Sigma-1R Protects Retinal Ganglion Cells in Optic Nerve Crush Model for Glaucoma.

Purpose: The purpose of this study was to determine the effects of the Sigma-1R (σ-1r) on retinal ganglion cell (RGC) survival following optic nerve crush (ONC) and the signaling mechanism involved in the σ-1r protection. Methods: The overall strategy was to induce injury by ONC and mitigate RGC death by increasing σ-1r expression and/or activate σ-1r activity in σ-1r K/O mice and wild type (WT) mice. AAV2-σ-1r vector was used to increase σ-1r expression and σ-1r agonist used to activate the σ-1r and RGCs were counted. Immunohistochemical and Western blot analysis determined phosphorylated (p)-c-Jun, c-Jun, and Caspase-3. Pattern electroretinography (PERG) determined RGC activity. Results: RGC counts and function were similar in pentazocine-treated WT mice when compared to untreated mice and in WT mice when compared with σ-1r K/O mice. Pentazocine-induced effects and the effects of σ-1r K/O were only observable after ONC. ONC resulted in decreased RGC counts and activity in both WT and σ-1r K/O mice, with σ-1r K/O mice experiencing significant decreases compared with WT mice. The σ-1r transgenic expression resulted in increased RGC counts and activity following ONC. In WT mice, treatment with σ-1r agonist pentazocine resulted in increased RGC counts and increased activity when compared with untreated WT mice. There were time-dependent increases in c-jun, p-c-jun, and caspase-3 expression in ONC mice that were mitigated with pentazocine-treatment. Conclusions: These findings suggest that the apoptotic pathway is involved in RGC losses seen in an ONC model. The σ-1r offers neuroprotection, as activation and/or transgenic expression of σ-1r attenuated the apoptotic pathway and restored RGCs number and function following ONC.

Preparing the Next Generation of Diverse Biomedical Researchers: The University of North Texas Health Science Center's Initiative for Maximizing Student Development (IMSD) Predoctoral Program.

The National Science Foundation (NSF) reports that underrepresented minority students are just as interested as their White counterparts in majoring in science upon entering college.1 However, the numbers of those receiving bachelors' degrees, attending graduate school, and earning doctorates remain lower than their White peers. To close this gap, the National Institutes of General Medical Science's (NIGMS) Initiative for Maximizing Student Development (IMSD) at University of Texas Health Science Center (UNTHSC) supports the timely completion of PhD degrees by underrepresented students and their transition into successful biomedical research careers. Throughout UNTHSC's IMSD training program, we have designed interventions anchored by the central hypothesis that PhD attainment requires attentiveness to multiple factors (knowledge, psychosocial, financial and self-efficacy). An assessment of program outcomes demonstrates a progressive increase in trainee retention. Importantly, not-withstanding quantitative measurable outcomes, trainee and mentor evaluations express the value in addressing multiple factors relevant to their success. Since 1996, our cumulative success of underrepresented minority students completing the doctorate increased from 64% (1996) to 84% completion (2018). Herein, we describe the UNTHSC IMSD training approach spanning its performance over two five-year cycles (2004-2008; 2009-2013) and new interventions created from lessons learned that influenced UNTHSC's newly awarded IMSD program (2017-2022).

Glucocorticoid-Induced Ocular Hypertension: Origins and New Approaches to Minimize.

Introduction: Glucocorticoids (GCs) have unique actions in their combined anti-inflammatory and immunosuppressive activities and are among the most commonly-prescribed drugs, particularly for inflammatory conditions. They are often used clinically to treat inflammatory eye diseases like uveitis, optic neuritis, conjunctivitis, keratitis and others, but are often accompanied by side effects, like ocular hypertension that can be vision threatening. Areas covered: The review will focus on the complex molecular mechanism of action of GCs that involve both transactivation and transrepression and their use therapeutically that can cause significant systemic side effects, particularly ocular hypertension that can lead to glaucoma. Expert Opinion: While we are still unclear as to all the mechanisms responsible for GC-induced ocular hypertension, however, there are potential novel therapies that are in development that can separate some of the anti-inflammatory therapeutic efficacy from their ocular hypertension side effect. This review provides some insight into these approaches.

Hybrid Compound SA-2 is Neuroprotective in Animal Models of Retinal Ganglion Cell Death.

