GluA2 trafficking is involved in apoptosis of retinal ganglion cells induced by activation of EphB/EphrinB reverse signaling in a rat chronic ocular hypertension model.

EphB1, expressed in Müller cells, and ephrinB2, expressed in both Müller cells and retinal ganglion cells (RGCs), constitute an EphB/ephrinB reverse signaling in RGCs. Whether and how this reverse signaling is involved in RGC apoptosis in a rat chronic ocular hypertension (COH) model was investigated. In the COH model, both EphB1 and ephrinB2 were significantly increased and the reverse signaling was activated, which was accompanied by increased protein levels of phosphorylated (p) src, GluA2, and p-GluA2. Intravitreal injection of EphB2-Fc, an activator of ephrinB2, induced an increase in TUNEL-positive signals in normal retinae. A coimmunoprecipitation assay demonstrated direct interactions among ephrinB2, p-src, and GluA2. Moreover, in COH rats the expression of GluA2 proteins on the surface of retinal cells was decreased. Such GluA2 endocytosis could be prevented by preoperational intravitreal injection of 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo [3,4-d] pyrimidine (PP2), an inhibitor of src family tyrosine kinases, and possibly involved the protein interacting with C kinase 1 and phosphorylation of GluA2. In normal rats, intravitreal injection of EphB2-Fc caused changes in these protein levels similar to those observed in COH rats, which all could be avoided by preinjection of PP2. Patch-clamp experiments further showed that the current-voltage relationship of AMPA receptor-mediated EPSCs of RGCs exhibited stronger inward rectification in EphB2-Fc-injected rats. Furthermore, preinjection of PP2 or N-[3-[[4-[(3-aminopropyl)amino]butyl]amino]propyl]-1-naphthaleneacetamide trihydrochloride) (Naspm), a Ca(2+)-permeable GluA2-lacking AMPA receptor inhibitor, remarkably inhibited RGC apoptosis in either EphB2-Fc-injected or COH rats. Together, elevated GluA2 trafficking induced by activated EphB2/ephrinB2 reverse signaling likely contributes to RGC apoptosis in COH rats.

Group I mGluR-mediated inhibition of Kir channels contributes to retinal Müller cell gliosis in a rat chronic ocular hypertension model.

Müller cell gliosis, which is characterized by upregulated expression of glial fibrillary acidic protein (GFAP), is a universal response in many retinal pathological conditions. Whether down-regulation of inward rectifying K+ (Kir) channels, which commonly accompanies the enhanced GFAP expression, could contribute to Müller cell gliosis is poorly understood. We investigated changes of Kir currents, GFAP and Kir4.1 protein expression in Müller cells in a rat chronic ocular hypertension (COH) model, and explored the mechanisms underlying Müller cell gliosis. We show that Kir currents and Kir4.1 protein expression in Müller cells were reduced significantly, while GFAP expression was increased in COH rats, and these changes were eliminated by MPEP, a group I metabotropic glutamate receptors (mGluR I) subtype mGluR5 antagonist. In normal isolated Müller cells, the mGluR I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) suppressed the Kir currents and the suppression was blocked by MPEP. The DHPG effect was mediated by the intracellular Ca2+ -dependent PLC/IP3-ryanodine/PKC signaling pathway, but the cAMP-PKA pathway was not involved. Moreover, intravitreal injection of DHPG in normal rats induced changes in Müller cells, similar to those observed in COH rats. The DHPG-induced increase of GFAP expression in Müller cells was obstructed by Ba2+, suggesting the involvement of Kir channels. We conclude that overactivation of mGluR5 by excessive extracellular glutamate in COH rats could contribute to Müller cell gliosis by suppressing Kir channels.

Enhanced expression of NR2B subunits of NMDA receptors in the inherited glaucomatous DBA/2J mouse retina.

DBA/2J mouse has been used as a model for spontaneous secondary glaucoma. Here, we investigated changes in expression of NMDA receptor (NMDAR) subunits and Cdk5/p35/NMDAR signaling in retinas of DBA/2J mice using Western blot technique. The protein levels of NR1 and NR2A subunits in retinas of DBA/2J mice at all ages (6-12 months) were not different from those in age-matched C57BL/6 mice. In contrast, the protein levels of NR2B subunits, in addition to age-dependent change, significantly increased with elevated intraocular pressure (IOP) in DBA/2J mice at 6 and 9 months as compared with age-matched controls. Moreover, expression of Cdk5, p35 and ratio of p-NR2A(S1232)/NR2A progressively increased with time in both strains, suggestive of activated Cdk5/p35 signaling pathway. However, the changes in these proteins were in the same levels in both strain mice, except a significant increase of p35 proteins at 6 months in DBA/2J mice. Meanwhile, the protein levels of Brn-3a, a retinal ganglion cell (RGC) maker, remarkably decreased at 9-12 months in DBA/2J mice, which was in parallel with the changes of NR2B expression. Our results suggest that elevated IOP-induced increase in expression of NR2B subunits of NMDARs may be involved in RGC degeneration of DBA/2J mice.

