May 2017
Volume 58, Issue 5
Open Access
Physiology and Pharmacology  |   May 2017
Sigma-1 Receptor Regulates Mitochondrial Function in Glucose- and Oxygen-Deprived Retinal Ganglion Cells
Author Affiliations & Notes
  • Dorette Z. Ellis
    Department of Pharmaceutical Sciences, University of North Texas Systems College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, United States
    North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Linya Li
    North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Yong Park
    North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Shaoqing He
    North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Brett Mueller
    Kentucky Lions Eye Center, University of Louisville, Louisville, Kentucky, United States
  • Thomas Yorio
    North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Correspondence: Dorette Z. Ellis, Department of Pharmaceutical Sciences and North Texas Eye Research Institute, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA; dorette.ellis@unthsc.edu
Investigative Ophthalmology & Visual Science May 2017, Vol.58, 2755-2764. doi:10.1167/iovs.16-19199
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      Dorette Z. Ellis, Linya Li, Yong Park, Shaoqing He, Brett Mueller, Thomas Yorio; Sigma-1 Receptor Regulates Mitochondrial Function in Glucose- and Oxygen-Deprived Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(5):2755-2764. doi: 10.1167/iovs.16-19199.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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.

Glaucoma is a group of heterogeneous optic neuropathies in which retinal ganglion cells (RGC) and their axons die. While elevated IOP is a risk factor for the development of primary open-angle glaucoma (POAG),1 neuropathy was also observed in normal tension glaucoma patients (IOP measurements of 6–10 mm Hg); suggesting that in addition to high IOP, there may be other common predisposing factors for the development of glaucoma. 
One possible predisposing factor is dysfunctional mitochondria that appears to contribute to the death of neuronal cells. Mitochondria serve important functions in cells particularly in maximizing the cell's energy production. Additionally, it has become increasingly clear, that mitochondria are controllers of cell death and are tasked with regulating the cells' response to oxidative stress and other toxic stimuli. Thus, perturbations in mitochondrial dynamics as a result of mutations in nuclear or mitochondrial genes involved in regulating these processes are linked to diseases, such as Leber hereditary optic neuropathy,2,3 dominant optic atrophy,4,5 and Parkinson's disease.6 Interestingly, mitochondrial dysfunction has also been implicated as a secondary causative factor in the development of glaucoma and in RGC death.7 There is also the presence of an increased accumulation of mitochondria in the optic nerve head in glaucomatous states,8 possibly as a result of impaired anterograde fast axonal transport in the optic nerve (ON).9,10 
Mitochondrial DNA (mtDNA) mutations and increased mtDNA content have been shown in POAG patients, suggesting increased pathologic changes in RGCs and the inability to provide increased response to oxidative stress.11 Furthermore, there was reduced mitochondrial respiration in a significant population of POAG patients.11,12 Taken together, these data suggest that the optic neurodegeneration observed in glaucoma may be associated with mitochondrial dysfunction. 
Recently, we13,14 and others,15,16 have described a unique neuroprotective pathway in the retina that is mediated by the sigma-1 receptor (σ-1r). The σ-1r is a 26-kD transmembrane, nonopioid receptor1719 localized at the endoplasmic reticular (ER) membrane.20 At the ER, the σ-1r complexes with the mitochondrion-associated ER membrane (MAM) and regulates calcium transport and homeostasis between the two organelles.21,22 The mechanisms by which σ-1r modulate cell function are relatively unknown. However, recent observations suggest that the σ-1r associates with and prevents the influx of calcium by L-type voltage-gated calcium channels in RGCs.14 It also inhibits glutamate-induced increases in intracellular calcium concentrations, and prevents the overexpression of the proapoptotic protein Bax, and activation of caspase-3.16,23 These findings suggest that the neuroprotective effects of σ-1r agonists include attenuation of targets in the intrinsic mitochondrial death signaling pathway; a pathway implicated in the pathogenesis of glaucoma.24,25 Recently, we demonstrated that 50% of the RGCs that were exposed to an hypoxic-like condition, produced death 4 to 6 hours post exposure to this insult.26 Pretreatment with pentazocine, the σ-1r agonist, rescued RGCs from this hypoxic-induced death. Therefore, in the current study we will use a similar hypoxic condition defined as oxygen and glucose deprivation (OGD), in isolated primary RGCs to determine the effects of OGD on mitochondrial function. We will also determine if activation of the σ-1r with an agonist or overexpression of the σ-1r will mitigate the effects of OGD. 
Methods
Primary RGCs
All procedures were done in accordance to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee at the University of North Texas Health Science Center. We isolated rat primary RGCs from postnatal day 4 to 7 from retinas using the two-step panning method of Ben Barres, PhD,27 that results in a purity of 98%.13,14,28 Briefly, retinas were isolated from Sprague Dawley rat pups (Charles River, Wilmington, MA, USA) and placed in 4.5 units/mL papain solution (Worthington, Lakewood, NJ, USA) to dissociate the cells. Retinal ganglion cells were isolated using sequential immunopanning techniques; anti-macrophage antibody (CLAD51240; Cedar Lane Laboratories, Hornby, ON, USA) was used to isolate macrophages, and Thy 1.1 antibody was used to isolate RGCs. After isolation, cells were allowed to mature for 7 to 10 days13,14,28 prior to performing experiments. 
Hypoxic-Like Treatment
Cells were incubated at 37°C at normoxia (10% CO2) or under hypoxia (10% CO2 and 0.5% O2) for 4 hours in Dulbecco's modified Eagle's medium (DMEM; no glucose, no phenol red and trophic factors; GIBCO, Grand Island, NY, USA). For normoxia, media contained 4.5 g/L glucose, brain-derived neurotrophic factor (BDNF) (1 μg/20 mL), Forskolin (84 μg/20 mL), and Ciliary neurotrophic factor (0.2 μg/20 mL). 
Mitochondria Membrane Potential (Δψm)
