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.

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.

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.

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.

Sigma-1 receptor stimulation attenuates calcium influx through activated L-type Voltage Gated Calcium Channels in purified retinal ganglion cells.

Sigma-1 receptors (σ-1rs) exert neuroprotective effects on retinal ganglion cells (RGCs) both in vivo and in vitro. This receptor has unique properties through its actions on several voltage-gated and ligand-gated channels. The purpose of this study was to investigate the role that σ-1rs play in regulating cell calcium dynamics through activated L-type Voltage Gated Calcium Channels (L-type VGCCs) in purified RGCs. RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy1.1 antibody. Calcium imaging was used to measure changes in intracellular calcium after depolarizing the cells with potassium chloride (KCl) in the presence or absence of two σ-1r agonists [(+)-SKF10047 and (+)-Pentazocine], one σ-1r antagonist (BD1047), and one L-type VGCC antagonist (Verapamil). Finally, co-localization studies were completed to assess the proximity of σ-1r with L-type VGCCs in purified RGCs. VGCCs were activated using KCl (20 mM). Pre-treatment with a known L-type VGCC blocker demonstrated a 57% decrease of calcium ion influx through activated VGCCs. Calcium imaging results also demonstrated that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Antagonist treatment using BD1047 demonstrated a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs, implying that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in purified RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in purified RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in purified RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in purified RGCs implied that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs.

Canonical transient receptor potential 6 (TRPC6), a redox-regulated cation channel.

This study examined the effect of H(2)O(2) on the TRPC6 channel and its underlying mechanisms using a TRPC6 heterologous expression system. In TRPC6-expressing HEK293T cells, H(2)O(2) significantly stimulated Ca(2+) entry in a dose-dependent manner. Electrophysiological experiments showed that H(2)O(2) significantly increased TRPC6 channel open probability and whole-cell currents. H(2)O(2) also evoked a robust inward current in A7r5 vascular smooth muscle cells, which was nearly abolished by knockdown of TRPC6 using a small interfering RNA. Catalase substantially attenuated arginine vasopressin (AVP)-induced Ca(2+) entry in cells co-transfected with TRPC6 and AVP V1 receptor. N-Ethylmaleimide and thimerosal were able to simulate the H(2)O(2) response. Dithiothreitol or glutathione-reduced ethyl ester significantly antagonized the response. Furthermore, both N-ethylmaleimide- and H(2)O(2)-induced TRPC6 activations were only observed in the cell-attached patches but not in the inside-out patches. Moreover, 1-oleoyl-2-acetyl-sn-glycerol effect on TRPC6 was significantly greater in the presence of H(2)O(2). Biotinylation assays revealed a significant increase in cell surface TRPC6 in response to H(2)O(2). Similarly, in cells transfected with TRPC6-EGFP, confocal microscopy showed a significant increase in fluorescence intensity in the region of the cell membrane and adjacent to the membrane. AVP also increased the fluorescence intensity on the surface of the cells co-transfected with TRPC6-EGFP and V1 receptor, and this response was inhibited by catalase. These data indicate that H(2)O(2) activates TRPC6 channels via modification of thiol groups of intracellular proteins. This cysteine oxidation-dependent pathway not only stimulates the TRPC6 channel by itself but also sensitizes the channels to diacylglycerol and promotes TRPC6 trafficking to the cell surface.

FKBP51 protects 661w cell culture from staurosporine-induced apoptosis.