Overexpression of erythropoietin-producing hepatocyte receptor B4 and ephrin-B2 is associated with estrogen receptor expression in endometrial adenocarcinoma.

Endometrial adenocarcinoma (EC) is one of the most frequently diagnosed types of endometrial cancer and is typically a consequence of continuous estrogen receptor stimulation. Erythropoietin-producing hepatocyte receptor B4 (EphB4) and its ligand ephrin-B2 have been reported to be overexpressed in EC cells; however, the function in EC remains unclear. The present study aimed to elucidate the role of EphB4 and ephrin-B2 in EC. The protein expression pattern of EphB4 and ephrin-B2 was analyzed through immunohistochemistry and western blot analysis in endometrium with adenomyosis or simple endometrial hyperplasia, atypical endometrial hyperplasia, double-positive estrogen receptor (ER)/progesterone receptor (PR) EC and double-negative ER/PR EC. The expression of EphB4 and ephrin-B2 was demonstrated to be increased in atypical EH and ER/PR-positive EC, but not ER/PR-negative EC. Furthermore, EphB4 and ephrin-B2 expression was positively associated with ER expression in EC tissue. The results of the present study suggest that the overexpression of EphB4 and ephrin-B2 in the endometrium serves a role in the pathogenesis of EC, in addition to being associated with ER expression.

Cannabinoid CB1 receptor signaling dichotomously modulates inhibitory and excitatory synaptic transmission in rat inner retina.

In the inner retina, ganglion cells (RGCs) integrate and process excitatory signal from bipolar cells (BCs) and inhibitory signal from amacrine cells (ACs). Using multiple labeling immunohistochemistry, we first revealed the expression of the cannabinoid CB1 receptor (CB1R) at the terminals of ACs and BCs in rat retina. By patch-clamp techniques, we then showed how the activation of this receptor dichotomously regulated miniature inhibitory postsynaptic currents (mIPSCs), mediated by GABAA receptors and glycine receptors, and miniature excitatory postsynaptic currents (mEPSCs), mediated by AMPA receptors, of RGCs in rat retinal slices. WIN55212-2 (WIN), a CB1R agonist, reduced the mIPSC frequency due to an inhibition of L-type Ca(2+) channels no matter whether AMPA receptors were blocked. In contrast, WIN reduced the mEPSC frequency by suppressing T-type Ca(2+) channels only when inhibitory inputs to RGCs were present, which could be in part due to less T-type Ca(2+) channels of cone BCs, presynaptic to RGCs, being in an inactivation state under such condition. This unique feature of CB1R-mediated retrograde regulation provides a novel mechanism for modulating excitatory synaptic transmission in the inner retina. Moreover, depolarization of RGCs suppressed mIPSCs of these cells, an effect that was eliminated by the CB1R antagonist SR141716, suggesting that endocannabinoid is indeed released from RGCs.

Group I metabotropic glutamate receptor agonist DHPG modulates Kir4.1 protein and mRNA in cultured rat retinal Müller cells.

Müller cell gliosis is a general response in a variety of pathological alternations of the retina, which is characterized by the upregulated expression of glial fibrillary acidic protein (GFAP) and the downregulation of membrane K(+) conductance. We have demonstrated that downregulation of Kir K(+) currents in Müller cells in an experimental glaucoma model is due to activation of group I metabotropic glutamate receptor (mGluR I) by glutamate, which contributes to Müller cell gliosis. Here, whether and how activation of mGluR I modulate membrane Kir4.1 protein internalization and Kir4.1 mRNA expression were investigated in purified cultured rat retinal Müller cells using immunocytochemistry, Western blot and real-time PCR techniques. DHPG (10µM, a selective mGluR I agonist) treatment induced Müller cell gliosis, as evidenced by enhanced GFAP expression. Although total Kir4.1 proteins extracted from the DHPG-treated cells kept unchanged, Kir4.1 proteins in the cell membrane compartment were significantly decreased, which was prior to the change of GFAP in time course. In addition, DHPG (10 and 100µM) treatment induced a transient decrease in Kir4.1 mRNA expression in the cells. All these results suggest that activation of mGluR I by DHPG may decrease the number of functional Kir4.1 channels in purified cultured rat retinal Müller cells through modulating Kir4.1 protein and mRNA, thus contributing to Müller cell gliosis.

Involvement of calpain/p35-p25/Cdk5/NMDAR signaling pathway in glutamate-induced neurotoxicity in cultured rat retinal neurons.