Purpose: Determine the toxicity, bioavailability in the retina, and neuroprotective effects of a hybrid antioxidant-nitric oxide donor compound SA-2 against oxidative stress-induced retinal ganglion cell (RGC) death in neurodegenerative animal models. Methods: Optic nerve crush (ONC) and ischemia reperfusion (I/R) injury models were used in 12-week-old C57BL/6J mice to mimic conditions of glaucomatous neurodegeneration. Mice were treated intravitreally with either vehicle or SA-2. Retinal thickness was measured by spectral-domain optical coherence tomography (SD-OCT). The electroretinogram and pattern ERG (PERG) were used to assess retinal function. RGC survival was determined by counting RBPMS-positive RGCs and immunohistochemical analysis of superoxide dismutase 1 (SOD1) levels was carried out in the retina sections. Concentrations of SA-2 in the retina and choroid were determined using HPLC and MS. In addition, the direct effect of SA-2 treatment on RGC survival was assessed in ex vivo rat retinal explants under hypoxic (0.5% O2) conditions. Results: Compound SA-2 did not induce any appreciable change in retinal thickness, or in a- or b-wave amplitude in naive animals. SA-2 was found to be bioavailable in both the retina and choroid after a single intravitreal injection (2% wt/vol). An increase in SOD1 levels in the retina of mice subjected to ONC and SA-2 treatment, suggests an enhancement in antioxidant activity. SA-2 provided significant (P < 0.05) RGC protection in all three of the tested RGC injury models in rodents. PERG amplitudes were significantly higher in both I/R and ONC mouse eyes following SA-2 treatment (P ≤ 0.001) in comparison with the vehicle and control groups. Conclusions: Compound SA-2 was effective in preventing RGC death and loss of function in three different rodent models of acute RGC injury: ONC, I/R, and hypoxia.

Neuroprotective Effects of Psalmotoxin-1, an Acid-Sensing Ion Channel (ASIC) Inhibitor, in Ischemia Reperfusion in Mouse Eyes.

PURPOSE: The purpose of the current study is to assess changes in the expression of Acid-Sensing Ion Channel (ASIC)1a and ASIC2 in retinal ganglion cells (RGCs) after retinal ischemia and reperfusion (I/R) injury and to test if inhibition of ASIC1a provides RGC neuroprotection. METHODS: Transient ischemia was induced in one eye of C57BL/6 mice by raising intraocular pressure to 120 mmHg for 60 min followed by retinal reperfusion by restoring normal pressure. RGC function was measured by Pattern electroretinography (PERG). In addition, retinal ASIC1a and ASIC2 were observed by immunohistochemistry and western blot. Changes in calpain, fodrin, heat shock protein 70 (HSP70), Brn3a, super oxide dismutase-1 (SOD1), catalase, and glutathione perioxidase-4 (GPX4) protein levels were assessed by western blot. RGC numbers were measured by immunohistochemistry on whole retinal flat mounts using anti-RNA binding protein with multiple splicing (RBPMS) antibodies. Intravitreal injection of psalmotoxin-1, a selective ASIC1a blocker, was used to assess the neuroprotective effect of ASIC1a inhibition. RESULTS: Levels of ASIC1a and ASIC2 after I/R increased in RGCs. Upregulation of ASIC1a but not ASIC2 was attenuated by intravitreal injection of psalmotoxin-1. I/R induced activation of calpain and degradation of fodrin, HSP70, and reduction in Brn3a. In contrast, while psalmotoxin-1 attenuated calpain activation and increased Brn3a levels, it failed to block HSP70 degradation. Unlike SOD1 protein which was reduced, catalase protein levels increased after I/R. Psalmotoxin-1, although not affecting SOD1 and GPX4, increased catalase levels significantly. Psalmotoxin-1 also increased RBPMS-labeled RGCs following I/R as judged by immunohistochemistry of retinal flat mounts. Finally, psalmotoxin-1 enhanced the amplitude of PERG following I/R, suggesting partial rescue of RGC function. CONCLUSION: Psalmotoxin-1 appears to exert a neuroprotective effect under ischemic insults and targeting inhibition of ASICs may represent a new therapeutic approach in ischemic retinal diseases.

Targets of Neuroprotection in Glaucoma.