PURPOSE: Neurodegenerative diseases and neurotraumas typically result in apoptosis of specific neurons leading to the pathology observed during the disease state. Existing treatments target the symptoms instead of preventing the death of these neurons. Although neuroprotective drugs should be useful as a treatment to prevent further loss of neurons, efficacious molecules are lacking. FK506 (tacrolimus), a widely used immunosuppressant drug, has significant neuroprotective and neuroregenerative properties throughout the central nervous system, including the eye. FK506 achieves these properties through interaction with FK506 binding proteins (FKBP), including FK506 binding protein 51 (FKBP51). In this study, we examine the effects of FKBP51 as a neuroprotective agent on a neuronal cell line. METHODS: We cultured 661w cell cultures with or without FK506, or stably transfected them with an FKBP51 expression vector. These cells were then exposed to the apoptosis-inducing agent staurosporine. Cell viability was determined using a calcein AM/propidium iodide assay. Protein levels and activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were determined by western immunoblot analysis. RESULTS: FKBP51 overexpression significantly protected 661w cell cultures from staurosporine-induced apoptosis. FKBP51 overexpression also significantly increased NF-κB p65 protein levels and activated NF-κB p65. FK506 treatment significantly protected 661w neuronal cultures from staurosporine-induced apoptosis. FK506 increased FKBP51, NF-κB p65, and levels of activated NF-κB p65 protein. CONCLUSIONS: These results suggest that FKBP51 protects 661w cell cultures from apoptosis induced by staurosporine. Additionally, FK506 protected 661w cell cultures from apoptosis and displayed a mechanism similar to that of FKBP51 overexpression. Both FK506 and FKBP51 appear to act through activation of NF-κB p65 protein, suggesting a common pathway for neuroprotection. These findings implicate FKBP51 as a protein important to neuronal cell culture survival. FKBP51 may be a potential therapeutic drug target for preventing the neurodegeneration and neurotrauma that occur during neurodegenerative diseases.

Endothelin, astrocytes and glaucoma.

It has become increasingly clear that astrocytes may play an important role in the genesis of glaucoma. Astrogliosis occurs in response to ocular stress or the presence of noxious stimuli. Agents that appear to stimulate reactive gliosis are becoming increasingly clear. One class of agents that is emerging is the endothelins (ETs; specifically, ET-1). In this review we examine the interactions of ET-1 with astrocytes and provide examples where ET-1 appears to contribute to activation of astrocytes and play a role in the neurodegenerative effects that accompany such reactivation resulting in astrogliosis. These actions are presented in the context of glaucoma although information is also presented with respect to ET-1's role in the central nervous system and brain. While much has been learned with respect to ET-1/astrocyte interactions, there are still a number of questions concerning the potential therapeutic implications of these findings. Hopefully this review will stimulate others to examine this potential.

Protein profiling of human nonpigmented ciliary epithelium cell secretome: the differentiation factors characterization for retinal ganglion cell line.

The purpose of this paper was to characterize proteins secreted from the human nonpigmented ciliary epithelial (HNPE) cells, which have differentiated a rat retinal ganglion cell line, RGC-5. Undifferentiated RGC-5 cells have been shown to express several marker proteins characteristic of retinal ganglion cells. However, RGC-5 cells do not respond to N-methyl-D aspartate (NMDA), or glutamate. HNPE cells have been shown to secrete numbers of neuropeptides or neuroproteins also found in the aqueous humor, many of which have the ability to influence the activity of neuronal cells. This paper details the profile of HNPE cell-secreted proteins by proteomic approaches. The experimental results revealed the identification of 132 unique proteins from the HNPE cell-conditioned SF-medium. The biological functions of a portion of these identified proteins are involved in cell differentiation. We hypothesized that a differentiation system of HNPE cell-conditioned SF-medium with RGC-5 cells can induce a differentiated phenotype in RGC-5 cells, with functional characteristics that more closely resemble primary cultures of rat retinal ganglion cells. These proteins may replace harsh chemicals, which are currently used to induce cell differentiation.

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.

Assessment of SNPs associated with the human glucocorticoid receptor in primary open-angle glaucoma and steroid responders.

