Search

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.

Background: Connexin43 (Cx43) is one of major gap junction proteins in glial cells. Mutation in the gap junction protein alpha 1 gene of Cx43 has been detected in human glaucomatous retinas, suggestive of involvement of Cx43 in pathogenesis of glaucoma. However, the role of Cx43 in glaucoma has not been clearly elucidated. Methods: A mouse model of chronic ocular hypertension (COH) was produced by injecting magnetic microbeads into the eye anterior chamber. To explore the role of Rac1 in regulating Cx43 function, Rac1 conditional knockout in astrocytes was generated by subretinal injection of AAV-GFAP-Cre in Rac1flox/flox mice. The hemichannel activity was assayed by ethidium bromide uptake. The level and source of ATP were assayed by a commercial ATP assay kit, ATP sensors and removing microglia. Results: In this study, we showed that Cx43 were mainly expressed in retinal astrocytes. Intraocular pressure (IOP) elevation induced astrocyte activation, as evidenced by increased expressions of c-Fos and glial fibrillary acidic protein (GFAP), which results in downregulation of Cx43 and changes of Cx43 phosphorylation at Ser373 and Ser368 sites. In the optic nerve head of COH mice, Cx43 expression in Gap43 (an activity-dependent plasticity protein) positive astrocytes was reduced. In COH retinas, Rac1, a member of the Rho family, was activated, which was consistent with the decrease of Cx43 expression in a time course. Pharmacological inhibition of Rac1 inhibited the activity of its downstream molecule PAK1, and reversed the reduction of Cx43 expression and astrocyte activation induced by IOP elevation. Co-immunoprecipitation experiments further demonstrated the interactions between Cx43 and active Rac1 or p-PAK1. Inhibition of Rac1 or conditional knockout of Rac1 in macroglial cells increased the ATP release through Cx43 hemichannels in astrocytes of COH retinas. Additionally, Rac1 deletion in astrocytes upregulated the expression of adenosine A3 receptor in retinal ganglion cells (RGCs) and promoted RGC survival, at least at early stage of IOP elevation through activating adenosine receptors.Conclusions: Our results showed that Rac1 in astrocytes regulated glaucomatous RGC survival through Cx43-mediated ATP release. These findings suggest that modulation of Rac1/PAK1/Cx43 pathway in astrocytes may be a potential strategy of neuroprotection in glaucoma.

Autophagy has a fundamental role in maintaining cell homeostasis. Although autophagy has been implicated in glaucomatous pathology, how it regulates retinal ganglion cell (RGC) injury is largely unknown. In the present work, we found that biphasic autophagy in RGCs occurred in a mouse model of chronic ocular hypertension (COH), accompanied by activation of Rac1, a member of the Rho family. Rac1 conditional knockout (Rac1 cKO) in RGCs attenuated RGC apoptosis, in addition to blocking the increase in the number of autophagosomes and the expression of autophagy-related proteins (Beclin1, LC3-II/I, and p62) in COH retinas. Electron micrograph and double immunostaining of LAMP1 and LC3B showed that Rac1 cKO accelerated autolysosome fusion in RGC axons of COH mice. Inhibiting the first autophagic peak with 3-methyladenine or Atg13 siRNA reduced RGC apoptosis, whereas inhibiting the second autophagic peak with 3-MA or blocking autophagic flux by chloroquine increased RGC apoptosis. Furthermore, Rac1 cKO reduced the number of autophagosomes and apoptotic RGCs induced by rapamycin injected intravitreally, which suggests that Rac1 negatively regulates mTOR activity. Moreover, Rac1 deletion decreased Bak expression and did not interfere with the interaction of Beclin1 and Bcl-2 or Bak in COH retinas. In conclusion, autophagy promotes RGC apoptosis in the early stages of glaucoma and results in autophagic cell death in later stages. Rac1 deletion alleviates RGC damage by regulating the cross talk between autophagy and apoptosis through mTOR/Beclin1-Bak. Interfering with the Rac1/mTOR signaling pathway may provide a new strategy for treating glaucoma.