Progressive neurodegeneration of the optic nerve and the loss of retinal ganglion cells is a hallmark of glaucoma, the leading cause of irreversible blindness worldwide, with primary open-angle glaucoma (POAG) being the most frequent form of glaucoma in the Western world. While some genetic mutations have been identified for some glaucomas, those associated with POAG are limited and for most POAG patients, the etiology is still unclear. Unfortunately, treatment of this neurodegenerative disease and other retinal degenerative diseases is lacking. For POAG, most of the treatments focus on reducing aqueous humor formation, enhancing uveoscleral or conventional outflow, or lowering intraocular pressure through surgical means. These efforts, in some cases, do not always lead to a prevention of vision loss and therefore other strategies are needed to reduce or reverse the progressive neurodegeneration. In this review, we will highlight some of the ocular pharmacological approaches that are being tested to reduce neurodegeneration and provide some form of neuroprotection.

A feed-forward regulation of endothelin receptors by c-Jun in human non-pigmented ciliary epithelial cells and retinal ganglion cells.

c-Jun, c-Jun N-terminal kinase(JNK) and endothelin B (ETB) receptor have been shown to contribute to the pathogenesis of glaucoma. Previously, we reported that an increase of c-Jun and CCAAT/enhancer binding protein ß (C/EBPß) immunohistostaining is associated with upregulation of the ETB receptor within the ganglion cell layer of rats with elevated intraocular pressure (IOP). In addition, both transcription factors regulate the expression of the ETB receptor in human non-pigmented ciliary epithelial cells (HNPE). The current study addressed the mechanisms by which ET-1 produced upregulation of ET receptors in primary rat retinal ganglion cells (RGCs) and HNPE cells. Treatment of ET-1 and ET-3 increased the immunocytochemical staining of c-Jun and C/EBPß in primary rat RGCs and co-localization of both transcription factors was observed. A marked increase in DNA binding activity of AP-1 and C/EBPß as well as elevated protein levels of c-Jun and c-Jun-N-terminal kinase (JNK) were detected following ET-1 treatment in HNPE cells. Overexpression of ETA or ETB receptor promoted the upregulation of c-Jun and also elevated its promoter activity. In addition, upregulation of C/EBPß augmented DNA binding and mRNA expression of c-Jun, and furthermore, the interaction of c-Jun and C/EBPß was confirmed using co-immunoprecipitation. Apoptosis of HNPE cells was identified following ET-1 treatment, and overexpression of the ETA or ETB receptor produced enhanced apoptosis. ET-1 mediated upregulation of c-Jun and C/EBPß and their interaction may represent a novel mechanism contributing to the regulation of endothelin receptor expression. Reciprocally, c-Jun was also found to regulate the ET receptors and C/EBPß appeared to play a regulatory role in promoting expression of c-Jun. Taken together, the data suggests that ET-1 triggers the upregulation of c-Jun through both ETA and ETB receptors, and conversely c-Jun also upregulates endothelin receptor expression, thereby generating a positive feed-forward loop of endothelin receptor activation and expression. This feed-forward regulation may contribute to RGC death and astrocyte proliferation following ET-1 treatment.

Sigma-1 Receptor Regulates Mitochondrial Function in Glucose- and Oxygen-Deprived Retinal Ganglion Cells.

Purpose: Understanding the role of mitochondria in retinal ganglion cells (RGCs) is relevant to human disease as studies have shown mitochondrial abnormalities in primary open-angle glaucoma patients. This study seeks to determine the effects of the sigma-1 receptor (σ-1r) and its agonists on mitochondrial function in oxygen- and glucose- deprived (OGD) purified neonatal RGCs. Methods: Retinal ganglion cells were isolated from rat pups and subjected to OGD in varying conditions in the presence or absence of σ-1r agonist and antagonist and following addition of an AAV2-σ-1r vector that was used to increase σ-1r expression. Western blots and immunofluorescence microscopy validated findings. Mitochondrial function was determined by measuring mitochondrial membrane potential (Δψm) using the dye, fluorescence tetraethylbenzimidazolylcarbocyanineiodide (JC-1), and determination of cytochrome c oxidase activity using a cytochrome c oxidase assay kit. Caspase 3 and 7 activities were also measured using a luminescent assay kit. Results: Oxygen and glucose deprivation in RGCs resulted in decreased mitochondrial membrane potential and cytochrome c oxidase activity when compared with normoxic RGCs. σ-1r agonists or overexpression of the σ-1r restored the mitochondrial membrane potential comparable to normoxic conditions, while σ-1r antagonists abolished these effects. Oxygen and glucose depreavtation induced decreases in cytochrome c activity were partially restored by overexpression or activation of σ-1r. Caspase activity was increased in response to OGD and was decreased by the addition of σ-1r agonist, pentazocine, and following σ-1r overexpression. Conclusions: These data suggest that activation and/or overexpression of σ-1r restores RGCs mitochondrial function following OGD and that mitochondrial function is vital to the function of RGCs.