We investigated possible involvement of a calpain/p35-p25/cyclin-dependent kinase 5 (Cdk5) signaling pathway in modifying NMDA receptors (NMDARs) in glutamate-induced injury of cultured rat retinal neurons. Glutamate treatment decreased cell viability and induced cell apoptosis, which was accompanied by an increase in Cdk5 and p-Cdk5(T15) protein levels. The Cdk5 inhibitor roscovitine rescued the cell viability and inhibited the cell apoptosis. In addition, the protein levels of both calpain 2 and calpain-specific alpha-spectrin breakdown products (SBDPs), which are both Ca(2+)-dependent, were elevated in glutamate-induced cell injury. The protein levels of Cdk5, p-Cdk5(T15), calpain 2 and SBDPs tended to decline with glutamate treatments of more than 9 h. Furthermore, the elevation of SBDPs was attenuated by either D-APV, a NMDAR antagonist, or CNQX, a non-NMDAR antagonist, but was hardly changed by the inhibitors of intracellular calcium stores dantrolene and xestospongin. Moreover, the Cdk5 co-activator p35 was significantly up-regulated, whereas its cleaved product p25 expression showed a transient increase. Glutamate treatment for less than 9 h also considerably enhanced the ratio of the Cdk5-phosphorylated NMDAR subunit NR2A at Ser1232 site (p-NR2A(S1232)) and NR2A (p-NR2A(S1232)/NR2A), and caused a translocation of p-NR2A(S1232) from the cytosol to the plasma membrane. The enhanced p-NR2A(S1232) was inhibited by roscovitine, but augmented by over-expression of Cdk5. Calcium imaging experiments further showed that intracellular Ca(2+) concentrations ([Ca(2+)](i)) of retinal cells were steadily increased following glutamate treatments of 2 h, 6 h and 9 h. All these results suggest that the activation of the calpain/p35-p25/Cdk5 signaling pathway may contribute to glutamate neurotoxicity in the retina by up-regulating p-NR2A(S1232) expression.

RGS2 and RGS4 modulate melatonin-induced potentiation of glycine currents in rat retinal ganglion cells.

Regulator of G-protein signaling (RGS) proteins 2 (RGS2) and 4 (RGS4) play an important role in regulating G(i/o)- and G(q)-coupled receptors. In the present study, we investigated the possible impact of RGS2 and RGS4 on modulation of glycine currents of rat retinal ganglion cells (RGCs) mediated by the G(i/o)-coupled melatonin MT(2) receptor, using immunohistochemistry, Western blot analysis and whole-cell patch-clamp techniques. By immunofluorescence labeling the expression profiles of RGS2 and RGS4 proteins were basically similar. Both of them were widely expressed in the rat retina, particularly in the inner plexiform layer (IPL) and the ganglion cell layer (GCL). In addition, sparse signals of RGS2 and RGS4 were also detected in the inner nuclear layer (INL). Double immunofluorescence labeling further showed that all of RGCs retrogradely labeled expressed both RGS2 and RGS4. Western blot analysis confirmed the presence of RGS2 and RGS4 proteins in the rat retina. Intracellular dialysis of RGCs with the antibody against RGS2/RGS4 to block RGS2/RGS4 function gradually increased glycine current amplitudes of these cells. In the presence of the RGS2/RGS4 antibody melatonin-induced potentiation of glycine currents of RGCs was not observable. These results suggest that RGS2/RGS4 are coupled to melatonin receptor signaling in rat RGCs and these proteins may regulate the MT(2) receptor to change melatonin-induced modulation of glycine currents in rat RGCs.

Protection by tetrahydroxystilbene glucoside against cerebral ischemia: involvement of JNK, SIRT1, and NF-kappaB pathways and inhibition of intracellular ROS/RNS generation.

Many natural polyphenolic compounds have been shown to attenuate reactive oxygen/nitrogen species (ROS/RNS) formation and protect against ischemia/reperfusion injury both in vitro and in vivo. 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside (TSG), an active component of the rhizome extract from Polygonum multiflorum, exhibits antioxidative and anti-inflammatory effects. Here, we used an in vitro ischemic model of oxygen-glucose deprivation followed by reperfusion (OGD-R) and an in vivo ischemic model of middle cerebral artery occlusion (MCAO) to investigate the neuroprotective effects of TSG on ischemia/reperfusion brain injury and the related mechanisms. We demonstrated that OGD-R-induced neuronal injury, intracellular ROS generation, and mitochondrial membrane potential dissipation were reversed by TSG. The elevation of H2O2-induced [Ca2+]i was also attenuated by TSG. Inhibition of the c-Jun N-terminal kinase (JNK) and Bcl-2 family-related apoptotic signaling pathway was involved in the neuroprotection afforded by TSG. Meanwhile, TSG inhibited iNOS mRNA expression induced by OGD-R, which may be mediated by the activation of SIRT1 and inhibition of NF-kappaB activation. In vivo studies further demonstrated that TSG significantly reduced the brain infarct volume and the number of positive cells by TUNEL staining in the cerebral cortex compared to the MCAO group. Our study indicates that TSG protects against cerebral ischemia/reperfusion injury through multifunctional cytoprotective pathways.