MicroRNA-146a-5p protects retinal ganglion cells through reducing neuroinflammation in experimental glaucoma.

Neuroinflammation plays important roles in retinal ganglion cell (RGC) degeneration in glaucoma. MicroRNA-146 (miR-146) has been shown to regulate inflammatory response in neurodegenerative diseases. In this study, whether and how miR-146 could affect RGC injury in chronic ocular hypertension (COH) experimental glaucoma were investigated. We showed that in the members of miR-146 family only miR-146a-5p expression was upregulated in COH retinas. The upregulation of miR-146a-5p was observed in the activated microglia and Müller cells both in primary cultured conditions and in COH retinas, but mainly occurred in microglia. Overexpression of miR-146a-5p in COH retinas reduced the levels pro-inflammatory cytokines and upregulated the levels of anti-inflammatory cytokines, which were further confirmed in the activated primary cultured microglia. Transfection of miR-146a-5p mimic increased the percentage of anti-inflammatory phenotype in the activated BV2 microglia, while transfection of miR-146a-5p inhibitor resulted in the opposite effects. Transfection of miR-146a-5p mimic/agomir inhibited the levels of interleukin-1 receptor associated kinase (IRAK1) and TNF receptor associated factor 6 (TRAF6) and phosphorylated NF-κB subunit p65. Dual luciferase reporter gene assay confirmed that miR-146a-5p could directly target IRAK1 and TRAF6. Moreover, downregulation of IRAK1 and TRAF6 by siRNA techniques or blocking NF-κB by SN50 in cultured microglia reversed the miR-146a-5p inhibitor-induced changes of inflammatory cytokines. In COH retinas, overexpression of miR-146a-5p reduced RGC apoptosis, increased RGC survival, and partially rescued the amplitudes of photopic negative response. Our results demonstrate that overexpression of miR-146a-5p attenuates RGC injury in glaucoma by reducing neuroinflammation through downregulating IRAK1/TRAF6/NF-κB signaling pathway in microglia.

EphA4/ephrinA3 reverse signaling mediated downregulation of glutamate transporter GLAST in Müller cells in an experimental glaucoma model.

Deficiency of glutamate transporter GLAST in Müller cells may be culpable for excessive extracellular glutamate, which involves in retinal ganglion cell (RGC) damage in glaucoma. We elucidated how GLAST was regulated in rat chronic ocular hypertension (COH) model. Western blot and whole-cell patch-clamp recordings showed that GLAST proteins and GLAST-mediated current densities in Müller cells were downregulated at the early stages of COH. In normal rats, intravitreal injection of the ephrinA3 activator EphA4-Fc mimicked the changes of GLAST in COH retinas. In purified cultured Müller cells, EphA4-Fc treatment reduced GLAST expression at mRNA and protein levels, which was reversed by the tyrosine kinase inhibitor PP2 or transfection with ephrinA3-siRNA (Si-EFNA3), suggesting that EphA4/ephrinA3 reverse signaling mediated GLAST downregulation. EphA4/ephrinA3 reverse signaling-induced GLAST downregulation was mediated by inhibiting PI3K/Akt/NF-κB pathways since EphA4-Fc treatment of cultured Müller cells reduced the levels of p-Akt/Akt and NF-κB p65, which were reversed by transfecting Si-EFNA3. In Müller cells with ephrinA3 knockdown, the PI3K inhibitor LY294002 still decreased the protein levels of NF-κB p65 in the presence of EphA4-Fc, and the mRNA levels of GLAST were reduced by LY294002 and the NF-κB inhibitor SN50, respectively. Pre-injection of the PI3K/Akt pathway activator 740 Y-P reversed the GLAST downregulation in COH retinas. Western blot and TUNEL staining showed that transfecting of Si-EFNA3 reduced Müller cell gliosis and RGC apoptosis in COH retinas. Our results suggest that activated EphA4/ephrinA3 reverse signaling induces GLAST downregulation in Müller cells via inhibiting PI3K/Akt/NF-κB pathways, thus contributing to RGC damage in glaucoma.

Modulation of Rac1/PAK1/connexin43-mediated ATP release from astrocytes contributes to retinal ganglion cell survival in experimental glaucoma.

