Neuroprotective effect of meglumine cyclic adenylate against ischemia/reperfusion injury via STAT3-Ser727 phosphorylation.

OBJECTIVES: Ischemia/reperfusion can induce neuronal apoptosis in the brain and lead to function deficits. The activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is neuroprotective against transient cerebral ischemia. The neuroprotective mechanisms of PKA mainly involve the regulation of gene transcription via the PKA/CREB pathway. The present study aims to investigate the neuroprotective effect of meglumine cyclic adenylate, an activator of PKA, under a rat model of global cerebral ischemia/reperfusion and to reveal the underlying mechanism involving signal transducer and activator of transcription 3 (STAT3)-Ser727 phosphorylation and mitochondrion modulation. MATERIALS AND METHODS: Male Sprague-Dawley rats were subjected to 15 min global cerebral ischemia, and meglumine cyclic adenylate was treated through tail intravenous injection 30 min before ischemia. Cresyl violet staining was used to evaluate neuron injury at 5 d of reperfusion. Western blotting was used to detect p-Ser727-STAT3, total STAT3, cytochrome c (Cyt c) and active caspase-3 in the tissues of hippocampal CA1 region at 6 h of reperfusion. STAT3-S727A was overexpressed in HT22 cells to reveal the significance of STAT3-Ser727 phosphorylation in the neuroprotective effect of meglumine cyclic adenylate. RESULTS: Pretreatment with meglumine cyclic adenylate not only significantly ameliorated neuron loss in CA1 region after global cerebral ischemia but also enhanced STAT3-Ser727 phosphorylation, increased mitochondrial STAT3, and decreased cytosolic Cyt c and active caspase-3. Overexpression of STAT3-S727A in HT22 cells eliminated meglumine cyclic adenylate-induced increase of p-Ser727-STAT3, mitochondrial STAT3, cytosolic Cyt c and active caspase-3. CONCLUSION: Meglumine cyclic adenylate protects neurons against ischemia/reperfusion injury via promoting p-Ser727-STAT3-associated mitochondrion modulation and inhibiting apoptosis pathway.

Neuronal activity-induced SUMOylation of Akt1 by PIAS3 is required for long-term potentiation of synaptic transmission.

Neuronal activity regulates spatial distribution of the SUMOylation system in cytosolic and dendritic sites, which has been implicated in learning, memory, and underlying synaptic structural and functional remodeling in the hippocampus. However, the functional target proteins for activated small ubiquitin-like modifiers (SUMOs) and downstream molecular consequences behind long-term potentiation (LTP) of synaptic plasticity remain to be elucidated. In this study, we showed that N-methyl-D-aspartate receptor-mediated neuronal activity induced the covalent modification of cytosolic Akt1 by small ubiquitin-like modifier 1 (SUMO1) in rat cortical and hippocampal CA1 neurons. Protein inhibitor of activated STAT3 (PIAS3) was involved in the activity-induced Akt1 SUMO1-ylation, and K64 and K276 residues were major SUMOylated sites. Importantly, Akt1 SUMOylation at K64 and K276 enhanced its enzymatic activity and facilitated T308 phosphorylation. Furthermore, the N-terminal SAP domain of PIAS3 bound Akt1 directly. The disruption of Akt1-PIAS3 interaction by Tat-SAP, a synthetic Tat-fused cell-permeable peptide containing PIAS3 SAP domain, inhibited neuronal activity-induced Akt1 SUMOylation and impaired LTP expression and late phase LTP maintenance in the hippocampus. Correlatedly, Tat-SAP not only blocked the LTP-related extracellular signal-regulated kinase (ERK)1/2-Elk-1-brain-derived neurotrophic factor (BDNF)/Arc signaling, but also disrupted mammalian target of rapamycin (mTOR)-eIF4E-binding protein 1 (4E-BP1) pathway. These findings reveal an activity-induced Akt1 SUMOylation by PIAS3 that contributes to ERK1/2-BDNF/Arc and mTOR-4E-BP1 cascades, and in turn, long-lasting excitatory synaptic responses.

PKCλ is critical in AMPA receptor phosphorylation and synaptic incorporation during LTP.