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 Muller cells. Our results showed that in the group I metabotropic glutamate receptor (mGluR I) agonist DHPG-activated cultured Muller 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 Muller cells decreased cell proliferation and induced cell apoptosis. In normal Muller 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 Muller 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 Muller cells still kept higher levels when Muller 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 Muller cells, thus aggravating RGC injury in glaucoma.

Previous studies have demonstrated that EphA4 participates in neuronal injury, and there is a strong interaction between ephrinA3 and EphA4. In this study, we showed that in a rat chronic ocular hypertension (COH) experimental glaucoma model, expression of EphA4 and ephrinA3 proteins was increased in retinal cells, including retinal ganglion cells (RGCs) and Muller cells, which may result in ephrinA3/EphA4 forward signaling activation on RGCs, as evidenced by increased p-EphA4/EphA4 ratio. Intravitreal injection of ephrinA3-Fc, an activator of EphA4, mimicked the effect of COH on p-EphA4/EphA4 and induced an increase in TUNEL-positive signals in normal retinas, which was accompanied by dendritic spine retraction and thinner dendrites in RGCs. Furthermore, Intravitreal injection of ephrinA3-Fc increased the levels of phosphorylated src and GluA2 (p-src and p-GluA2). Co-immunoprecipitation assay demonstrated interactions between EphA4, p-src and GluA2. Intravitreal injection of ephrinA3-Fc reduced the expression of GluA2 proteins on the surface of normal retinal cells, which was prevented by intravitreal injection of PP2, an inhibitor of src-family tyrosine kinases. Pre-injection of PP2 or the Ca2+-permeable GluA2-lacking AMPA receptor inhibitor Naspm significantly and partially reduced the number of TUNEL-positive RGCs in the ephrinA3-Fc-injected and COH retinas. Our results suggest that activated ephrinA3/EphA4 forward signaling promoted GluA2 endocytosis, then resulted in dendritic spine retraction of RGCs, thus contributing to RGC apoptosis in COH rats. Attenuation of the strength of ephrinA/EphA signaling in an appropriate manner may be an effective way for preventing the loss of RGCs in glaucoma.

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, Ca(2+)/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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12264-019-00431-3) contains supplementary material, which is available to authorized users.

Endocannabinoid signaling system is involved in regulating multiple neuronal functions in the central nervous system by activating G-protein coupled cannabinoid CB1 and CB2 receptors (CB1Rs and CB2Rs). Growing evidence has shown that CB1Rs and CB2Rs are extensively expressed in retinal ganglion cells (RGCs). Here, modulation of L- and T-types Ca 2+ channels by activating CB1Rs and CB2Rs in RGCs was investigated. Triple immunofluorescent staining showed that L-type subunit Ca V 1.2 was co-localized with T-type subunits (Ca V 3.1, Ca V 3.2 and Ca V 3.3) in rat RGCs. In acutely isolated rat RGCs, the CB1R agonist WIN55212-2 suppressed both peak and steady-state Ca 2+ currents in a dose-dependent manner, with IC 50 being 9.6 μM and 8.4 μM, respectively. It was further shown that activation of CB1Rs by WIN55212-2 or ACEA, another CB1R agonist, significantly suppressed both L- and T-type Ca 2+ currents, and shifted inactivation curve of T-type one toward hyperpolarization direction. While the effect on L-type Ca 2+ channels was mediated by intracellular cAMP/protein kinase A (PKA), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and calcium/calmodulin-dependent protein kinase II (CaMKII) signaling pathways, only CaMKII signaling pathway was involved in the effect on T-type Ca 2+ channels. Furthermore, CB65 and HU308, two specific CB2R agonists, significantly suppressed T-type Ca 2+ channels, which was mediated by intracellular cAMP/PKA and CaMKII signaling pathways, but had no effect on L-type channels. These results imply that endogenous cannabinoids may modulate the excitability and the output of RGCs by differentially suppressing the activity of L- and T-type Ca 2+ channels through activation of CB1Rs and CB2Rs. This article is part of the Special Issue entitled “A New Dawn in Cannabinoid Neurobiology”.

Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of the Rho GTPase family which plays important roles in dendritic spine morphology and plasticity, is a key regulator of cytoskeletal reorganization in dendrites and spines. Here, we investigated whether and how Rac1 modulates synaptic transmission in mouse retinal ganglion cells (RGCs) using selective conditional knockout of Rac1 (Rac1-cKO). Rac1-cKO significantly reduced the frequency of AMPA receptor-mediated miniature excitatory postsynaptic currents, while glycine/GABA(A) receptor-mediated miniature inhibitory postsynaptic currents were not affected. Although the total GluA1 protein level was increased in Rac1-cKO mice, its expression in the membrane component was unchanged. Rac1-cKO did not affect spine-like branch density in single dendrites, but significantly reduced the dendritic complexity, which resulted in a decrease in the total number of dendritic spine-like branches. These results suggest that Rac1 selectively affects excitatory synaptic transmission in RGCs by modulating dendritic complexity. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12264-019-00353-0) contains supplementary material, which is available to authorized users.

It was previously shown that EphB/ephrinB reverse signaling in retinal ganglion cells (RGCs) is activated and involved in RGC apoptosis in a rat chronic ocular hypertension (COH) model. In the present work, we first show that ephrinB/EphB forward signaling was activated in COH retinas, and RGC apoptosis in COH retinas was reduced by PP2, an inhibitor of ephrinB/EphB forward signaling. We further demonstrate that treatment of cultured Müller cells with ephrinB1-Fc, an EphB1 activator, or intravitreal injection of ephrinB1-Fc in normal rats induced an increase in phosphorylated EphB levels in these cells, indicating the activation of ephrinB/EphB forward signaling, similar to those in COH retinas. The ephrinB1-Fc treatment did not induce Müller cell gliosis, as evidenced by unchanged GFAP expression, but significantly up-regulated mRNA and protein levels of tumor necrosis factor-α (TNF-α) in Müller cells, thereby promoting RGC apoptosis. Production of TNF-α induced by the activation of ephrinB/EphB forward signaling was mediated by the NR2B subunit of NMDA receptors, which was followed by a distinct PI3K/Akt/NF-κB signaling pathway, as pharmacological interference of each step of this pathway caused a reduction of TNF-α production, thus attenuating RGC apoptosis. Functional analysis of forward and reverse signaling in such a unique system, in which ephrin and Eph exist respectively in a glial element and a neuronal element, is of theoretical importance. Moreover, our results also raise a possibility that suppression of ephrinB/EphB forward signaling may be a new strategy for ameliorating RGC apoptosis in glaucoma.

Group I metabotropic glutamate receptor (mGluR I) activation exerts a slow postsynaptic excitatory effect in the CNS. Here, the issues of whether and how this receptor is involved in regulating retinal ganglion cell (RGC) excitability were investigated in retinal slices using patch-clamp techniques. Under physiological conditions, RGCs displayed spontaneous firing. Extracellular application of LY367385 (10 µM)/MPEP (10 µM), selective mGluR1 and mGluR5 antagonists, respectively, significantly reduced the firing frequency, suggesting that glutamate endogenously released from bipolar cells constantly modulates RGC firing. DHPG (10 µM), an mGluR I agonist, significantly increased the firing and caused depolarization of the cells, which were reversed by LY367385, but not by MPEP, suggesting the involvement of the mGluR1 subtype. Intracellular Ca2+-dependent PI-PLC/PKC and calcium/calmodulin-dependent protein kinase II (CaMKII) signaling pathways mediated the DHPG-induced effects. In the presence of cocktail synaptic blockers (CNQX, D-AP5, bicuculline, and strychnine), which terminated the spontaneous firing in both ON and OFF RGCs, DHPG still induced depolarization and triggered the cells to fire. The DHPG-induced depolarization could not be blocked by TTX. In contrast, Ba2+, an inwardly rectifying potassium channel (Kir) blocker, and Cs+ and ZD7288, hyperpolarization-activated cation channel (I h) blockers, mimicked the effect of DHPG. Furthermore, in the presence of Ba2+/ZD7288, DHPG did not show further effects. Moreover, Kir and I h currents could be recorded in RGCs, and extracellular application of DHPG indeed suppressed these currents. Our results suggest that activation of mGluR I regulates the excitability of rat RGCs by inhibiting Kir and I h.