Connexin43 (Cx43) is a major gap junction protein in glial cells. Mutations have been found in the gap-junction alpha 1 gene encoding Cx43 in glaucomatous human retinas, suggestive of the involvement of Cx43 in the pathogenesis of glaucoma. However, how Cx43 is involved in glaucoma is still unknown. We showed that increased intraocular pressure in a glaucoma mouse model of chronic ocular hypertension (COH) downregulated Cx43, which was mainly expressed in retinal astrocytes. Astrocytes in the optic nerve head where they gather and wrap the axons (optic nerve) of retinal ganglion cells (RGCs) were activated earlier than neurons in COH retinas and the alterations in astrocytes plasticity in the optic nerve caused a reduction in Cx43 expression. A time course showed that reductions of Cx43 expression were correlated with the activation of Rac1, a member of the Rho family. Co-immunoprecipitation assays showed that active Rac1, or the downstream signaling effector PAK1, negatively regulated Cx43 expression, Cx43 hemichannel opening and astrocyte activation. Pharmacological inhibition of Rac1 stimulated Cx43 hemichannel opening and ATP release, and astrocytes were identified to be one of the main sources of ATP. Furthermore, conditional knockout of Rac1 in astrocytes enhanced Cx43 expression and ATP release, and promoted RGC survival by upregulating the adenosine A3 receptor in RGCs. Our study provides new insight into the relationship between Cx43 and glaucoma, and suggests that regulating the interaction between astrocytes and RGCs via the Rac1/PAK1/Cx43/ATP pathway may be used as part of a therapeutic strategy for managing glaucoma.

Soluble tumor necrosis factor-alpha-induced hyperexcitability contributes to retinal ganglion cell apoptosis by enhancing Nav1.6 in experimental glaucoma.

BACKGROUND: Neuroinflammation plays an important role in the pathogenesis of glaucoma. Tumor necrosis factor-alpha (TNF-α) is a major pro-inflammatory cytokine released from activated retinal glial cells in glaucoma. Here, we investigated how TNF-α induces retinal ganglion cell (RGC) hyperexcitability and injury. METHODS: Whole-cell patch-clamp techniques were performed to explore changes in spontaneous firing and evoked action potentials, and Na+ currents in RGCs. Both intravitreal injection of TNF-α and chronic ocular hypertension (COH) models were used. Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (q-PCR), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) techniques were employed to investigate the molecular mechanisms of TNF-α effects on RGCs. RESULTS: Intravitreal injection of soluble TNF-α significantly increased the spontaneous firing frequencies of RGCs in retinal slices. When the synaptic transmissions were blocked, more than 90% of RGCs still showed spontaneous firing; both the percentage of cells and firing frequency were higher than the controls. Furthermore, the frequency of evoked action potentials was also higher than the controls. Co-injection of the TNF-α receptor 1 (TNFR1) inhibitor R7050 eliminated the TNF-α-induced effects, suggesting that TNF-α may directly act on RGCs to induce cell hyperexcitability through activating TNFR1. In RGCs acutely isolated from TNF-α-injected retinas, Na+ current densities were upregulated. Perfusing TNF-α in RGCs of normal rats mimicked this effect, and the activation curve of Na+ currents shifted toward hyperpolarization direction, which was mediated through p38 MAPK and STAT3 signaling pathways. Further analysis revealed that TNF-α selectively upregulated Nav1.6 subtype of Na+ currents in RGCs. Similar to observations in retinas of rats with COH, intravitreal injection of TNF-α upregulated the expression of Nav1.6 proteins in both total cell and membrane components, which was reversed by the NF-κB inhibitor BAY 11-7082. Inhibition of TNFR1 blocked TNF-α-induced RGC apoptosis. CONCLUSIONS: TNF-α/TNFR1 signaling induces RGC hyperexcitability by selectively upregulating Nav1.6 Na+ channels, thus contributing to RGC apoptosis in glaucoma.

Interplay between Müller cells and microglia aggravates retinal inflammatory response in experimental glaucoma.