Direct phosphorylation of GluA1 by PKC controls α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor (AMPAR) incorporation into active synapses during long-term potentiation (LTP). Numerous signalling molecules that involved in AMPAR incorporation have been identified, but the specific PKC isoform(s) participating in GluA1 phosphorylation and the molecule triggering PKC activation remain largely unknown. Here, we report that the atypical isoform of PKC, PKCλ, is a critical molecule that acts downstream of phosphatidylinositol 3-kinase (PI3K) and is essential for LTP expression. PKCλ activation is required for both GluA1 phosphorylation and increased surface expression of AMPARs during LTP. Moreover, p62 interacts with both PKCλ and GluA1 during LTP and may serve as a scaffolding protein to place PKCλ in close proximity to facilitate GluA1 phosphorylation by PKCλ. Thus, we conclude that PKCλ is the critical signalling molecule responsible for GluA1-containing AMPAR phosphorylation and synaptic incorporation at activated synapses during LTP expression.

Phosphorylation of 4.1N by CaMKII Regulates the Trafficking of GluA1-containing AMPA Receptors During Long-term Potentiation in Acute Rat Hippocampal Brain Slices.

OBJECTIVE: GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) inserted into postsynaptic membranes are key to the process of long-term potentiation (LTP). Some evidence has shown that 4.1N plays a critical role in the membrane trafficking of AMPARs. However, the underlying mechanism behind this is still unclear. We investigated the role of 4.1N-mediated membrane trafficking of AMPARs during theta-burst stimulation long-term potentiation (TBS-LTP), to illustrate the molecular mechanism behind LTP. METHODS: LTP was induced by TBS in rat hippocampal CA1 neuron. Tat-GluA1 (MPR), which disrupts the association of 4.1N-GluA1, and autocamtide-2-inhibitory peptide, myristoylated (Myr-AIP), a CaMKII antagonist, were used to explore the role of 4.1N in the AMPARs trafficking during TBS-induced LTP. Immunoprecipitation (IP) and immunoblotting (IB)were used to detect protein expression, phosphorylation, and the interaction of p-CaMKII-4.1N-GluA1. RESULTS: We found that Myr-AIP attenuated increases of p-CaMKII (T286), p-GluA1 (ser831), and 4.1N phosphorylation after TBS-LTP, and decreased the association of p-CaMKII-4.1N-GluA1, along with the expression of GluA1, at postsynaptic densities during TBS-LTP. We also designed interfering peptides to disrupt the interaction between 4.1N and GluA1, which showed that Tat-GluA1 (MPR) or Myr-AIP inhibited TBS-LTP and attenuated increases of GluA1 at postsynaptic sites, while Tat-GluA1 (MPR) or Myr-AIP had no effects on miniature excitatory postsynaptic currents (mEPSCs) in non-stimulated hippocampal CA1 neurons. CONCLUSION: Active CaMKII enhanced the phosphorylation of 4.1N and facilitated the association of p-CaMKII with 4.1N-GluA1, which in turn resulted in GluA1 trafficking during TBS-LTP. The association of 4.1N-GluA1 is required for LTP, but not for basal synaptic transmission.

A selective M1 and M3 receptor antagonist, penehyclidine hydrochloride, prevents postischemic LTP: involvement of NMDA receptors.

Our previous and other studies have confirmed that a selective M1 and M3 receptor antagonist, Penehyclidine hydrochloride (PHC), has neuroprotection activity in cerebral ischemia. However, the precise mechanisms of protection of PHC are still elusive. In this study we analyzed PHC-mediated neuroprotection on a model of brain ischemia (oxygen and glucose deprivation), named postischemic LTP (i-LTP). We found that the activation of NMDA receptor was required for the induction of i-LTP. Compared with scopolamine, PHC could prevent it due to selectively blocking M1 receptor, not M2 receptor, to decrease NMDAR activation. Our findings further showed that the inhibition of SK2 channels occluded the prevention of PHC on NMDAR activation. Furthermore, we confirmed that PHC exerted its roles through directly disinhibition of SK2 channels by blocking M1 receptor and subsequent restricting PKC activation. Moreover, our studies further revealed the critical roles of SK2 channels in i-LTP. Thus, the mechanisms of PHC in brain protection may be involved in suppression of NMDAR by regulation of SK2 channels. Our results obtained in effects of PHC on i-LTP further provided a better understanding of the therapy strategy during stroke and identified potential therapeutic targets to prevent development of ischemia.

