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. Author manuscript; available in PMC: 2013 Sep 16.
Published in final edited form as: Brain Res Mol Brain Res. 2002 Nov 15;107(2):97–107. doi: 10.1016/s0169-328x(02)00444-8

Analysis of Sigma Receptor (σR1) expression in retinal ganglion cells cultured under hyperglycemic conditions and in diabetic mice

M Shamsul Ola 1,*, Pamela Moore 1,*, Dennis Maddox 1, Amira El-Sherbeny 1, Wei Huang 1,3, Penny Roon 1, Neeraj Agarwal 2, Vadivel Ganapathy 3, Sylvia B Smith 1,4,**
PMCID: PMC3773709  NIHMSID: NIHMS380060  PMID: 12425939

Summary

The type 1 sigma receptor (σR1) is a nonopiate and nonphencyclidine binding site that has numerous pharmacological and physiological functions. In some studies, agonists for σR1 have been shown to afford neuroprotective against overstimulation of the NMDA receptor. σR1 expression has been demonstrated recently in retinal ganglion cells (RGC). RGCs undergo apoptosis early in diabetic retinopathy via NMDA receptor overstimulation. In the present study we asked whether RGCs cultured under hyperglycemic conditions and RGCs of diabetic mice continue to express σ1. RGCs were cultured 48 h in RPMI medium containing either 45 mM glucose or 11 mM glucose plus 34 mM mannitol (osmolar control). C57BL/6 mice were made diabetic using streptozotocin. The retina was dissected from normal and streptozotocin-induced diabetic mice 3, 6 and 12 weeks post-onset of diabetes. σR1 was analyzed in cells using semiquantitative RT-PCR and in tissues σR1 by semiquantitative RT-PCR, in situ hybridization, western blot analysis and immunolocalization. The RT-PCR analysis of cultured RGCs showed that σR1 mRNA is expressed under hyperglycemic conditions at levels similar to control cells. Similarly, analysis of retinas of diabetic mice showed no difference in levels of mRNA encoding σR1 compared to retinas of control mice. In situ hybridization analysis showed that expression patterns of σR1 mRNA in the ganglion cell layer were similar between diabetic and control mice. Western blot analysis suggested that levels of σR1 in retina were similar between diabetic and control retinas. Immunohistochemical analysis of σR1 showed a similar pattern of σR1 protein expression between control and diabetic retina. These studies demonstrate that σR1 is expressed under hyperglycemic conditions in vitro and in vivo.

Keywords: sigma receptor, retinal ganglion cells, retina, diabetes, diabetic retinopathy

Introduction

Diabetic retinopathy is a major sight-threatening disease and is the leading cause of blindness among working-aged Americans [48]. The best known clinical feature of non-proliferative diabetic retinopathy is microangiopathy. This develops in patients with Type I and Type II diabetes after about 10–15 years of disease onset [30]. The microangiopathy becomes sight threatening when it leads to macular edema and/or retinal ischemia with subsequent unregulated new vessel formation [10, 11]. In addition to its effects on retinal vasculature, diabetes also affects the neuronal cells in the retina [27]. ERG data obtained in diabetic patients suggest that abnormalities in the ganglion and inner retinal cell layers occur within the first year of disease onset [13, 39]. Indeed, Bresnick proposed that diabetic retinopathy is a primary neurosensory disorder that precedes vasculopathy by many years [7]. Barber and colleagues demonstrated increased apoptosis in cells of the retinal ganglion cell layer early in diabetes, both in a streptozotocin-induced rat model of diabetes and in retinas of patients with diabetes [3]. We have evidence that retinal ganglion cells die by apoptosis also in streptozotocin-induced diabetic mice (Moore et al, unpublished results).

The retinal ganglion cell death in diabetic retinopathy is thought to be mediated by overstimulation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor leading to excessive levels of intracellular calcium [26]. The increase in intracellular Ca2+ acts as a second messenger that sets in motion the cascade leading to cell death. Glutamate is the primary excitotoxin that activates the NMDA receptor and diabetes is associated with elevated levels of glutamate in the vitreous body [1]. Glutamate levels are elevated also in the retinas of rats with experimentally induced diabetes [23]. Under normal conditions, glutamate levels in the retina are kept in check by the enzyme glutamine synthetase produced by retinal Müller cells. Recent studies have shown that Müller cells are affected early in experimental and human diabetes and the expression of glutamine synthetase by these cells is decreased [33].