Mitochondrial membrane potential was measured in purified neonatal RGCs by using a fluorescence tetraethylbenzimidazolylcarbocyanineiodide (JC-1) assay kit (Abcam, Cambridge, United Kingdom). Purified RGCs were seeded in a 96-well black frame clear flat-bottom plate (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at density of 10,000 cells/well. Cells exposed to the JC-1 dye (1 μM)29 were imaged using a fluorescent plate reader (Infinite m200; Tecan Group Ltd., Männedorf, Switzerland). The JC-1 dye exhibits potential-dependent accumulation in the mitochondria that is indicated by a fluorescence emission shift from green (∼529 nm) to red (∼590 nm). Alteration in the ionic equilibrium results in mitochondria depolarization and is indicated by a decrease in the red/green fluorescent ratio. Data is represented as the red/green fluorescent ratio. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP30; 100 nM), a protonophore, served as a positive control for the JC-1 dye/mitochondria depolarization. 
RGC AAV2 Transduction
Three days after RGC isolation, purified RGCs were transduced with AAV2-CAG-σ-1r-GFP (AAV2-σ-1r-GFP) and AAV2-CAG-EGFP at 105 multiplicity of infection (MOI), respectively, for 10 days after which experimental conditions were applied. 
Western Blots
Purified RGCs were seeded onto 6-well plates (∼300,000 RGCs/well). Following treatment, RGCs were homogenized in 65 μL of ice cold RIPA buffer containing 50-mM Tris, pH 7.4, 10-mM Mg2+, 1-mM EDTA, 1-mM EGTA, 10-mM benzamide, 0.08-mM sodium molybdate, 0.01% Triton X-100, 10-μM okadaic acid, 100-ng/mL leupeptin, 100-ng/mL aprotinin, and 2-mM sodium pyrophosphate. Lysed aliquots were sonicated and protein concentration determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Samples were then electrophoresed on a precast ready-made 12% SDS-PAGE gradient gel (Bio-Rad Laboratories). The protein was then transferred onto a polyvinilydene diflouride (PVDF) membrane blot (Millipore, Billerica, MA, USA) overnight at 4°C. Membrane blots were then blocked with 4% nonfat dried milk for 1 hour (Bio-Rad Laboratories), and then probed with 1:1000 dilution of primary mouse monoclonal antibody against σ-1r overnight at 4°C. (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membrane blots were then incubated at room temperature for 1 hour with a horseradish peroxidase conjugated secondary antibody (Bio-Rad Laboratories) at a dilution of 1:10000 in 0.4% nonfat dried milk. The proteins were visualized (ChemiDoc XRS+ System with Image Lab Software; Bio-Rad Laboratories) using ECL detection reagent SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL, USA). 
Immunofluorescence
Purified RGCs were grown on 12-mm coverslips and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Antigen retrieval involved the use of 6-M urea in 0.1-M Tris, pH 9.5 at 80°C for 10 minutes. Then, cells were permeabilized with 0.1% Triton X-100 for 5 minutes. These cells were then blocked with 5% normal donkey serum and 5% BSA for 1 hour at room temperature. Blocking solution was removed and cells were then incubated with primary antibodies: affinity-purified σ-1r monoclonal antibody (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or polyclonal antibody (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-voltage-dependent anion channel (VDAC) polyclonal antibody (1:200 dilution; EMD Millipore); RNA binding protein with multiple splicing (RBPMS) polyclonal antibody (1:100 dilution; GeneTex, Inc., Irvine, CA, USA) at 4°C overnight. Coverslips were then washed three times with PBS, and a 1:1000 dilution of secondary antibodies donkey anti-rabbit IgG or anti-goat (Alexa Fluor 647; Invitrogen, Carlsbad, CA, USA) conjugate and donkey anti-mouse IgG or anti-rabbit IgG (Alexa Fluor 546; Invitrogen) conjugate were added and incubated for 1 hour in the dark at room temperature. After incubation, the coverslips were washed three times with PBS. Mounting was performed on glass slides using antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; P36931 Prolong Gold; Invitrogen) and allowed to dry for overnight in the dark. Cells were viewed on a confocal laser scanning microscope (LSM 510; Zeiss, Thornwood, NY, USA) at ×40. 
Caspase 3/7 Activity Assay
Caspases 3 and 7 activity were measured in RGCs by using a luminescent assay kit (G8091; Promega, Madison, WI, USA). Purified RGCs were seeded (∼10,000 RGCs/well) in 96-well opaque plate (BD Falcon, Franklin Lakes, NJ, USA). After the appropriate treatments, 50 μL of Caspase–Glo 3/7 assay reagent were added into each well, shaken for 30 seconds on an orbital shaker, and incubated for 1 hour at room temperature. The total volumes of 100 μL of each well were then transferred into a white-walled 96-well plate (BD Falcon). Luciferase signal was then read by a luminescent plate reader (Infinite m200; Tecan Group Ltd., Männedorf, Switzerland). 
Cytochrome C Oxidase Activity
Cytochrome c oxidase activity was measured using a cytochrome c oxidase assay kit (Sigma-Aldrich Corp., St. Louis, MO, USA), according to the manufacturer's protocol as previously described.28 Briefly, treated purified RGCs were exposed to conditions in which the cytochrome c oxidase within the cells converts ferrocytochrome c to ferrocytochrome c. This is a colorimetric assay in which ferrocytochrome c absorbs at 550 nm; decreased absorbance at 550 nm indicates cytochrome c oxidase–induced oxidation of ferrocytochrome c. Approximately 350,000 RGCs were seeded in each well of a 6-well dish and subjected to appropriate treatments. Cells were trypsinized, and supernatant was removed after a spin at 200g for 5 minutes. Ferrocytochrome c was measured using a spectrophotometer (GENESYS10 uv; Thermo Scientific). 
Statistical Analysis
Statistical analysis was performed using ANOVA, followed by Tukey's all pairwise multiple comparison test method for comparison of significant difference among different means. 
Results
Localization and Expression of the σ-1r
The purification of RGCs was validated using RBPMS antibody, considered to be a protein marker for RGCs.31,32 Figure 1A. Distribution of the endogenous σ-1r in RGCs was assessed using the σ-1r antibody. Figure 1B depicts localization of the endogenous σ-1r in RGC soma and neurites and in the periphery of the nucleus. We also tested the transduction efficiency of the AAV2-σ-1r in RGCs. Figure 2A demonstrates more intense immunostaining in AAV2-σ-1r-green fluorescence protein (GFP) transduced cells when compared with nontransduced cells or AAV2-empty vector (red staining). Green fluorescence protein validated transduction of AAV2 vector into RGCs and DAPI staining validated nuclei. Western blots (Fig. 2B) confirmed these findings. Using the σ-1r antibody (σ-1R), we demonstrated that GFP/σ-1r fusion protein stained at molecular weigh of 53 kDa, while there was no σ-1r staining in control or empty vector containing GFP. In samples containing endogenous σ-1r, exposure to the σ-1r antibody resulted in staining at 26 kDa, demonstrating endogenous σ-1r in all samples tested. β-Tubulin served as loading controls. 
Figure 1
 