Protein kinase C promotes N-methyl-D-aspartate (NMDA) receptor trafficking by indirectly triggering calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation.

Regulation of neuronal NMDA receptor (NMDAR) is critical in synaptic transmission and plasticity. Protein kinase C (PKC) promotes NMDAR trafficking to the cell surface via interaction with NMDAR-associated proteins (NAPs). Little is known, however, about the NAPs that are critical to PKC-induced NMDAR trafficking. Here, we showed that calcium/calmodulin-dependent protein kinase II (CaMKII) could be a NAP that mediates the potentiation of NMDAR trafficking by PKC. PKC activation promoted the level of autophosphorylated CaMKII and increased association with NMDARs, accompanied by functional NMDAR insertion, at postsynaptic sites. This potentiation, along with PKC-induced long term potentiation of the AMPA receptor-mediated response, was abolished by CaMKII antagonist or by disturbing the interaction between CaMKII and NR2A or NR2B. Further mutual occlusion experiments demonstrated that PKC and CaMKII share a common signaling pathway in the potentiation of NMDAR trafficking and long-term potentiation (LTP) induction. Our results revealed that PKC promotes NMDA receptor trafficking and induces synaptic plasticity through indirectly triggering CaMKII autophosphorylation and subsequent increased association with NMDARs.

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.

Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome.

Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity, and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal activation. In exploring the basis of these N-glycosylation alterations, we discovered that they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed during neuronal excitation were in close association with endoplasmic reticulum (ER) exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrite's satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction, and disease.

Metaplastic regulation of long-term potentiation/long-term depression threshold by activity-dependent changes of NR2A/NR2B ratio.

In vivo experience induces changes in synaptic NMDA receptor (NMDAR) subunit components, which are correlated with subsequent modifications of synaptic plasticity. However, little is known about how these subunit changes regulate the induction threshold of subsequent plasticity. At hippocampal Schaffer collateral-CA1 synapses, we first examined whether a recent history of neuronal activity could affect subsequent synaptic plasticity through its actions on NMDAR subunit components. We found that prior activity history produced by priming stimulations (PSs) across a wide range of frequencies (1-100 Hz) could induce bidirectional changes in the NR2A/NR2B ratio, which governs the threshold for subsequent long-term potentiation/long-term depression (LTP/LTD). Manipulating the NR2A/NR2B ratio through partial NR2 subunit blockade mimicked the PS regulation of the LTP/LTD threshold. Our results demonstrate that activity-dependent changes in the NR2A/NR2B ratio can be critical factors in metaplastic regulation of the LTP/LTD threshold.

Distinct trafficking and expression mechanisms underlie LTP and LTD of NMDA receptor-mediated synaptic responses.

Although an increasing number of studies have demonstrated the plasticity of NMDA receptor-mediated synaptic transmission, little is known about the molecular mechanisms that underlie this neurologically important process. In a study of NMDAR-mediated synaptic responses in hippocampal Schaffer-CA1 synapses whose AMPA receptor (AMPAR) activity is totally blocked, we uncovered differences between the trafficking mechanisms that underlie the long-term potentiation (LTP) and long-term depression (LTD) that can be induced in these cells under these conditions. The LTP-producing protocol failed to induce a change in the amplitude of NMDAR-mediated postsynaptic currents (NMDAR EPSCs) in the first 5-10 min, but induced gradual enhancement of NMDAR EPSCs thereafter that soon reached a stable magnitude. This "slow" LTP of NMDAR EPSCs (LTP(NMDA)) was blocked by inhibiting exocytosis or actin polymerization in postsynaptic cells. By contrast, LTD of NMDAR EPSCs (LTD(NMDA)) was immediately inducible, and, although it was blocked by the actin stabilizer, it was unaffected by exocytosis or endocytosis inhibitors. Furthermore, concomitant changes in the decay time of NMDAR EPSCs suggested that differential switches in NR2 subunit composition accompanied LTP(NMDA) and LTD(NMDA), and these changes were blocked by the calcium buffer BAPTA or an mGluR antagonist. Our results suggest that LTP(NMDA) and LTD(NMDA) utilize different NMDAR trafficking pathways and express different ratios of NMDAR subunits on the postsynaptic surface.

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.

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.