BACKGROUND: Glaucoma, the leading cause of irreversible blindness, is a retinal neurodegenerative disease, which results from progressive apoptotic death of retinal ganglion cells (RGCs). Although the mechanisms underlying RGC apoptosis in glaucoma are extremely complicated, an abnormal cross-talk between retinal glial cells and RGCs is generally thought to be involved. However, how interaction of Müller cells and microglia, two types of glial cells, contributes to RGC injury is largely unknown. METHODS: A mouse chronic ocular hypertension (COH) experimental glaucoma model was produced. Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (q-PCR), transwell co-culture of glial cells, flow cytometry assay, ELISA, Ca2+ image, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) techniques were employed to investigate the interaction of Müller cells and microglia, and its underlying mechanisms in COH retina. RESULTS: We first showed that Müller cell activation in mice with COH induced microglia activation through the ATP/P2X7 receptor pathway. The activation of microglia resulted in a significant increase in mRNA and protein levels of pro-inflammatory factors, such as tumor necrosis factor-α and interleukin-6. These inflammatory factors in turn caused the up-regulation of mRNA expression of pro-inflammatory factors in Müller cells through a positive feedback manner. CONCLUSIONS: These findings provide robust evidence, for the first time, that retinal inflammatory response may be aggravated by an interplay between activated two types of glial cells. These results also suggest that to reduce the interplay between Müller cells and microglia could be a potential effective strategy for preventing the loss of RGCs in glaucoma.

Tumor necrosis factor-alpha aggravates gliosis and inflammation of activated retinal Müller cells.

Tumor necrosis factor-alpha (TNF-α), a major inflammatory factor released from activated retinal glial cells, is implicated in the pathogenesis of glaucoma. In this study, we investigated whether and how TNF-α may affect functional conditions of activated retinal Müller cells. Our results showed that in the group I metabotropic glutamate receptor (mGluR I) agonist DHPG-activated cultured Müller cells, TNF-α treatment aggravated cell gliosis, as evidenced by significantly increased expression of glial fibrillary acidic protein (GFAP). TNF-α treatment of the DHPG-activated Müller cells decreased cell proliferation and induced cell apoptosis. In normal Müller cells, TNF-α treatment increased the mRNA levels of leukocyte inhibitory factor (LIF), intercellular cell adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and chemokine C-C-motif ligand 2 (CCL2), which could be significantly attenuated when Müller cells were pre-activated. However, TNF-α-induced elevation in mRNA levels of inflammatory factors, such as TNF-α, inducible nitric oxide synthase (iNOS), and interleukin-6 (IL-6), in normal Müller cells still kept higher levels when Müller cells were pre-activated. Furthermore, the TNF-α-induced changes of cytokines were partially mediated by NF-κB signaling pathway. Our results suggest that TNF-α may promote gliosis and inflammatory response of activated Müller cells, thus aggravating RGC injury in glaucoma.

Dopamine D1 receptor-mediated upregulation of BKCa currents modifies Müller cell gliosis in a rat chronic ocular hypertension model.

Müller cell gliosis is a common response in many retinal pathological conditions. We previously demonstrated that downregulation of Kir channels contributes to Müller cell gliosis in a rat chronic ocular hypertension (COH) model. Here, the possible involvement of outward K+ currents in Müller cell gliosis was investigated. Outward K+ current densities in Müller cells isolated from COH rats, as compared with those in normal rats, showed a significant increase, which was mainly contributed by large-conductance Ca2+ -activated K+ (BKCa ) channels. The involvement of BKCa channels in Müller cell gliosis is suggested by the fact that glial fibrillary acidic protein (GFAP) levels were augmented in COH retinas when these channels were suppressed by intravitreal injections of iberiotoxin. In COH retinas an increase in dopamine (DA) D1 receptor (D1R) expression in Müller cells was revealed by both immunohistochemistry and Western blotting. Moreover, protein levels of tyrosine hydroxylase were also increased, and consistent to this, retinal DA contents were elevated. SKF81297, a selective D1R agonist, enhanced BKCa currents of normal Müller cells through intracellular cAMP-PKA signaling pathway. Furthermore, GFAP levels were increased by the D1R antagonist SCH23390 injected intravitreally through eliminating the BKCa current upregulation in COH retinas, but partially reduced by SKF81297. All these results strongly suggest that the DA-D1R system may be activated to a stronger extent in COH rat retinas, thus increasing BKCa currents of Müller cells. The upregulation of BKCa channels may antagonize the Kir channel inhibition-induced depolarization of Müller cells, thereby attenuating the gliosis of these cells.