PURPOSE: While chronic glucocorticoid (GC) therapy leads to ocular hypertension in about one third of individuals, almost all primary open-angle glaucoma (POAG) patients show this response and are called "steroid responders." Two differentially spliced isoforms of the glucocorticoid receptor (GR), GRalpha and GRbeta, regulate GC responsiveness in trabecular meshwork (TM) cells. GRbeta acts as a dominant negative regulator of GC activity and is expressed at lower levels in glaucomatous TM cells, making them more sensitive to GCs. Several arginine/serine-rich splicing factor (SR) proteins have been implicated in alternative splicing of the GR. We have previously demonstrated that immunophilins FKBP5 and FKBP4 are required for GRalpha and GRbeta translocation into the nucleus, which is essential for their biologic activity. The purpose of the present study was to use single nucleotide polymorphism (SNP) genotyping to determine whether there are any allele frequency differences in GR, FKBP4/5, or SR genes between normal control, POAG, and steroid responder populations. METHODS: Clinically characterized individuals (400 normal controls, 197 POAG, and 107 steroid responders) were recruited from the U. Iowa Ophthalmology Clinics after IRB approved consent. Genotyping of DNA samples for 48 SNPs in SFRS3, SFRS5, SFRS9, FKBP4, FKBP5, and NR3C1 was done at GeneSeek using a mass spectroscopy based system. RESULTS: All 48 SNPs displayed high call rates (99%). There were no significant differences in allele frequencies or genotypes in SNPs for SFRS5, SFRS9, FKBP4, FKBP5, and NR3C1 between the 3 groups. Up to three SNPs in SFRS3 had p-values <0.05 when comparing controls to POAG or steroid responders, but this statistical significance was lost when the p values were adjusted for multiple measures. CONCLUSIONS: Although these 6 genes may be involved in the pathogenesis of GC-induced ocular hypertension, it does not appear that major heritable risk alleles in these genes are responsible for the development of GC-induced ocular hypertension or POAG.

Changes in ocular aquaporin expression following optic nerve crush.

PURPOSE: Changes in the expression of water channels (aquaporins; AQP) have been reported in several diseases. However, such changes and mechanisms remain to be evaluated for retinal injury after optic nerve crush (ONC). This study was designed to analyze changes in the expression of AQP4 (water selective channel) and AQP9 (water and lactate channel) following ONC in the rat. METHODS: Rat retinal ganglion cells (RGCs) were retrogradely labeled by applying FluoroGold onto the left superior colliculus 1 week before ONC. Retinal injuries were induced by ONC unilaterally. Real-time PCR was used to measure changes in AQP4, AQP9, thy-1, Kir4.1 (K(+) channel), and beta-actin messages. Changes in AQP4, AQP9, Kir4.1, B cell lymphoma-x (bcl-xl), and glial fibrillary acidic protein (GFAP) expression were measured in total retinal extracts using western blotting. RESULTS: The number of RGCs labeled retrogradely from the superior colliculus was 2,090+/-85 cells/mm(2) in rats without any treatment, which decreased to 1,091+/-78 (47% loss) and 497+/-87 cells/mm(2) (76% loss) on days 7 and 14, respectively. AQP4, Kir4.1, and thy-1 protein levels decreased at days 2, 7, and 14, which paralleled a similar reduction in mRNA levels, with the exception of Kir4.1 mRNA at day 2 showing an apparent upregulation. In contrast, AQP9 mRNA and protein levels showed opposite changes to those observed for the latter targets. Whereas AQP9 mRNA increased at days 2 and 14, protein levels decreased at both time points. AQP9 mRNA decreased at day 7, while protein levels increased. GFAP (a marker of astrogliosis) remained upregulated at days 2, 7, and 14, while bcl-xl (anti-apoptotic) decreased. CONCLUSIONS: The reduced expression of AQP4 and Kir4.1 suggests dysfunctional ion coupling in retina following ONC and likely impaired retinal function. The sustained increase in GFAP indicates astrogliosis, while the decreased bcl-xl protein level suggests a commitment to cellular death, as clearly shown by the reduction in the RGC population and decreased thy-1 expression. Changes in AQP9 expression suggest a contribution of the channel to retinal ganglion cell death and response of distinct amacrine cells known to express AQP9 following traumatic injuries.

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.

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).