Localization of endogenous σ-1r in isolated primary RGCs from rat pups. (A) Immunofluorescence detection of RGCs using RBPMS staining confirms that these cells are RGCs. Images were taken at ×40, zoom 1. Scale bars: 50 μM. (B) Immunohistochemical detection of endogenous σ-1r in RGCs. Differential interference contrast imaging (DIC) of the same cell was performed. Images are representative of three to five experiments. Images were taken at ×40, zoom 2. Scale bars: 50 μM.
Figure 1
 
Localization of endogenous σ-1r in isolated primary RGCs from rat pups. (A) Immunofluorescence detection of RGCs using RBPMS staining confirms that these cells are RGCs. Images were taken at ×40, zoom 1. Scale bars: 50 μM. (B) Immunohistochemical detection of endogenous σ-1r in RGCs. Differential interference contrast imaging (DIC) of the same cell was performed. Images are representative of three to five experiments. Images were taken at ×40, zoom 2. Scale bars: 50 μM.
Figure 2
 
Immunofluorescence and Western blots of σ-1r in RGCs transduced with AAV2-σ-1r-GFP. (A) σ-1R denotes σ-1r antibody and AAV2-GFP denotes empty vector. Images were taken at ×40, zoom 1. Scale bars: 50 μM. (B) For blots 50 μg of protein homogenates was used and β tubulin served as loading controls. Images are representative of three experiments.
Figure 2
 
Immunofluorescence and Western blots of σ-1r in RGCs transduced with AAV2-σ-1r-GFP. (A) σ-1R denotes σ-1r antibody and AAV2-GFP denotes empty vector. Images were taken at ×40, zoom 1. Scale bars: 50 μM. (B) For blots 50 μg of protein homogenates was used and β tubulin served as loading controls. Images are representative of three experiments.
Activation of Endogenous σ-1r Receptor Restores Δψm Following OGD
Primary RGCs were exposed to OGD and normoxic conditions in the presence of JC-1 (1 μM) dye for 4 hours and Δψm was measured. FCCP served as a control for Δψm. There were significant decreases in Δψm in cells treated with OGD when compared with normoxic conditions (Fig. 3A). To test the involvement of the σ-1r in Δψm, varying concentrations of pentazocine were used to determine efficacy. Cells were exposed to pentazocine (0.1–100 μM) and subjected to OGD for 4 hours. Figure 3B demonstrates that there is a concentration-dependent increase in Δψm following OGD. There were significant increases in Δψm in cells exposed to OGD and then treated with 10- and 100-μM pentazocine, when compared with OGD-treated cells, or OGD-treated with 0.1 to 1 μM pentazocine. Δψm was not significantly different in cells treated with 10- and 100-μM pentazocine, suggesting that 10-μM pentazocine produced a maximum effect. To confirm the involvement of the σ-1r in restoring Δψm in OGD-treated cells, RGCs were pretreated with the σ-1r antagonist, BD 1047 and pentazocine was subsequently added. BD 1047 abolished the pentazocine-induced increase in Δψm following OGD (Fig. 3C). 
Figure 3
 