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.

Rapamycin alleviates brain edema after focal cerebral ischemia reperfusion in rats.

Brain edema is a major consequence of cerebral ischemia reperfusion. However, few effective therapeutic options are available for retarding the brain edema progression after cerebral ischemia. Recently, rapamycin has been shown to produce neuroprotective effects in rats after cerebral ischemia reperfusion. Whether rapamycin could alleviate this brain edema injury is still unclear. In this study, the rat stroke model was induced by a 1-h left transient middle cerebral artery occlusion using an intraluminal filament, followed by 48 h of reperfusion. The effects of rapamycin (250 µg/kg body weight, intraperitoneal; i.p.) on brain edema progression were evaluated. The results showed that rapamycin treatment significantly reduced the infarct volume, the water content of the brain tissue and the Evans blue extravasation through the blood-brain barrier (BBB). Rapamycin treatment could improve histological appearance of the brain tissue, increased the capillary lumen space and maintain the integrity of BBB. Rapamycin also inhibited matrix metalloproteinase 9 (MMP9) and aquaporin 4 (AQP4) expression. These data imply that rapamycin could improve brain edema progression after reperfusion injury through maintaining BBB integrity and inhibiting MMP9 and AQP4 expression. The data of this study provide a new possible approach for improving brain edema after cerebral ischemia reperfusion by administration of rapamycin.

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.

Activation of retinal glial cells contributes to the degeneration of ganglion cells in experimental glaucoma.

Elevation of intraocular pressure (IOP) is a major risk factor for neurodegeneration in glaucoma. Glial cells, which play an important role in normal functioning of retinal neurons, are well involved into retinal ganglion cell (RGC) degeneration in experimental glaucoma animal models generated by elevated IOP. In response to elevated IOP, mGluR I is first activated and Kir4.1 channels are subsequently inhibited, which leads to the activation of Müller cells. Müller cell activation is followed by a complex process, including proliferation, release of inflammatory and growth factors (gliosis). Gliosis is further regulated by several factors. Activated Müller cells contribute to RGC degeneration through generating glutamate receptor-mediated excitotoxicity, releasing cytotoxic factors and inducing microglia activation. Elevated IOP activates microglia, and following morphological and functional changes, these cells, as resident immune cells in the retina, show adaptive immune responses, including an enhanced release of pro-inflammatory factors (tumor neurosis factor-α, interleukins, etc.). These ATP and Toll-like receptor-mediated responses are further regulated by heat shock proteins, CD200R, chemokine receptors, and metabotropic purinergic receptors, may aggravate RGC loss. In the optic nerve head, astrogliosis is initiated and regulated by a complex reaction process, including purines, transmitters, chemokines, growth factors and cytokines, which contributes to RGC axon injury through releasing pro-inflammatory factors and changing extracellular matrix in glaucoma. The effects of activated glial cells on RGCs are further modified by the interplay among different types of glial cells. This review is concluded by presenting an in-depth discussion of possible research directions in this field in the future.

EphA4/ephrinA3 reverse signaling induced Müller cell gliosis and production of pro-inflammatory cytokines in experimental glaucoma.