While activation of cannabinoid CB1 receptor (CB1R) regulates a variety of retinal neuronal functions by modulating ion channels in these cells, effect of activated cannabinoid receptors on Ca(2+) channels in retinal Müller cells is still largely unknown. In the present work we show that three subunits of T-type Ca(2+) channels, CaV3.1, CaV3.2 and CaV3.3, as well as one subunit of L-type Ca(2+) channels, CaV1.2, were expressed in rat Müller cells by immunofluorescent staining. Consistently, nimodipine- and mibefradil-sensitive Na(+) currents through L- and T-type Ca(2+) channels could be recorded electrophysiologically. The cannabinoid receptor agonist WIN55212-2 significantly suppressed Ca(2+) channel currents, mainly the T-type one, in acutely isolated rat Müller cells in a dose-dependent manner, with an IC50 of 3.98μM. The WIN55212-2 effect was not blocked by AM251/SR141716, specific CB1R antagonists. Similar suppression of the currents was observed when anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), endogenous ligands of cannabinoid receptors, were applied. Moreover, even though CB2 receptors (CB2Rs) were expressed in rat Müller cells, the effects of WIN55212-2 and 2-AG on Ca(2+) channel currents were not blocked by AM630, a selective CB2R antagonist. However, the effect of AEA could be partially rescued by AM630. These results suggest that WIN55212-2 and 2-AG receptor-independently suppressed the Ca(2+) channel currents in Müller cells, while AEA suppressed the currents partially through CB2Rs. The existence of receptor-dependent and -independent mechanisms suggests that cannabinoids may modulate Müller cell functions through multiple pathways.

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 orN-[3-[[4-[(3-aminopropyl)amino]butyl]amino]propyl]-1-naphthaleneacetamide trihydrochloride) (Naspm), a Ca2+-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.

By challenging specific receptors, melatonin synthesized and released by photoreceptors regulates various physiological functions in the vertebrate retina. Here, we studied modulatory effects of melatonin on K+ currents of rod-dominant ON type bipolar cells (Rod-ON-BCs) in rat retinal slices by patch-clamp techniques. Double immunofluorescence experiments conducted in isolated cell and retinal section preparations showed that the melatonin MT2 receptor was expressed in somata, dendrites and axon terminals of rat Rod-ON-BCs. Electrophysiologically, application of melatonin selectively inhibited the tetraethylammonium (TEA)-sensitive K+ current component, but did not show any effect on the 4-aminopyridine (4-AP)-sensitive component. Consistent with the immunocytochemical result, the melatonin effect was blocked by co-application of 4-phenyl-2-propionamidotetralin (4-P-PDOT), a specific MT2 receptor antagonist. Neither protein kinase A (PKA) nor protein kinase G (PKG) seemed to be involved because both the PKA inhibitor Rp-cAMP and the PKG inhibitor KT5823 did not block the melatonin-induced suppression of the K+ currents. In contrast, application of the phospholipase C (PLC) inhibitor U73122 or the protein kinase C (PKC) inhibitor bisindolylmaleimide IV (Bis IV) eliminated the melatonin effect, and when the Ca2+ chelator BAPTA-containing pipette was used, melatonin failed to inhibit the K+ currents. These results suggest that suppression of the TEA-sensitive K+ current component via activation of MT2 receptors expressed on rat Rod-ON-BCs may be mediated by a Ca2+-dependent PLC/inositol 1,4,5-trisphosphate (IP3)/PKC signaling pathway.