Amyloid ß causes excitation/inhibition imbalance through dopamine receptor 1-dependent disruption of fast-spiking GABAergic input in anterior cingulate cortex.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly. At the early stages of AD development, the soluble ß-amyloid (Aß) induces synaptic dysfunction, perturbs the excitation/inhibition balance of neural circuitries, and in turn alters the normal neural network activity leading to cognitive decline, but the underlying mechanisms are not well established. Here by using whole-cell recordings in acute mouse brain slices, we found that 50 nM Aß induces hyperexcitability of excitatory pyramidal cells in the cingulate cortex, one of the most vulnerable areas in AD, via depressing inhibitory synaptic transmission. Furthermore, by simultaneously recording multiple cells, we discovered that the inhibitory innervation of pyramidal cells from fast-spiking (FS) interneurons instead of non-FS interneurons is dramatically disrupted by Aß, and perturbation of the presynaptic inhibitory neurotransmitter gamma-aminobutyric acid (GABA) release underlies this inhibitory input disruption. Finally, we identified the increased dopamine action on dopamine D1 receptor of FS interneurons as a key pathological factor that contributes to GABAergic input perturbation and excitation/inhibition imbalance caused by Aß. Thus, we conclude that the dopamine receptor 1-dependent disruption of FS GABAergic inhibitory input plays a critical role in Aß-induced excitation/inhibition imbalance in anterior cingulate cortex.

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.

Selective and regulated trapping of nicotinic receptor weak base ligands and relevance to smoking cessation.

To better understand smoking cessation, we examined the actions of varenicline (Chantix) during long-term nicotine exposure. Varenicline reduced nicotine upregulation of α4ß2-type nicotinic receptors (α4ß2Rs) in live cells and neurons, but not for membrane preparations. Effects on upregulation depended on intracellular pH homeostasis and were not observed if acidic pH in intracellular compartments was neutralized. Varenicline was trapped as a weak base in acidic compartments and slowly released, blocking 125I-epibatidine binding and desensitizing α4ß2Rs. Epibatidine itself was trapped; 125I-epibatidine slow release from acidic vesicles was directly measured and required the presence of α4ß2Rs. Nicotine exposure increased epibatidine trapping by increasing the numbers of acidic vesicles containing α4ß2Rs. We conclude that varenicline as a smoking cessation agent differs from nicotine through trapping in α4ß2R-containing acidic vesicles that is selective and nicotine-regulated. Our results provide a new paradigm for how smoking cessation occurs and suggest how more effective smoking cessation reagents can be designed.

Geldanamycin inhibits Fas signaling pathway and protects neurons against ischemia.

The inhibitor of Heat shock proteins 90, geldanamycin (GA), has been reported neuroprotective against both global and focal brain ischemia. To understand the mechanisms underlies the neuroprotection effect of GA, we investigated the relationship between GA pretreatment and Fas signaling pathway in rat global brain ischemia/reperfusion model in the present study. Results showed that GA attenuated neuron loss significantly in hippocampal CA1 region. Upon GA pretreatment, Mixed Lineage Kinase 3 (MLK3) expression and activation and FasL expression was decreased, the assembly of death-inducing signaling complex and activation of downstream apoptosis-associating proteins were inhibited along with neuroprotection. Based on the facts that MLK3 is one client protein of HSP90 and MLK3 pathway induces FasL expression in ischemic brain injury, our study suggests one of the mechanisms of neuroprotection against brain ischemia from GA.

PDZ1 inhibitor peptide protects neurons against ischemia via inhibiting GluK2-PSD-95-module-mediated Fas signaling pathway.

Respecting the selective inhibition of peptides on protein-protein interactions, they might become potent methods in ischemic stroke therapy. In this study, we investigated the effect of PDZ1 inhibitor peptide on ischemic neuron apoptosis and the relative mechanism. Results showed that PDZ1 inhibitor peptide, which significantly disrupted GluK2-PSD-95 interaction, efficiently protected neuron from ischemia/reperfusion-induced apoptosis. Further, PDZ1 inhibited FasL expression, DISC assembly and activation of Caspase 8, Bid, Caspase 9 and Caspase 3 after global brain ischemia. Based on our previous report that GluK2-PSD-95 pathway increased FasL expression after global brain ischemia, the neuron protection effect of PDZ1 inhibitor peptide was considered to be achieved by disrupting GluK2-PSD-95 interaction and subsequently inhibiting FasL expression and Fas apoptosis pathway.