σ-1r regulation of Δψm in RGCs; Δψm is expressed JC-1 ratio of aggregate/monomers. (A) Retinal ganglion cells were exposed to OGD and normoxic conditions and Δψm was measured. Values for Δψm represent the mean ± SEM for an average of three samples in six experiments. *Significantly different from normoxic conditions at P < 0.05 by ANOVA, and Tukey's all pairwise multiple comparison test. (B) Pentazocine (PTZ) protection from OGD is concentration-dependent. Retinal ganglion cells were incubated in OGD conditions with or without varying concentrations of PTZ; 0.1 to 100 μM. Δψm was measured. FCCP served as positive control for the JC-1 dye. Values for Δψm represent the mean ± SEM for an average of three samples in four experiments. *Significantly different from OGD without PTZ at P < 0.05 by ANOVA, and Tukey's all pairwise multiple comparison test. (C) σ-1r antagonist, BD1047 inhibits PTZ action. Oxygen- and glucose-deprived conditions were significantly (*) different from normoxic conditions at P < 0.05, Δψm measured in the presence of PTZ/OGD/ BD1047 was significantly (#) different from PTZ/OGD at P < 0.05 and Δψm measured in PTZ/OGD was significantly (##) different from OGD condition at P < 0.05. Values for Δψm represent the mean ± SEM for an average of three samples in four experiments (by ANOVA, and Tukey's all pairwise multiple comparison test).
Figure 3
 
σ-1r regulation of Δψm in RGCs; Δψm is expressed JC-1 ratio of aggregate/monomers. (A) Retinal ganglion cells were exposed to OGD and normoxic conditions and Δψm was measured. Values for Δψm represent the mean ± SEM for an average of three samples in six experiments. *Significantly different from normoxic conditions at P < 0.05 by ANOVA, and Tukey's all pairwise multiple comparison test. (B) Pentazocine (PTZ) protection from OGD is concentration-dependent. Retinal ganglion cells were incubated in OGD conditions with or without varying concentrations of PTZ; 0.1 to 100 μM. Δψm was measured. FCCP served as positive control for the JC-1 dye. Values for Δψm represent the mean ± SEM for an average of three samples in four experiments. *Significantly different from OGD without PTZ at P < 0.05 by ANOVA, and Tukey's all pairwise multiple comparison test. (C) σ-1r antagonist, BD1047 inhibits PTZ action. Oxygen- and glucose-deprived conditions were significantly (*) different from normoxic conditions at P < 0.05, Δψm measured in the presence of PTZ/OGD/ BD1047 was significantly (#) different from PTZ/OGD at P < 0.05 and Δψm measured in PTZ/OGD was significantly (##) different from OGD condition at P < 0.05. Values for Δψm represent the mean ± SEM for an average of three samples in four experiments (by ANOVA, and Tukey's all pairwise multiple comparison test).
Overexpression of the σ-1r Restores Δψm After OGD
Because σ-1r activation restored mitochondria Δψm, we tested if overexpression of the σ-1r in RGCs resulted in increased Δψm in RGCs exposed to OGD. Retinal ganglion cells were transduced with AAV2-σ-1r vector or AAV2-empty vector for 10 days and then exposed to OGD for 4 hours and Δψm was measured. Normoxia served as control for OGD conditions. Oxygen and glucose deprivation exposure resulted in significant decreases in Δψm when compared with normoxic conditions. However, in RGCs transduced with AAV2-σ-1r vector, these decreases were ameliorated, and Δψm significantly increased when compared with the cells containing AAV2-empty vector or cells treated with OGD conditions (Fig. 4A). Addition of pentazocine significantly increased the Δψm of RGC transduced with the AAV2-σ-1r vector when compared with AAV2-σ-1r vector without pentazocine (Fig. 4B). 
Figure 4
 
AAV2-σ-1r vector increases Δψm in RGCs exposed to OGD. (A) Δψm was measured in RGCs transduced with AAV2-σ-1r vector. Oxygen- and glucose-deprived conditions were significantly (*) different from normoxic conditions at P < 0.05, and OGD/AAV- σ-1r vector was significantly (#) different from OGD at P < 0.05. Values for Δψm represent the mean ± SEM for an average of three samples in three experiments by ANOVA and Tukey's all pairwise multiple comparison test. (B) Δψm was measured in RGCs transduced with AAV2-σ-1r vector, AAV2-empty vector served as controls. Values for Δψm represent the mean ± SEM for an average of three samples in three experiments. *Significantly different from control conditions at P < 0.05 and OGD + sigma-1 receptor virus without PTZ at P < 0.05 (by ANOVA and Tukey's all pairwise multiple comparison test).
Figure 4
 