Truncated tau is widely detected in Alzheimer's disease brain, and caspase-3 has been considered as a major executioner for tau truncation at aspartate421 (D421), according to its capability of cleaving recombinant tau in vitro. Here we investigated the relationship between D421 truncated tau and caspase-3 in two transgenic mouse models for tauopathies. In adult transgenic mice, activated caspase-3 could not be detected in neurons containing truncated tau, with the exception of a few glia-like cells or neurons in postnatal mice. Caspase-3 expression exhibited a dramatic decrease at the early development stage, and kept at constantly low levels during adult stages in both wild type and transgenic mice. On the other hand, co-incubating brain homogenates from adult tau transgenic mice and ethanol-treated postnatal mice promoted tau truncation at D421, which was mildly reduced by caspase inhibitor, but completely suppressed by phosphatase inhibitor, indicating that hyperphosphorylated tau becomes a poor substrate for truncation at D421. Taken together, our study shows that insufficient caspase-3 expression and hyperphosphorylated status of tau in the adult transgenic mouse brain restrict caspase-3 as an efficient enzyme for tau truncation in vivo. Clearly, there is a caspase-3 independent mechanism responsible for tau truncation at D421 in these models.

Ghrelin is a novel brain-gut peptide and the endogenous ligand of growth hormone secretagogue receptor 1a (GHSR-1a). Evidences have been shown that ghrelin inhibited cell apoptosis in cardiocytes, endotheliocytes, osteoblasts, and so on. Recently, it was reported that ghrelin inhibited neuronal apoptosis of hypothalamus and hippocampus. However, little is known about the effects of ghrelin on cortical neurons during focal ischemia/reperfusion injury. In the present study, we showed that ghrelin (i.v.) prevented cortical neurons from injury induced by ischemia/reperfusion in vivo and by LPS, glutamate, NMDA and H(2)O(2) in vitro. We found that the expression of ghrelin's receptor (GHSR-1a) in rat cerebral cortex were obviously decreased by ischemia/reperfusion injury and increased by ghrelin (i.v.). Ghrelin up-regulated the expression of Bcl-2/Bax and HSP70, and inhibited caspase8, 9, 3 through GHSR-1a, which was contributed to the neuroprotective mechanism of ghrelin. Ghrelin might be an important regulator in therapeutic strategy of cortex injury.

Dopamine plays an important role in regulating neuronal functions in the central nervous system by activating the specific G-protein coupled receptors. Both D1 and D2 dopamine receptors are extensively distributed in the retinal neurons. In the present study, we investigated the effects of D1 receptor signaling on outward K(+) currents in acutely isolated rat retinal ganglion cells (RGCs) by patch-clamp techniques. Extracellular application of SKF81297 (10 μM), a specific D1 receptor agonist, significantly and reversibly suppressed outward K(+) currents of the cells, which was reversed by SCH23390 (10 μM), a selective D1 receptor antagonist. We further showed that SKF81297 mainly suppressed the glybenclamide (Gb)- and 4-aminopyridine (4-AP)-sensitive K(+) current components, but did not show effect on the tetraethylammonium (TEA)-sensitive one. Both protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) signaling pathways were likely involved in the SKF81297-induced suppression of the K(+) currents since either Rp-cAMP (10 μM), a cAMP/PKA signaling inhibitor, or KN-93 (10 μM), a specific CaMKII inhibitor, eliminated the SKF81297 effect. In contrast, neither protein kinase C (PKC) nor mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway seemed likely to be involved because both the PKC inhibitor bisindolylmaleimide IV (Bis IV) (10 μM) and the MAPK/ERK1/2 inhibitor U0126 (10 μM) did not block the SKF81297-induced suppression of the K(+) currents. These results suggest that activation of D1 receptors suppresses the Gb- and 4-AP-sensitive K(+) current components in rat RGCs through the intracellular PKA and CaMKII signaling pathways, thus modulating the RGC excitability.