Previous work showed that ephrinA3/EphA4 forward signaling contributed to retinal ganglion cell (RGC) damage in experimental glaucoma. Since up-regulated patterns of ephrinA3 and EphA4 were observed in Müller cells and RGCs, an EphA4/ephrinA3 reverse signaling may exist in Müller cells of chronic ocular hypertension (COH) retina. We investigated effects of EphA4/ephrinA3 reverse signaling activation on Müller cells in COH retina. Intravitreal injection of the ephrinA3 agonist EphA4-Fc increased glial fibrillary acidic protein (GFAP) levels in normal retinas, suggestive of Müller cell gliosis, which was confirmed in purified cultured Müller cells treated with EphA4-Fc. These effects were mediated by intracellular STAT3 signaling pathway as phosphorylated STAT3 (p-STAT3) levels and ratios of p-STAT3/STAT3 were significantly increased in both COH retinas and EphA4-Fc intravitreally injected retinas, as well as in EphA4-Fc treated purified cultured Müller cells. The increase of GFAP protein levels in EphA4-Fc-injected retinas and EphA4-Fc treated purified cultured Müller cells could be partially eliminated by stattic, a selective STAT3 blocker. Co-immunoprecipitation results testified to the presence of interaction between ephrinA3 and STAT3/p-STAT3. In addition, intravitreal injection of EphA4-Fc or EphA4-Fc treatment of cultured Müller cells significantly up-regulated mRNA and protein contents of pro-inflammatory cytokines. Moreover, intravitreal injection of EphA4-Fc increased the number of apoptotic RGCs, which could be reversed by the tyrosine kinase blocker PP2. Overall, EphA4/ephrinA3 reverse signaling may induce Müller cell gliosis and increases release of pro-inflammatory factors, which could contribute to RGC death in glaucoma. Inhibition of EphA4/ephrinA3 signaling may provide an effective neuroprotection in glaucoma.

L- and T-type Ca2+ channels dichotomously contribute to retinal ganglion cell injury in experimental glaucoma.

Retinal ganglion cell apoptotic death is the main pathological characteristic of glaucoma, which is the leading cause of irreversible blindness. Disruption of Ca2+ homeostasis plays an important role in glaucoma. Voltage-gated Ca2+ channel blockers have been shown to improve vision in patients with glaucoma. However, whether and how voltage-gated Ca2+ channels are involved in retinal ganglion cell apoptotic death are largely unknown. In this study, we found that total Ca2+ current densities in retinal ganglion cells were reduced in a rat model of chronic ocular hypertension experimental glaucoma, as determined by whole-cell patch-clamp electrophysiological recordings. Further analysis showed that L-type Ca2+ currents were downregulated while T-type Ca2+ currents were upregulated at the later stage of glaucoma. Western blot assay and immunofluorescence experiments confirmed that expression of the CaV1.2 subunit of L-type Ca2+ channels was reduced and expression of the CaV3.3 subunit of T-type Ca2+ channels was increased in retinas of the chronic ocular hypertension model. Soluble tumor necrosis factor-α, an important inflammatory factor, inhibited the L-type Ca2+ current of isolated retinal ganglion cells from control rats and enhanced the T-type Ca2+ current. These changes were blocked by the tumor necrosis factor-α inhibitor XPro1595, indicating that both types of Ca2+ currents may be mediated by soluble tumor necrosis factor-α. The intracellular mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and nuclear factor kappa-B signaling pathway mediate the effects of tumor necrosis factor-α. TUNEL assays revealed that mibefradil, a T-type calcium channel blocker, reduced the number of apoptotic retinal ganglion cells in the rat model of chronic ocular hypertension. These results suggest that T-type Ca2+ channels are involved in disrupted Ca2+ homeostasis and apoptosis of retinal ganglion cells in glaucoma, and application of T-type Ca2+ channel blockers, especially a specific CaV3.3 blocker, may be a potential strategy for the treatment of glaucoma.

Dopamine receptor-mediated roles on retinal ganglion cell hyperexcitability and injury in experimental glaucoma.

Extraordinary excitability (hyperexcitability) is closely related to retinal ganglion cell (RGC) injury in glaucoma. Dopamine (DA) and its receptors are involved in modulating RGC excitability. We investigated how DA system affects RGC injury in chronic ocular hypertension (COH) experimental glaucoma model. Western blotting and immunohistochemistry results revealed that expression of DA D2-like receptor (D2R) in RGCs was increased in COH retinas. Patch-clamp recordings showed that outward K+ currents were downregulated, while Na+ currents and NaV1.6 expression were upregulated in RGCs of COH retinas, which could be reversed by intravitreal pre-injection of the D2R antagonist sulpiride, but not by the D1-like receptor (D1R) antagonist SCH23390. However, pre-injection of the D1R agonist SKF81297 could partially reverse the increased expression of NaV1.6 proteins. Consistently, the numbers of evoked action potentials induced by current injections were increased in RGCs of COH retinas, indicating that RGCs may be in a condition of hyperexcitability. The increased frequency of evoked action potentials could be partially block by pre-injection of sulpiride, SKF81297 or DA, respectively. Furthermore, the increased number of TUNEL-positive RGCs in COH retinas could be partially reduced by intravitreal pre-injection of sulpiride, but not by pre-injection of SCH23390. Moreover, pre-injection of SKF81297 or DA could reduce the number of TUNEL-positive RGCs in COH retinas. All these results indicate that in COH retina, activation of D2R enhances RGC hyperexcitability and injury, while activation of D1R results in the opposite effects. Selective inhibition of D2R or activation of D1R may be an effective strategy for treatment of glaucoma.

