-95) expression on the increased tyrosine phosphorylation of N-methyl-D-aspartate receptor subunit NR2A and interactions of Src and Fyn with NR2A after brain ischemia were investigated by immunoprecipitation and immunoblotting. Transient (15 min) brain ischemia was induced by the four-vessel occlusion method in Sprague-Dawley rats. Intracerebroventricular infusion of PSD-95 antisense oligonucleotides (every 24 h for 3 days before ischemia), but not missense oligonucleotides or vehicle, not only markedly decreased the protein level of PSD-95 but also attenuated the elevated tyrosine phosphorylation of NR2A and interactions of Src and Fyn with NR2A induced by 6 h of reperfusion following ischemia in the hippocampus. The protein levels of NR2A, Src and Fyn had no differences under the above conditions. These data suggested that PSD-95 is critical for facilitating NR2A tyrosine phosphorylation by Src family kinases in postischemic brain.

Regulation of c-Jun N-terminal kinase activation in hydrogen peroxide induced neurotoxicity.

C-Jun N-terminal kinase 1 and 2 (JNK1/2) have been shown to be transiently activated and involved in neurotoxicity. We searched for possible upstream molecules, which are responsible for the regulation of hydrogen peroxide-(H2O2) induced JNK1/2 activation and JNK1/2-mediated apoptotic-like cell death in cultured rat cortical neurons. The results showed that JNK1/2 activation (monitored by anti-diphosphorylated JNK1/2 antibody) was largely prevented by elimination of extracellular Ca2+ or blockage of NMDA-receptors (NMDA-R), and was weakly but significantly decreased by blockage of L-type voltage-gated calcium channel (L-VGCC); furthermore, JNK1/2 activation was largely prevented by inhibition of Ca2+/calmodulin-dependent protein kinase-II (CaMKII) and protein-tyrosine kinases (PTK). We also found that H2O2-induced apoptotic-like cell death was partially prevented by elimination of extracellular Ca2+, or by inhibition of NMDA-R, L-VGCC, PTK and CaMKII, respectively. The above results suggest that in H2O2-induced neurotoxicity, JNK1/2 activation is mainly mediated by NMDA-R and L-VGCC. Consequently, PTK and CaMKII are critical intermediaries in JNK1/2 activation and are mainly responsible for JNK1/2-mediated apoptotic-like cell death.