Somatostatin (SRIF) modulates neurotransmitter release by activating the specific receptors (sst1-sst5). Our previous study showed that sst5 receptors are expressed in rat retinal GABAergic amacrine cells. Here, we investigated modulation of GABA release by SRIF in cultured amacrine cells, using patch-clamp techniques. The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the amacrine cells was significantly reduced by SRIF, which was partially reversed by BIM 23056, an sst5 receptor antagonist, and was further rescued by addition of CYN-154806, an sst2 receptor antagonist. Both nimodipine, an L-type Ca2+ channel blocker, and ω-conotoxin GVIA, an N-type Ca2+ channel blocker, suppressed the sIPSC frequency, and in the presence of nimodipine and ω-conotoxin GVIA, SRIF failed to further suppress the sIPSC frequency. Extracellular application of forskolin, an activator of adenylate cyclase, increased the sIPSC frequency, while the membrane permeable protein kinase A (PKA) inhibitor Rp-cAMP reduced it, and in the presence of Rp-cAMP, SRIF did not change sIPSCs. However, SRIF persisted to suppress the sIPSCs in the presence of KT5823, a protein kinase G (PKG) inhibitor. Moreover, pre-incubation with Bis IV, a protein kinase C (PKC) inhibitor, or pre-application of xestospongin C, an inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor, SRIF still suppressed the sIPSC frequency. All these results suggest that SRIF suppresses GABA release from the amacrine cells by inhibiting presynaptic Ca2+ channels, in part through activating sst5/sst2 receptors, a process that is mediated by the intracellular cAMP–PKA signaling pathway.

Cannabinoid CB1 receptor (CB1R) signaling system is extensively distributed in the vertebrate retina. Activation of CB1Rs regulates a variety of functions of retinal neurons through modulating different ion channels. In the present work we studied effects of this receptor signaling on K(+) channels in retinal ganglion cells by patch-clamp techniques. The CB1R agonist WIN55212-2 (WIN) suppressed outward K(+) currents in acutely isolated rat retinal ganglion cells in a dose-dependent manner, with an IC50 of 4.7 μM. We further showed that WIN mainly suppressed the tetraethylammonium (TEA)-sensitive K(+) current component. While CB1Rs were expressed in rat retinal ganglion cells, the WIN effect on K(+) currents was not blocked by either AM251/SR141716, specific CB1R antagonists, or AM630, a selective CB2R antagonist. Consistently, cAMP-protein kinase A (PKA) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways were unlikely involved in the WIN-induced suppression of the K(+) currents because both PKA inhibitors H-89/Rp-cAMP and MAPK/ERK1/2 inhibitor U0126 failed to block the WIN effects. WIN-induced suppression of the K(+) currents was not observed when WIN was intracellularly applied. Furthermore, an endogenous ligand of the cannabinoid receptor anandamide, the specific CB1R agonist ACEA and the selective CB2R agonist CB65 also suppressed the K(+) currents, and the effects were not blocked by AM251/SR141716 or AM630 respectively. All these results suggest that the WIN-induced suppression of the outward K(+) currents in rat retinal ganglion cells, thereby regulating the cell excitability, were not through CB1R/CB2R signaling pathways.

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.

The microtubule-associated protein tau has been implicated in β-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°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 3β. 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. © 2009 Wiley-Liss, Inc.

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), which has been originally isolated from rat stomach. It has been reported that ghrelin inhibited apoptosis in several cells, such as cardiomyocytes, endothelial cells, adipocyte, adrenal zona glomerulosa cells, pancreatic beta-cells, osteoblastic MC3T3-E1 cells, intestinal epithelial cells and hypothalamic neurons. However, it is unknown whether heat-shock protein 70 (HSP70) or apoptosis signal-regulating kinase 1 (ASK1) is the important target molecule which mediates the anti-apoptotic effects of ghrelin. We show that ghrelin inhibited ASK1 activity induced by sodium nitroprusside (SNP), inhibited ASK1-mediated caspase 3 activation and apoptosis in PC12 cells. Ghrelin promoted expression of HSP70. Quercetin, an inhibitor of HSP70, blocked the effects of ghrelin on ASK1 activity. Thus, ghrelin inhibits ASK1-mediated apoptosis and ASK1 activation by a mechanism involving induction of HSP70 expression. The results of the present study suggest the therapeutic potential of ghrelin for some pathological processes or disorders.

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.