Principles of Ocular Pharmacology.

Recently, in a poll by Research America, a significant number of individuals placed losing their eyesight as having the greatest impact on their lives more so than other conditions, such as limb loss or memory loss. When they were also asked to rank which is the worst disease that could happen to them, blindness was ranked first by African-Americans and second by Caucasians, Hispanics, and Asians. Therefore, understanding the mechanisms of disease progression in the eye is extremely important if we want to make a difference in people's lives. In addition, developing treatment programs for these various diseases that could affect our eyesight is also critical. One of the most effective treatments we have is in the development of specific drugs that can be used to target various components of the mechanisms that lead to ocular disease. Understanding basic principles of the pharmacology of the eye is important if one seeks to develop effective treatments. As our population ages, the incidence of devastating eye diseases increases. It has been estimated that more than 65 million people suffer from glaucoma worldwide (Quigley and Broman. Br J Ophthalmol 90:262-267, 2006). Add to this the debilitating eye diseases of age-related macular degeneration, diabetic retinopathy, and cataract, the number of people effected exceeds 100 million. This chapter focuses on ocular pharmacology with specific emphasis on basic principles and outlining where in the various ocular sites are drug targets currently in use with effective drugs but also on future drug targets.

Glucocorticoid therapy and ocular hypertension.

The projected number of people who will develop age-related macular degeneration in estimated at 2020 is 196 million and is expected to reach 288 million in 2040. Also, the number of people with Diabetic retinopathy will grow from 126.6 million in 2010 to 191.0 million by 2030. In addition, it is estimated that there are 2.3 million people suffering from uveitis worldwide. Because of the anti-inflammatory properties of glucocorticoids (GCs), they are often used topically and/or intravitreally to treat ocular inflammation conditions or edema associated with macular degeneration and diabetic retinopathy. Unfortunately, ocular GC therapy can lead to severe side effects. Serious and sometimes irreversible eye damage can occur as a result of the development of GC-induced ocular hypertension causing secondary open-angle glaucoma. According to the world health organization, glaucoma is the second leading cause of blindness in the world and it is estimated that 80 million will suffer from glaucoma by 2020. In the current review, mechanisms of GC-induced damage in ocular tissue, GC-resistance, and enhancing GC therapy will be discussed.

Involvement of AMPA Receptor and Its Flip and Flop Isoforms in Retinal Ganglion Cell Death Following Oxygen/Glucose Deprivation.

PURPOSE: The α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors (AMPAR) subunits can be posttranscriptionally modified by alternative splicing forming flip and flop isoforms. We determined if an ischemia-like insult to retinal ganglion cells (RGCs) increases AMPAR susceptibility to s-AMPA-mediated excitotoxicity through changes in posttranscriptional modified isoforms. METHODS: Purified neonatal rat RGCs were subjected to either glucose deprivation (GD) or oxygen/glucose deprivation (OGD) conditions followed by treatment with either 100 µM s-AMPA or Kainic acid. A live-dead assay and caspase 3 assay was used to assess cell viability and apoptotic changes, respectively. We used JC-1 dye and dihydroethidium to measure mitochondria depolarization and reactive oxygen species (ROS), respectively. Calcium imaging with fura-2AM was used to determine intracellular calcium, while the fluorescently-labeled probe, Nanoprobe1, was used to detect calcium-permeable AMPARs. Quantitative PCR (qPCR) analysis was done to determine RNA editing sites AMPAR isoforms. RESULTS: Glucose deprivation, as well as an OGD insult followed by AMPAR stimulation, produced a significant increase in RGC death. Retinal ganglion cell death was independent of caspase 3/7 activity, but was accompanied by increased mitochondrial depolarization and increased ROS production. This was associated with an elevated intracellular Ca(2+) and calcium permeable-AMPARs. The mRNA expression of GLUA2 and GLUA3 flop isoform decreased significantly, while no appreciable changes were found in the corresponding flip isoforms. There were no changes in the Q/R editing of GLUA2, while R/G editing of GLUA2 flop declined under these conditions. CONCLUSIONS: Following oxidative injury, RGCs become more susceptible to AMPAR-mediated excitotoxicity. RNA editing and changes in alternative spliced flip and flop isoforms of AMPAR subunits may contribute to increased RGC death.