Suppression of Pyk2 attenuated the increased tyrosine phosphorylation of NMDA receptor subunit 2A after brain ischemia in rat hippocampus.

Effects of suppressing the protein expression of Pyk2 on increased tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A) after brain ischemia in rat hippocampus were studied with immunoprecipitation and immunoblot. Transient (15 min) brain ischemia and reperfusion (I/R) was induced by four-vessel occlusion in Sprague-Dawley (SD) rats. I/R led to increases of tyrosine phosphorylation of NR2A and interaction of Pyk2 and Src kinase with NR2A after 6 h of reperfusion. The increases were attenuated by Pyk2 antisense oligonucleotides intracerebroventricularly infused every 24 h for 4 days before ischemia, but not missense oligonucleotides or vehicle. The antisense also inhibited the increased auto-phosphorylation of Pyk2 and Src kinase, while the protein expression of NR2A or Src kinase had no obvious change under the above conditions. The data suggested that Pyk2 may be involved in facilitating NR2A tyrosine phosphorylation by Src kinase after I/R.

Suppression of postsynaptic density protein 95 by antisense oligonucleotides diminishes postischemic pyramidal cell death in rat hippocampal CA1 subfield.

Our previous investigation has shown that postsynaptic density protein 95 (PSD-95) is critical for the Src family kinases-mediated tyrosine phosphorylation of N-methyl-d-aspartate receptor subunit 2A (NR2A) in the postischemic hippocampus. To clarify the roles of PSD-95 in the ischemic brain damage, histological method was performed to examine the effects of PSD-95 antisense oligonucleotides (AS) on the postischemic delayed cell death in rat hippocampus. Transient (15 min) brain ischemia was induced by the four-vessel occlusion method in Sprague-Dawley rats. Five days of reperfusion following brain ischemia (I/R5d) led to hippocampal CA1 pyramidal cell death upward of 90%. Intracerebroventricular infusion of AS (every 24 h for 3 days before ischemia) not only decreased the PSD-95 expression but also increased the number of surviving pyramidal neurons, while missense oligonucleotides (MS) had no effects. To further investigate the mechanisms underlying the neuroprotection of PSD-95 deficiency, the interaction of proline-rich tyrosine kinase 2 (Pyk2) with NR2A as well as autophosphorylation (Tyr402) of Pyk2 were detected. Immunoprecipitation and immunoblot analysis showed that preischemic treatment with AS, but not MS or vehicle, attenuated the I/R6h-induced increases in Pyk2-NR2A association and Pyk2 autophosphorylation. The protein levels of NR2A and Pyk2 had no differences under the above conditions. Our data suggest that the recruitments of ion channels and signaling molecules may be involved in the PSD-95 neurotoxicity in the postischemic hippocampus.

Lithium suppressed Tyr-402 phosphorylation of proline-rich tyrosine kinase (Pyk2) and interactions of Pyk2 and PSD-95 with NR2A in rat hippocampus following cerebral ischemia.

It has been indicated that Pyk2/Src signaling pathway is involved in modulation of N-methyl-D-aspartate-type (NMDA) glutamate receptor activity. Lithium protects against glutamate-induced excitotoxicity in cultured neurons and in animal models of diseases. The neuroprotection against excitotoxicity afforded by lithium is time-dependent, requiring treatment for 6-7 days for maximal effect. In this study, we examined the time-course and the effect of lithium on Tyr-402 phosphorylation of Pyk2 and Tyr-416 phosphorylation of Src as well as the association of Pyk2 and NMDA receptor subunit 2A (NR2A) mediated by postsynaptic density protein 95 kDa (PSD-95) in the condition of cerebral ischemia, which was induced by occlusion of the four vessels in Sprague-Dawley rats. At 6 h of reperfusion following 15 min of ischemia (I/R), the effects induced by chronic lithium were observed, including the decrease in enhanced Tyr-402 phosphorylation of Pyk2, the inhibition in increased Tyr-416 phosphorylation of Src and the attenuation in enhanced interactions of Pyk2 and PSD-95 with NR2A. Our results further suggest that the activated Pyk2 potentiates NMDA receptor function during transient brain ischemia followed by reperfusion and the above inhibition induced by lithium is likely to result in the inactivation of NMDA receptor and contributes to the neuroprotection against excitotoxicity.