AAV2-σ-1r vector increases Δψm in RGCs exposed to OGD. (A) Δψm was measured in RGCs transduced with AAV2-σ-1r vector. Oxygen- and glucose-deprived conditions were significantly (*) different from normoxic conditions at P < 0.05, and OGD/AAV- σ-1r vector was significantly (#) different from OGD at P < 0.05. Values for Δψm represent the mean ± SEM for an average of three samples in three experiments by ANOVA and Tukey's all pairwise multiple comparison test. (B) Δψm was measured in RGCs transduced with AAV2-σ-1r vector, AAV2-empty vector served as controls. Values for Δψm represent the mean ± SEM for an average of three samples in three experiments. *Significantly different from control conditions at P < 0.05 and OGD + sigma-1 receptor virus without PTZ at P < 0.05 (by ANOVA and Tukey's all pairwise multiple comparison test).
Cytochrome C Oxidase Activity is Restored by σ-1r Following OGD Treatment
For a more robust validation of the involvement of the σ-1r in mitochondria health, we tested the effects of σ-1r on cytochrome c oxidase activity following OGD stress. There were significant decreases in cytochrome c oxidase activity following OGD when compared with normoxic conditions (Fig. 5). Activation or overexpression of σ-1r resulted in partial restoration of cytochrome c oxidase activity that was significantly different from cytochrome c oxidase activity in OGD conditions (Fig. 5). 
Figure 5
 
Cytochrome C oxidase activity is restored by σ-1r. Retinal ganglion cells were exposed to OGD conditions for 4 hours and either transduced with AAV-σ-1r vector or treated with pentazocine. Oxygen- and glucose-deprived conditions were significantly (*) different from normoxic conditions at P < 0.05, and OGD/AAV- σ-1r vector and OGD/10 μM PTZ were significantly (#) different from OGD at P < 0.05. Values represent the mean ± SD for four experiments by ANOVA and Tukey's all pairwise multiple comparison test.
Figure 5
 
Cytochrome C oxidase activity is restored by σ-1r. Retinal ganglion cells were exposed to OGD conditions for 4 hours and either transduced with AAV-σ-1r vector or treated with pentazocine. Oxygen- and glucose-deprived conditions were significantly (*) different from normoxic conditions at P < 0.05, and OGD/AAV- σ-1r vector and OGD/10 μM PTZ were significantly (#) different from OGD at P < 0.05. Values represent the mean ± SD for four experiments by ANOVA and Tukey's all pairwise multiple comparison test.
OGD-Induced Activation of Caspase 3/7 is Attenuated by Activation and Overexpression of the σ-1r
We examined the potential mechanism by which the σ-1r would restore Δψm in RGCs exposed to OGD. To overexpress σ-1r, cells were transduced with AAV2-σ-1r vector and exposed to OGD for 4 hours. To activate the σ-1r, cells were treated with pentazocine (10 μM) and exposed to OGD for 4 hours. Normoxia served as controls and caspase 3/7 activity was measured. There was a significant increase in caspase 3/7 activity in RGC exposed to OGD when compared with normoxic conditions (Fig. 6). Overexpression of σ-1r and pentazocine treatment resulted in significant decreases in caspase activity and restored activity to normoxic conditions (Fig. 6). 
Figure 6
 
Caspase activity in σ-1r–transduced RGCs. Data are expressed as relative fold change of caspase activity in OGD conditions when compared with normoxia. Retinal ganglion cells were exposed to AAV-σ-1r vector or PTZ and caspase activity was measured. Data representative of two experiments performed in triplicates. Oxygen- and glucose-deprived *significantly different from normoxia, and #significantly different from OGD/AAV-σ-1r, and OGD/PTZ at P < 0.05 (by ANOVA and Tukey's all pairwise multiple comparison test).
Figure 6
 
Caspase activity in σ-1r–transduced RGCs. Data are expressed as relative fold change of caspase activity in OGD conditions when compared with normoxia. Retinal ganglion cells were exposed to AAV-σ-1r vector or PTZ and caspase activity was measured. Data representative of two experiments performed in triplicates. Oxygen- and glucose-deprived *significantly different from normoxia, and #significantly different from OGD/AAV-σ-1r, and OGD/PTZ at P < 0.05 (by ANOVA and Tukey's all pairwise multiple comparison test).
Association of the σ-1r With the Voltage Dependent Anion Channel
We assessed association of the σ-1r with respect to the mitochondria in RGCs using the voltage-dependent anion channel (VDAC) as the mitochondrial membrane marker. When exposed to a VDAC antibody, RGC showed staining in the soma and neurites. Merged images with the σ-1r showed bright-colored staining in the cytoplasm that was more intense with overexpression of the σ-1r. As with Figure 1, there was σ-1r staining in the cytoplasm and around the periphery of the nucleus (Fig. 7). We then examined the effects of activation of the σ-1r by pentazocine. Figure 8A demonstrates that in the presence of pentazocine, endogenous σ-1r staining is more intense in the soma, neurites, and nucleus when compared with untreated cells. In merged images, there were more yellow color in the cytoplasm, suggesting close contact or association of σ-1r and VDAC. Because the σ-1r is a stress-response protein, we examined the effects of OGD on σ-1r association with VDAC. Retinal ganglion cells were exposed to OGD conditions and then stained with the σ-1r and VDAC antibodies. Under OGD conditions, both the σ-1r and VDAC staining were more punctate throughout the soma and neurites. Activation of the σ-1r with pentazocine resulted in brighter and less punctate staining of both the σ-1r and VDAC and merged images showed bright orange/yellow color suggesting close contact or association of the σ-1r with VDAC in RGCs (Fig. 8B). 
Figure 7
 