Design and synthesis of novel hybrid sydnonimine and prodrug useful for glaucomatous optic neuropathy.

Synthesis and bioactivity of novel dual acting nitric oxide releasing and reactive oxygen scavenging hybrid compound SA-2 is described. The hybrid molecule SA-2 significantly increased the superoxide dismutase enzyme level and protected the photoreceptor cells from H2O2 induced oxidative stress. Synthesis of ocular esterase sensitive aceloxy alkyl carbamate prodrug SA-4 with improved aqueous half-life is achieved to aid topical ocular formulation. This class of hybrid molecule and prodrug may have dual potential of improved IOP lowering and neuroprotection in glaucomatous optic neuropathy.

Endothelin-Mediated Changes in Gene Expression in Isolated Purified Rat Retinal Ganglion Cells.

PURPOSE: A growing body of evidence suggests that the vasoactive peptides endothelins (ETs) and their receptors (primarily the ETB receptor) are contributors to neurodegeneration in glaucoma. However, actions of ETs in retinal ganglion cells (RGCs) are not fully understood. The purpose of this study was to determine the effects of ETs on gene expression in primary RGCs. METHODS: Primary RGCs isolated from rat pups were treated with 100 nM of ET-1, ET-2, or ET-3 for 24 hours. Total RNA was extracted followed by cDNA synthesis. Changes in gene expression in RGCs were detected using Affymetrix Rat Genome 230 2.0 microarray and categorized by DAVID analysis. Real-time PCR was used to validate gene expression, and immunocytochemistry and immunoblotting to confirm the protein expression of regulated genes. RESULTS: There was more than 2-fold upregulation of 328, 378, or 372 genes, and downregulation of 48, 33, or 28 genes with ET-1, ET-2, or ET-3 treatment, respectively, compared to untreated controls. The Bcl-2 family, S100 family, matrix metalloproteinases, c-Jun, and ET receptors were the major genes or proteins that were regulated by endothelin treatment. Immunocytochemical staining revealed a significant increase in ETA receptor, ETB receptor, growth associated protein 43 (GAP-43), phosphorylated c-Jun, c-Jun, and Bax with ET-1 treatment. Protein levels of GAP-43 and c-Jun were confirmed by immunoblotting. CONCLUSIONS: Expression of key proteins having regulatory roles in apoptosis, calcium homeostasis, cell signaling, and matrix remodeling were altered by treatment with endothelins. The elucidation of molecular mechanisms underlying endothelins' actions in RGCs will help understand endothelin-mediated neurodegenerative changes during ocular hypertension.

AMPA receptor desensitization is the determinant of AMPA receptor mediated excitotoxicity in purified retinal ganglion cells.