P2X7/P2X4 Receptors Mediate Proliferation and Migration of Retinal Microglia in Experimental Glaucoma in Mice.

Microglia are involved in the inflammatory response and retinal ganglion cell damage in glaucoma. Here, we investigated how microglia proliferate and migrate in a mouse model of chronic ocular hypertension (COH). In COH retinas, the microglial proliferation that occurred was inhibited by the P2X7 receptor (P2X7R) blocker BBG or P2X7R knockout, but not by the P2X4R blocker 5-BDBD. Treatment of primary cultured microglia with BzATP, a P2X7R agonist, mimicked the effects of cell proliferation and migration in COH retinas through the intracellular MEK/ERK signaling pathway. Transwell migration assays showed that the P2X4R agonist CTP induced microglial migration, which was completely blocked by 5-BDBD. In vivo and in vitro experiments demonstrated that ATP, released from activated Müller cells through connexin43 hemichannels, acted on P2X7R to induce microglial proliferation, and acted on P2X4R/P2X7R (mainly P2X4R) to induce microglial migration. Our results suggest that inhibiting the interaction of Müller cells and microglia may attenuate microglial proliferation and migration in glaucoma.

Elevation of p-NR2A(S1232) by Cdk5/p35 contributes to retinal ganglion cell apoptosis in a rat experimental glaucoma model.

Glaucoma, mainly caused by high intraocular pressure (IOP), is characterized by apoptotic death of retinal ganglion cells (RGCs). We investigated the possible involvement of cyclin-dependent kinase 5 (Cdk5) and its activator p35, which have been implicated in a variety of neurological disorders, in RGC apoptosis in a rat experimental glaucoma model reproduced by blocking episcleral veins. Cholera toxin B subunit (CTB) retrogradely labeled RGCs displayed a dramatic reduction in number both in the central and peripheral retina on day 14 (D14) (P<0.05 vs. control), D21 (P<0.01 vs. control) and D28 (P<0.001 vs. control) after operation. Terminal dUTP nick end labeling (TUNEL)-positive cells were detected on D14 both in the central and peripheral regions, and numerous TUNEL-positive cells were found on D21 and D28 in both the regions (P all<0.001 vs. control). As compared with the control eyes, the expression level of Cdk5 was significantly increased on D21 (P<0.001), whereas that of p35 displayed a marked increase on D14 (P<0.01) and D21 (P<0.001). Meanwhile, both NR2A and p-NR2A(S1232) increased from D14 onwards (P<0.01 to 0.001). Co-immunoprecipitation indicated a direct interaction between Cdk5 and p-NR2A(S1232). Intraperitoneal injection of the Cdk5 inhibitor roscovitine remarkably inhibited RGC apoptosis (P<0.001 vs. vehicle group) and increased the number of CTB-labeled RGCs (P<0.05 to 0.01 vs. vehicle group) in whole flat-mounted retinas, which was accompanied by a significant decrease in expression levels of p35 and p-NR2A(S1232) (P all<0.01 vs. vehicle group). Our results suggest that elevation of p-NR2A(S1232) by Cdk5/p35 contributes to RGC apoptotic death in experimental glaucoma rats, which could be effectively ameliorated by inhibiting Cdk5/p35.

Melatonin potentiates glycine currents through a PLC/PKC signalling pathway in rat retinal ganglion cells.