Immunofluorescence detection of the σ-1r (red) and VDAC (pink; because of the GFP) in RGCs. Confocal laser scanning microscope image of the RGCs transduced with AAV-σ-1r demonstrate more intense staining of σ-1r than in control (endogenous σ-1r). Merged images suggest a bright fusion of colors (not red and not pink) in the cytoplasm. Figure is representative of three images. Images were taken at ×40, zoom 2. Scale bars: 50 μM.
Figure 7
 
Immunofluorescence detection of the σ-1r (red) and VDAC (pink; because of the GFP) in RGCs. Confocal laser scanning microscope image of the RGCs transduced with AAV-σ-1r demonstrate more intense staining of σ-1r than in control (endogenous σ-1r). Merged images suggest a bright fusion of colors (not red and not pink) in the cytoplasm. Figure is representative of three images. Images were taken at ×40, zoom 2. Scale bars: 50 μM.
Discussion
In the present study, the relationship between mitochondrial function and σ-1r was examined in RGCs. These studies demonstrated that the σ-1r is expressed in RGC soma and neurites, and that there is a functional association of the σ-1r with the mitochondria. When the σ-1r is overexpressed or is activated using a σ-1r agonist there is restoration of the mitochondrial Δψm and restoration of cytochrome c oxidase activity following OGD. Activation or overexpression of the σ-1r also prevented caspase activation. Our current study supports previous observations that demonstrated σ-1r expression in isolated RGCs33 and in rat inner nuclear layer and ganglion cell layer34 supporting a role of σ-1r in RGCs function. 
The current studies suggest an association of σ-1r to functional activity of mitochondria. There was overlap in σ-1r/VDAC expression and a qualitative increase in σ-1r/VDAC staining in response to activation of the σ-1r by pentazocine. Others have determined that σ-1r at MAM regulates mitochondria metabolic activities.35 Furthermore, additional studies have suggested an association of σ-1r with the mitochondria of rat brains.36 While we used VDAC as a mitochondrial membrane marker, others have reported localization of VDAC to the plasma membrane. We consistently observed σ-1r staining in the periphery of the nucleus. These findings are consistent with electron microscopy studies that demonstrated σ-1r expression in RGC nuclear envelope and endoplasmic reticulum.37 While the σ-1r is known to be located at MAM, its ability to translocate to other parts of the cell38,39 allows for its ability to inhibit stress-induced cell death pathways.23,40,41 Such activities support a protective effect of σ-1r. 
Activation and/or over expression of the σ-1r resulted in decreased caspase activity in the time course corresponding to changes in Δψm produced following OGD. These results are of interest as caspase 3/7 are important markers indicating that the cells have entered into the apoptotic signaling pathway (reviewed in Ref. 42). Other studies, using a live-dead assay, have demonstrated that activation of the σ-1r results in preservation of RGCs exposed to OGD26 in a time course that corresponds to changes in caspase activity. The mechanisms by which σ-1r inhibits caspase activity are currently unknown. However, previous studies have demonstrated that ERK/MAPK directly phosphorylates caspase-9, an upstream activator of caspase 3, leading to the inhibition of caspase 9 and subsequent inhibition of caspase 3.43 Interestingly, OGD inactivates ERK1/2 by reducing its phosphorylated state and in pentazocine-treated RGCs the phosphorylated state is restored in the time course corresponding to the σ-1r induced restoration from cell death.26 
The sequences of events leading to cell death are not entirely clear and continue to be studied. However, it is believed that under stress, as would be induced by OGD, dissipation of the Δψm could result in BAK/BAX-induced release of cytochrome c from the mitochondria inter-membrane space into the cytoplasm.44 In fact, studies have demonstrated σ-1r induced abrogation of cytochrome c release, and alters Bax/Bcl-2 ratios in oxidative stress models45 supports the role of the σ-1r in regulating cell viability under stress conditions. The importance of the σ-1r in maintaining proper mitochondrial function and overall cell viability is further validated as recent reports suggest that dysfunction of the gene encoding the σ-1r, SIGMAR1, results in motor neuron deficits including intracellular calcium signaling and mitochondrial function.46 Additionally, missense mutations in SIGMAR1 were identified in juvenile familial amyotrophic lateral sclerosis patients47 and functional studies in Neuro2A cells overexpressing a protein of the SIGMAR1 variant, demonstrated dysfunction at MAM contacts and aberrations in mitochondrial adenosine triphosphate production and subsequent cell death.48,49 Taken together, we and others have demonstrated the importance of the functional roles of the σ-1r in regulating physiological cell viability and in preventing cell dysfunction under stress conditions. The σ-1r thus may be a useful target for a potential therapeutic neuroprotective strategy. 
Figure 8
 
Pentazocine treatment demonstrates association between endogenous σ-1r and mitochondria (VDAC staining). Retinal gangliion cells were exposed to OGD conditions in the presence or absence of PTZ. Retinal ganglion cells were labeled with σ-1r antibody (red), VDAC (green), and DAPI (blue). Images were taken at ×40, zoom 2. Scale bars: 50 μM.
Figure 8
 