The ionotropic glutamate receptors (iGLuR) have been hypothesized to play a role in neuronal pathogenesis by mediating excitotoxic death. Previous studies on iGluR in the retina have focused on two broad classes of receptors: NMDA and non-NMDA receptors including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and kainate receptor. In this study, we examined the role of receptor desensitization on the specific excitotoxic effects of AMPAR activation on primary retinal ganglion cells (RGCs). Purified rat RGCs were isolated from postnatal day 4-7 Sprague-Dawley rats. Calcium imaging was used to identify the functionality of the AMPARs and selectivity of the s-AMPA agonist. Phosphorylated CREB and ERK1/2 expression were performed following s-AMPA treatment. s-AMPA excitotoxicity was determined by JC-1 mitochondrial membrane depolarization assay, caspase 3/7 luciferase activity assay, immunoblot analysis for α-fodrin, and Live (calcein AM)/Dead (ethidium homodimer-1) assay. RGC cultures of 98% purity, lacking Iba1 and GFAP expression were used for the present studies. Isolated prenatal RGCs expressed calcium permeable AMPAR and s-AMPA (100 µM) treatment of cultured RGCs significantly increased phosphorylation of CREB but not that of ERK1/2. A prolonged (6 h) AMPAR activation in purified RGCs using s-AMPA (100 µM) did not depolarize the RGC mitochondrial membrane potential. In addition, treatment of cultured RGCs with s-AMPA, both in the presence and absence of trophic factors (BDNF and CNTF), did not increase caspase 3/7 activities or the cleavage of α-fodrin (neuronal apoptosis marker), as compared to untreated controls. Lastly, a significant increase in cell survival of RGCs was observed after s-AMPA treatment as compared to control untreated RGCs. However, preventing the desensitization of AMPAR with the treatment with either kainic acid (100 µM) or the combination of s-AMPA and cyclothiazide (50 µM) significantly reduced cell survivability. Activation of the AMPAR in RGCs does not appear to activate a signaling cascade to apoptosis, suggesting that RGCs in vitro are not susceptible to AMPA excitotoxicity as previously hypothesized. Conversely, preventing AMPAR desensitization through differential agonist activation caused AMPAR mediated excitotoxicity. Activation of the AMPAR in increasing CREB phosphorylation was dependent on the presence of calcium, which may help explain this action in increasing RGC survival.

Neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma.

PURPOSE: Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP), leading to optic nerve head (ONH) cupping, axon loss, and apoptosis of retinal ganglion cells (RGCs), which could ultimately result in blindness. Brn3b is a class-4 POU domain transcription factor that plays a key role in RGC development, axon outgrowth, and pathfinding. Previous studies suggest that a decrease in Brn3b levels occurs in animal models of glaucoma. The goal of this study was to determine if adeno-associated virus (AAV)-directed overexpression of the Brn3b protein could have neuroprotective effects following elevated IOP-mediated neurodegeneration. METHODS: Intraocular pressure was elevated in one eye of Brown Norway rats (Rattus norvegicus), following which the IOP-elevated eyes were intravitreally injected with AAV constructs encoding either the GFP (rAAV-CMV-GFP and rAAV-hsyn-GFP) or Brn3b (rAAV-CMV-Brn3b and rAAV-hsyn-Brn3b). Retina sections through the ONH were stained for synaptic plasticity markers and neuroprotection was assessed by RGC counts and visual acuity tests. RESULTS: Adeno-associated virus-mediated expression of the Brn3b protein in IOP-elevated rat eyes promoted an upregulation of growth associated protein-43 (GAP-43), actin binding LIM protein (abLIM) and acetylated α-tubulin (ac-Tuba) both posterior to the ONH and in RGCs. The RGC survival as well as axon integrity score were significantly improved in IOP-elevated rAAV-hsyn-Brn3b-injected rats compared with those of the IOP-elevated rAAV-hsyn-GFP- injected rats. Additionally, intravitreal rAAV-hsyn-Brn3b administration significantly restored the visual optomotor response in IOP-elevated rat eyes. CONCLUSIONS: Adeno-associated virus-mediated Brn3b protein expression may be a suitable approach for promoting neuroprotection in animal models of glaucoma.

Methylene blue promotes quiescence of rat neural progenitor cells.

Neural stem cell-based treatment holds a new therapeutic opportunity for neurodegenerative disorders. Here, we investigated the effect of methylene blue on proliferation and differentiation of rat neural progenitor cells (NPCs) both in vitro and in vivo. We found that methylene blue inhibited proliferation and promoted quiescence of NPCs in vitro without affecting committed neuronal differentiation. Consistently, intracerebroventricular infusion of methylene blue significantly inhibited NPC proliferation at the subventricular zone (SVZ). Methylene blue inhibited mTOR signaling along with down-regulation of cyclins in NPCs in vitro and in vivo. In summary, our study indicates that methylene blue may delay NPC senescence through enhancing NPCs quiescence.

Sigma-1 receptor stimulation protects retinal ganglion cells from ischemia-like insult through the activation of extracellular-signal-regulated kinases 1/2.