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.

sigma-1 receptors protect RGC-5 cells from apoptosis by regulating intracellular calcium, Bax levels, and caspase-3 activation.

PURPOSE: sigma-1 Receptor ligands prevent neuronal death associated with glutamate excitotoxicity both in vitro and in vivo. However, the molecular mechanism of the neuroprotective effect remains to be elucidated. The present study was undertaken to determine whether sigma-1 receptor agonists provide neuroprotection by decreasing glutamate-induced calcium mobilization and preventing apoptotic gene expression. METHODS: Cell death was measured by using a calcein-AM/propidium iodide cell-survival assay. Western blot analysis determined the expression levels of Bax in normal RGC-5 cells. Caspase-3 activation after glutamate treatment was determined with a carboxyfluorescein caspase-3 detection kit. Glutamate-induced intracellular calcium mobilization was measured by using ratiometric calcium imaging. RESULTS: sigma-1 Receptor-overexpressing RGC-5 (RGC-5-S1R) cells had lower glutamate-induced intracellular calcium mobilization than did normal RGC-5 cells, and the sigma-1 receptor ligand (+)-SKF10047 reduced the glutamate calcium response in normal and RGC-5-S1R cells. (+)-SKF10047 protected RGC-5 cells from glutamate-induced cell death, and the RGC-5-S1R cells showed a significant resistance to glutamate-induced apoptosis compared with the control RGC-5 cells. BD1047, a sigma-1 receptor antagonist, blocked the protective effect of (+)-SKF10047. Western blot analysis showed that (+)-SKF10047 inhibited the increase in Bax after glutamate treatments. Glutamate-mediated cell death involved activation of caspase-3, and sigma-1 receptor activation prevented an increase in caspase-3 expression. CONCLUSIONS: The results suggest that sigma-1 receptors regulate intracellular calcium levels and prevent activation of proapoptotic genes, thus promoting retinal ganglion cell survival. The sigma-1 ligands appear to be neuroprotective and are a potential target for neuroprotective therapeutics.

FK506-binding protein 51 regulates nuclear transport of the glucocorticoid receptor beta and glucocorticoid responsiveness.

PURPOSE: A spliced variant of the human glucocorticoid receptor GRbeta has been implicated in glucocorticoid responsiveness in glaucoma. Over-expression of the FK506-binding immunophilin FKBP51 also causes a generalized state of glucocorticoid resistance. In the present study, the roles of FKBP51 in the nuclear transport of GRbeta and glucocorticoid responsiveness were investigated. METHODS: Human trabecular meshwork cells (GTM3 and TM5) and HeLa cells were treated with dexamethasone (DEX) and FK506 and transfected with GRbeta and FKBP51 expression vectors. Coimmunoprecipitation and Western blot analyses were performed to study interactions of FKBP51 and FKBP52 with GRalpha, GRbeta, Hsp90, or dynein. The cells were transfected with a GRE-luciferase reporter to evaluate the effects of DEX and FK506 and the overexpression of GRbeta and FKBP51 on glucocorticoid-mediated gene expression. RESULTS: FKBP51 was involved in constitutive nuclear transport of both GRalpha and -beta in the absence of ligands. FKBP52 appeared to be solely responsible for the nuclear transport of ligand-activated GRalpha. DEX stimulated the translocation of GRalpha but not GRbeta. Overexpression of either GRbeta or FKBP51 stimulated GRbeta translocation and reduced DEX-induced luciferase in HeLa cells. FK506 did not alter DEX-induced translocation of GRalpha. However, FK506 increased the association of FKBP51 with GRbeta and stimulated DEX-induced translocation of GRbeta in normal TM cells, but not in glaucoma TM cells. Increased nuclear GRbeta significantly inhibited glucocorticoid responsiveness in TM cells. CONCLUSIONS: Nuclear transport of GRbeta represents a novel mechanism through which FKBP51 alters GC sensitivity. GRbeta and FKBP51 may be responsible for increased responsiveness in steroid-induced ocular hypertensive individuals as well as in patients with glaucoma.