Pentazocine treatment demonstrates association between endogenous σ-1r and mitochondria (VDAC staining). Retinal gangliion cells were exposed to OGD conditions in the presence or absence of PTZ. Retinal ganglion cells were labeled with σ-1r antibody (red), VDAC (green), and DAPI (blue). Images were taken at ×40, zoom 2. Scale bars: 50 μM.
Acknowledgments
Supported by the Department of Defense (W81XH-10-2-0003) and the University of North Texas Systems College of Pharmacy. 
Disclosure: D.Z. Ellis, None; L. Li, None; Y. Park, None; S. He, None; B. Mueller, None; T. Yorio, None 
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Figure 1
 
Localization of endogenous σ-1r in isolated primary RGCs from rat pups. (A) Immunofluorescence detection of RGCs using RBPMS staining confirms that these cells are RGCs. Images were taken at ×40, zoom 1. Scale bars: 50 μM. (B) Immunohistochemical detection of endogenous σ-1r in RGCs. Differential interference contrast imaging (DIC) of the same cell was performed. Images are representative of three to five experiments. Images were taken at ×40, zoom 2. Scale bars: 50 μM.
Figure 1
 
Localization of endogenous σ-1r in isolated primary RGCs from rat pups. (A) Immunofluorescence detection of RGCs using RBPMS staining confirms that these cells are RGCs. Images were taken at ×40, zoom 1. Scale bars: 50 μM. (B) Immunohistochemical detection of endogenous σ-1r in RGCs. Differential interference contrast imaging (DIC) of the same cell was performed. Images are representative of three to five experiments. Images were taken at ×40, zoom 2. Scale bars: 50 μM.
Figure 2
 
Immunofluorescence and Western blots of σ-1r in RGCs transduced with AAV2-σ-1r-GFP. (A) σ-1R denotes σ-1r antibody and AAV2-GFP denotes empty vector. Images were taken at ×40, zoom 1. Scale bars: 50 μM. (B) For blots 50 μg of protein homogenates was used and β tubulin served as loading controls. Images are representative of three experiments.
Figure 2
 
Immunofluorescence and Western blots of σ-1r in RGCs transduced with AAV2-σ-1r-GFP. (A) σ-1R denotes σ-1r antibody and AAV2-GFP denotes empty vector. Images were taken at ×40, zoom 1. Scale bars: 50 μM. (B) For blots 50 μg of protein homogenates was used and β tubulin served as loading controls. Images are representative of three experiments.
Figure 3
 
σ-1r regulation of Δψm in RGCs; Δψm is expressed JC-1 ratio of aggregate/monomers. (A) Retinal ganglion cells were exposed to OGD and normoxic conditions and Δψm was measured. Values for Δψm represent the mean ± SEM for an average of three samples in six experiments. *Significantly different from normoxic conditions at P < 0.05 by ANOVA, and Tukey's all pairwise multiple comparison test. (B) Pentazocine (PTZ) protection from OGD is concentration-dependent. Retinal ganglion cells were incubated in OGD conditions with or without varying concentrations of PTZ; 0.1 to 100 μM. Δψm was measured. FCCP served as positive control for the JC-1 dye. Values for Δψm represent the mean ± SEM for an average of three samples in four experiments. *Significantly different from OGD without PTZ at P < 0.05 by ANOVA, and Tukey's all pairwise multiple comparison test. (C) σ-1r antagonist, BD1047 inhibits PTZ action. Oxygen- and glucose-deprived conditions were significantly (*) different from normoxic conditions at P < 0.05, Δψm measured in the presence of PTZ/OGD/ BD1047 was significantly (#) different from PTZ/OGD at P < 0.05 and Δψm measured in PTZ/OGD was significantly (##) different from OGD condition at P < 0.05. Values for Δψm represent the mean ± SEM for an average of three samples in four experiments (by ANOVA, and Tukey's all pairwise multiple comparison test).
Figure 3
 
σ-1r regulation of Δψm in RGCs; Δψm is expressed JC-1 ratio of aggregate/monomers. (A) Retinal ganglion cells were exposed to OGD and normoxic conditions and Δψm was measured. Values for Δψm represent the mean ± SEM for an average of three samples in six experiments. *Significantly different from normoxic conditions at P < 0.05 by ANOVA, and Tukey's all pairwise multiple comparison test. (B) Pentazocine (PTZ) protection from OGD is concentration-dependent. Retinal ganglion cells were incubated in OGD conditions with or without varying concentrations of PTZ; 0.1 to 100 μM. Δψm was measured. FCCP served as positive control for the JC-1 dye. Values for Δψm represent the mean ± SEM for an average of three samples in four experiments. *Significantly different from OGD without PTZ at P < 0.05 by ANOVA, and Tukey's all pairwise multiple comparison test. (C) σ-1r antagonist, BD1047 inhibits PTZ action. Oxygen- and glucose-deprived conditions were significantly (*) different from normoxic conditions at P < 0.05, Δψm measured in the presence of PTZ/OGD/ BD1047 was significantly (#) different from PTZ/OGD at P < 0.05 and Δψm measured in PTZ/OGD was significantly (##) different from OGD condition at P < 0.05. Values for Δψm represent the mean ± SEM for an average of three samples in four experiments (by ANOVA, and Tukey's all pairwise multiple comparison test).
Figure 4
 