Sigma-1 receptor (σ-1) activation and mitogen-activated protein kinases (MAPKs) have been shown to protect retinal ganglion cells (RGCs) from cell death. The purpose of this study was to determine if σ-1 receptor stimulation with pentazocine could promote neuroprotection under conditions of an ischemia-like insult (oxygen glucose deprivation (OGD)) through the phosphorylation of extracellular signal regulated kinase (pERK)1/2. Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using Thy1.1 antibodies. RGCs were cultured for 7 days before subjecting the cells to an OGD insult (0.5% oxygen in glucose-free medium) for 6 h. During the OGD, RGCs were treated with pentazocine (σ-1 receptor agonist) with or without BD 1047 (σ-1 receptor antagonist). In other experiments, primary RGCs were treated with pentazocine in the presence or absence of an MEK1/2 inhibitor, PD098059. Cell survival/death was assessed by staining with the calcein-AM/ethidium homodimer reagent. Levels of pERK1/2, total ERK1/2, and beta tubulin expression were determined by immunoblotting and immunofluorescence staining. RGCs subjected to OGD for 6 h induced 50% cell death in primary RGCs (p < 0.001) and inhibited pERK1/2 expression by 65% (p < 0.001). Cell death was attenuated when RGCs were treated with pentazocine under OGD (p < 0.001) and pERK1/2 expression was increased by 1.6 fold (p < 0.05) compared to OGD treated RGCs without pentazocine treatment. The co-treatment of PD098059 (MEK1/2 inhibitor) with pentazocine significantly abolished the protective effects of pentazocine on the RGCs during this OGD insult. Activation of the σ-1 receptor is a neuroprotective target that can protect RGCs from an ischemia-like insult. These results also established a direct relationship between σ-1 receptor stimulation and the neuroprotective effects of the ERK1/2 pathway in purified RGCs subjected to OGD. These findings suggest that activation of the σ-1 receptor may be a therapeutic target for neuroprotection particularly relevant to ocular neurodegenerative diseases that effect RGCs.

Role of the alternatively spliced glucocorticoid receptor isoform GRß in steroid responsiveness and glaucoma.

Glucocorticoid (GC)-induced ocular hypertension (OHT) is a serious side effect of GC therapy in susceptible individuals. This OHT is due to increased aqueous humor (AH) outflow resistance in the trabecular meshwork (TM) caused by GC-mediated changes in TM structure and function. GCs may also play a role in the development of primary open-angle glaucoma (POAG). Elevated cortisol levels in the AH or enhanced GC sensitivity may be one of the reasons for elevated intraocular pressure in POAG patients. The GC OHT responder population is at greater risk of developing POAG compared with non-responders. We recently have gained insight into the molecular mechanisms responsible for this differential GC responsiveness, which is attributed to differences in GC receptor isoform expression in the TM. This article summarizes current knowledge on alternative GC receptor splicing to generate GC receptor alpha (GRα) and GRß and their roles in the regulation of GC responsiveness in normal and glaucoma TM.

A forensic path to RGC-5 cell line identification: lessons learned.

In 2001, a transformed cell line RGC-5 was developed from the rat retina that was thought to be of retinal ganglion cell origin. Since that time many investigators have used this line in a wide variety of studies to understand better retinal ganglion cell activity, cell signaling, and neuroprotection. Recently, a publication emerged that claimed that this RGC-5 cell line was derived from mouse and not rat, and other studies also indicated the expression of certain proteins that typically were not associated with retinal ganglion cells. This certainly came as a shock not only to the originators of this cell line, but also to others who have been using this as an in vitro model of rat retinal ganglion cells. As a result, we undertook experiments to determine if the RGC-5 cell line currently in use may have been mischaracterized. We, indeed, found that the RGC-5 cell line was of mouse and not rat origin, as was claimed originally in the original research report. We further determined whether these cells were of retinal ganglion origin. Our findings showed conclusively that RGC-5 cells were, indeed, of mouse origin and, using additional cytogenetic profile testing, karyotyping, and genetic and protein profiling, we concluded that these cells were not of retinal ganglion cell origin, but were the cell line 661W, a mouse SV-40 T antigen transformed photoreceptor cell line. The 661W cell line also was present in the laboratory of the originating laboratory and probably resulted in cross-contamination. The present study reviews some of the errors that were made in misidentifying the RGC-5 cell line and offers some insight as to how this may have happened, and ways one can avoid mischaracterization of a potentially important cell line.