Changes in ocular aquaporin-4 (AQP4) expression following retinal injury.

PURPOSE: Changes in the expression of water channels or aquaporins (AQP) have been reported in several diseases. However, such changes and mechanisms remain to be evaluated for retinal injury. This study was designed to analyze changes in the expression of AQP4 following elevation of intraocular pressure (IOP) and after intravitreal endothelin-1 injection and the potential involvement of the ubiquitin-dependent proteasome. METHODS: Retinal injuries were induced by the elevation of intraocular pressure in rat eyes using the Morrison model or following endothelin-1 intravitreal injection. Immunohistochemistry using a combination of glial fibrillary acidic protein (GFAP) and aquaporin-4 antibodies were employed to follow changes in the optic nerve head astrocytes. Real-time quantitative PCR (Q-PCR) was used for measuring changes in AQP4, ubiquitin hydrolase L1 (UCH-L1), and beta-actin messages. Changes in AQP4, caspase-3, thy-1, ubiquitination, and GFAP expression were also followed in total retinal extracts using western blotting. An S5a column was used to purify ubiquitinated proteins. RESULTS: In retinas of both injury models, there was an upregulation of GFAP (a marker of astrogliosis), caspase-3, and downregulation of thy-1, a marker for retinal ganglion cell stress, and decreased retinal AQP4 mRNA and protein levels as determined by Q-PCR, and western blotting, respectively. By contrast, IOP enhanced expression and co-localization of GFAP and AQP4 in optic nerve astrocytes. AQP4 was detected in affinity-purified ubiquitinated proteins using S5a column, suggesting that AQP4 is a target for degradation by the ubiquitin-dependent proteasome. While elevation of IOP induced an increase in ubiquitination in retinal extracts, it decreased ubiquitination in optic nerve extracts as detected by western blotting. Enhanced ubiquitination and decreased ubiquitination appear to correlate with AQP4 expression. IOP decreased UCH-L1 (or protein gene protein [PGP9.5]) in retinal extracts as judged by Q-PCR. CONCLUSIONS: The enhanced expression of AQP4 in optic nerve astrocytes following elevation of IOP may explain the astrocytic hypertrophy normally seen in glaucoma patients and may involve alteration in the activity of ubiquitin-dependent proteasomal degradation system. The decreased ubiquitination in the optic nerve may lead to increased levels of proapoptotic proteins known to be degraded by the proteasome, and thus to axonal degeneration in glaucoma.

The post-baccalaureate premedical certification program at the University of North Texas Health Science Center strengthens admission qualifications for entrance into medical school.

The Post-Baccalaureate (postbac) Premedical Certification Program at the University of North Texas Health Science Center provides an opportunity for individuals to enhance their credentials for entry into medical school by offering a challenging biomedical science core curriculum in graduate biochemistry, cell biology, physiology, and pharmacology. In addition, students (called postbacs) receive instruction in human gross anatomy, histology, and embryology with first-year medical students. More than 90% of the students accepted into the postbac program have applied to medical school previously but have been rejected by admission committees at least once, primarily because of low cognitive scores. In spring 2001, seven postbacs completed the program, of which only one was admitted into the Texas College of Osteopathic Medicine (TCOM), the medical school affiliated with the University of North Texas Health Science Center. Three postbacs went to other medical schools. Thirty-one completed the program by spring 2006, of whom 13 were admitted to TCOM, and eight to other medical schools. After six years, 101 postbacs have completed the program, and 70 have been accepted into medical schools. Postbacs admitted into TCOM have performed well compared with their medical school classmates. Overall, average scores for postbacs are above those of their medical school classmates. In addition, postbacs have taken class leadership positions, served as tutors and mentors, and have served as school ambassadors for new applicants. The postbac premedical program has proven to be very successful in preparing students for the rigors of a medical school curriculum by allowing these students to develop the skills and confidence necessary to compete.

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.