In vertebrate retina, melatonin regulates various physiological functions. In this work we investigated the mechanisms underlying melatonin-induced potentiation of glycine currents in rat retinal ganglion cells (RGCs). Immunofluorescence double labelling showed that rat RGCs were solely immunoreactive to melatonin MT(2) receptors. Melatonin potentiated glycine currents of RGCs, which was reversed by the MT(2) receptor antagonist 4-P-PDOT. The melatonin effect was blocked by intracellular dialysis of GDP-beta-S. Either preincubation with pertussis toxin or application of the phosphatidylcholine (PC)-specific phospholipase C (PLC) inhibitor D609, but not the phosphatidylinositol (PI)-PLC inhibitor U73122, blocked the melatonin effect. The protein kinase C (PKC) activator PMA potentiated the glycine currents and in the presence of PMA melatonin failed to cause further potentiation of the currents, whereas application of the PKC inhibitor bisindolylmaleimide IV abolished the melatonin-induced potentiation. The melatonin effect persisted when [Ca(2+)](i) was chelated by BAPTA, and melatonin induced no increase in [Ca(2+)](i). Neither cAMP-PKA nor cGMP-PKG signalling pathways seemed to be involved because 8-Br-cAMP or 8-Br-cGMP failed to cause potentiation of the glycine currents and both the PKA inhibitor H-89 and the PKG inhibitor KT5823 did not block the melatonin-induced potentiation. In consequence, a distinct PC-PLC/PKC signalling pathway, following the activation of G(i/o)-coupled MT(2) receptors, is most likely responsible for the melatonin-induced potentiation of glycine currents of rat RGCs. Furthermore, in rat retinal slices melatonin potentiated light-evoked glycine receptor-mediated inhibitory postsynaptic currents in RGCs. These results suggest that melatonin, being at higher levels at night, may help animals to detect positive or negative contrast in night vision by modulating inhibitory signals largely mediated by glycinergic amacrine cells in the inner retina.

Deletion of tau attenuates heat shock-induced injury in cultured cortical neurons.

The microtubule-associated protein tau has been implicated in beta-amyloid- and glutamate-induced neurotoxicity. However, the potential role of tau in response to other insults to neurons remains unclear. In this study, we examined whether deletion of tau would change cell injury induced by heat shock in primary cultures of cortical neurons. After 30 min of a 45 degrees C heat shock, lactate dehydrogenase (LDH) release increased, reaching a peak at 6 hr in wild-type (WT) neurons. A significantly lower LDH release, with a peak delayed by 24 hr, was detected in tau knockout (TKO) neurons. After heat shock treatment, MAP-2 and tubulin staining of the processes of WT neurons revealed more dramatic abnormalities than in TKO neurons. Both WT and TKO neurons exhibited a similar elevation of HSP70 level but different time courses of Akt phosphorylation. In contrast to an early, brief response in WT neurons, TKO neurons displayed a late, but long-lasting increase in phosphorylation of Akt and its downstream target, glycogen synthase kinase 3beta. Additionally, inhibition of Akt activity aggravated the cell morbidity caused by heat shock exposure in both WT and TKO neurons, indicating a protective role of Akt against cell injury. In conclusion, our results demonstrate that deletion of tau attenuated heat shock-induced neuronal injury. Enhanced Akt response in the absence of endogenous tau is suggested to represent a compensatory mechanism for regulating cell reactions to stress stimuli.

Dopamine D2 Receptor-Mediated Modulation of Rat Retinal Ganglion Cell Excitability.

Ganglion cells (RGCs) are the sole output neurons of the retinal circuity. Here, we investigated whether and how dopamine D2 receptors modulate the excitability of dissociated rat RGCs. Application of the selective D2 receptor agonist quinpirole inhibited outward K+ currents, which were mainly mediated by glybenclamide- and 4-aminopyridine-sensitive channels, but not the tetraethylammonium-sensitive channel. In addition, quinpirole selectively enhanced Nav1.6 voltage-gated Na+ currents. The intracellular cAMP/protein kinase A, Ca2+/calmodulin-dependent protein kinase II, and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathways were responsible for the effects of quinpirole on K+ and Na+ currents, while phospholipase C/protein kinase C signaling was not involved. Under current-clamp conditions, the number of action potentials evoked by positive current injection was increased by quinpirole. Our results suggest that D2 receptor activation increases RGC excitability by suppressing outward K+ currents and enhancing Nav1.6 currents, which may affect retinal visual information processing.