AAV2-σ-1r vector increases Δψm in RGCs exposed to OGD. (A) Δψm was measured in RGCs transduced with AAV2-σ-1r vector. Oxygen- and glucose-deprived conditions were significantly (*) different from normoxic conditions at P < 0.05, and OGD/AAV- σ-1r vector was significantly (#) different from OGD at P < 0.05. Values for Δψm represent the mean ± SEM for an average of three samples in three experiments by ANOVA and Tukey's all pairwise multiple comparison test. (B) Δψm was measured in RGCs transduced with AAV2-σ-1r vector, AAV2-empty vector served as controls. Values for Δψm represent the mean ± SEM for an average of three samples in three experiments. *Significantly different from control conditions at P < 0.05 and OGD + sigma-1 receptor virus without PTZ at P < 0.05 (by ANOVA and Tukey's all pairwise multiple comparison test).
Figure 4
 
AAV2-σ-1r vector increases Δψm in RGCs exposed to OGD. (A) Δψm was measured in RGCs transduced with AAV2-σ-1r vector. Oxygen- and glucose-deprived conditions were significantly (*) different from normoxic conditions at P < 0.05, and OGD/AAV- σ-1r vector was significantly (#) different from OGD at P < 0.05. Values for Δψm represent the mean ± SEM for an average of three samples in three experiments by ANOVA and Tukey's all pairwise multiple comparison test. (B) Δψm was measured in RGCs transduced with AAV2-σ-1r vector, AAV2-empty vector served as controls. Values for Δψm represent the mean ± SEM for an average of three samples in three experiments. *Significantly different from control conditions at P < 0.05 and OGD + sigma-1 receptor virus without PTZ at P < 0.05 (by ANOVA and Tukey's all pairwise multiple comparison test).
Figure 5
 
Cytochrome C oxidase activity is restored by σ-1r. Retinal ganglion cells were exposed to OGD conditions for 4 hours and either transduced with AAV-σ-1r vector or treated with pentazocine. Oxygen- and glucose-deprived conditions were significantly (*) different from normoxic conditions at P < 0.05, and OGD/AAV- σ-1r vector and OGD/10 μM PTZ were significantly (#) different from OGD at P < 0.05. Values represent the mean ± SD for four experiments by ANOVA and Tukey's all pairwise multiple comparison test.
Figure 5
 
Cytochrome C oxidase activity is restored by σ-1r. Retinal ganglion cells were exposed to OGD conditions for 4 hours and either transduced with AAV-σ-1r vector or treated with pentazocine. Oxygen- and glucose-deprived conditions were significantly (*) different from normoxic conditions at P < 0.05, and OGD/AAV- σ-1r vector and OGD/10 μM PTZ were significantly (#) different from OGD at P < 0.05. Values represent the mean ± SD for four experiments by ANOVA and Tukey's all pairwise multiple comparison test.
Figure 6
 
Caspase activity in σ-1r–transduced RGCs. Data are expressed as relative fold change of caspase activity in OGD conditions when compared with normoxia. Retinal ganglion cells were exposed to AAV-σ-1r vector or PTZ and caspase activity was measured. Data representative of two experiments performed in triplicates. Oxygen- and glucose-deprived *significantly different from normoxia, and #significantly different from OGD/AAV-σ-1r, and OGD/PTZ at P < 0.05 (by ANOVA and Tukey's all pairwise multiple comparison test).
Figure 6
 
Caspase activity in σ-1r–transduced RGCs. Data are expressed as relative fold change of caspase activity in OGD conditions when compared with normoxia. Retinal ganglion cells were exposed to AAV-σ-1r vector or PTZ and caspase activity was measured. Data representative of two experiments performed in triplicates. Oxygen- and glucose-deprived *significantly different from normoxia, and #significantly different from OGD/AAV-σ-1r, and OGD/PTZ at P < 0.05 (by ANOVA and Tukey's all pairwise multiple comparison test).
Figure 7
 
Immunofluorescence detection of the σ-1r (red) and VDAC (pink; because of the GFP) in RGCs. Confocal laser scanning microscope image of the RGCs transduced with AAV-σ-1r demonstrate more intense staining of σ-1r than in control (endogenous σ-1r). Merged images suggest a bright fusion of colors (not red and not pink) in the cytoplasm. Figure is representative of three images. Images were taken at ×40, zoom 2. Scale bars: 50 μM.
Figure 7
 
Immunofluorescence detection of the σ-1r (red) and VDAC (pink; because of the GFP) in RGCs. Confocal laser scanning microscope image of the RGCs transduced with AAV-σ-1r demonstrate more intense staining of σ-1r than in control (endogenous σ-1r). Merged images suggest a bright fusion of colors (not red and not pink) in the cytoplasm. Figure is representative of three images. Images were taken at ×40, zoom 2. Scale bars: 50 μM.
Figure 8
 
Pentazocine treatment demonstrates association between endogenous σ-1r and mitochondria (VDAC staining). Retinal gangliion cells were exposed to OGD conditions in the presence or absence of PTZ. Retinal ganglion cells were labeled with σ-1r antibody (red), VDAC (green), and DAPI (blue). Images were taken at ×40, zoom 2. Scale bars: 50 μM.
Figure 8
 
Pentazocine treatment demonstrates association between endogenous σ-1r and mitochondria (VDAC staining). Retinal gangliion cells were exposed to OGD conditions in the presence or absence of PTZ. Retinal ganglion cells were labeled with σ-1r antibody (red), VDAC (green), and DAPI (blue). Images were taken at ×40, zoom 2. Scale bars: 50 μM.
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