Numerous studies have demonstrated robust neuroprotective properties of a group of ligands specific for type 1 sigma receptors (σR1). Sigma receptors were discovered in 1976 and were originally thought to be opiate receptors [32]. More recent studies have shown that they are nonopiate and nonphencyclidine binding sites [34]. Various antiexcitotoxic mechanisms for σR1 ligands have been postulated including inhibition of ischemia-induced glutamate release [28, 29], attenuation of postsynaptic glutamate-evoked Ca2+ influx [9, 20], depressed neuronal responsivity to NMDA receptor stimulation [4, 8, 49], and reduced nitric oxide production [14]. σR1 has been cloned recently and its molecular identity established [15, 43]. σR1 expression has been demonstrated in many tissues (e.g. liver, spleen, gastrointestinal tract, adrenal gland, testis, ovary, placenta, brain, and lacrimal gland 9,28]). Studies from our lab and others suggest that σR1 is expressed in retina [38, 42]. Senda et al [42] used binding assays and demonstrated the presence of σ binding sites in the bovine retina, though the molecular subtype and nature of the binding site and the identity of the cells that express σR were not elucidated. Recently, we carried out in situ hybridization and immunohistochemical studies and demonstrated that σR1 mRNA is expressed abundantly in the retina and the σR1 protein is detectable in normal retinal ganglion cells [38]. These data suggest that RGCs may be amenable to the neuroprotective effect of σR1 agonists under conditions of neurotoxicity such as occurs in diabetes. If these agonists are to be tested, it is imperative to determine whether σR1 continues to be expressed in the retina during diabetes. To address this question we examined σR1 expression in retinal ganglion cells cultured under hyperglycemic conditions and in intact retinas of diabetic mice. Our data suggest that σR1 continues to be expressed under hyperglycemic conditions in vitro and in vivo.

Materials and Methods

Reagents

DMEM:F12 medium, TRIzol reagent, and penicillin-streptomycin were purchased from Life Technologies (Rockville, MD). Fetal bovine serum, streptozotocin, D-(+)-glucose and all other chemicals were purchased from Sigma (St. Louis, MO). RNAWIZ reagent was purchased from Ambion (Austin, TX). RNA PCR core kit was from Perkin-Elmer (Boston, MA). The digoxigenin-labeling kit, the alkaline phosphatase-coupled anti-digoxigenin antibody (anti-DIG-AP) and the NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate) stock solutions were from Boehringer-Mannheim (Indianapolis, IN). Tissue-Tek OCT embedding compound was from Miles Laboratories (Elkhart, IN). Urine Strip Tests (Diascreen G) were purchased from American Diagnostics (Pendleton, IN). The Prestige Glucose Monitoring System was from Home Diagnostics (Ft. Lauderdale, FL). ECL Western detection system was from Amersham (Piscataway, NJ). Imject maleimide activated mariculture keyhole limpet hemocyanin was from Pierce (Rockford, IL). The FITC-conjugated AffiniPure goat anti-rabbit IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA). Complete Mini protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Mannheim, Germany.) Lipofectin and pSPORT plasmid were purchased from Gibco-Life Technologies (Gaithersburg, MD).

Cell Culture

The retinal ganglion cells used in these studies were the RGC-5 cell line. The development of this rat ganglion cell line has been described [24]. Rat ganglion cells were maintained at 37°C in a humidified chamber of 5% CO2. They were maintained in 75 cm2 flasks with Dulbecco’s modified Eagle’s medium: nutrient mixture F12 (DMEM:F12), supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. The culture medium was replaced with fresh medium every other day. For studies of the effects of high glucose levels, cells were grown in RPMI medium supplemented with glucose. Confluent cultures were exposed for 48 h to either high glucose medium, which contained 45 mM glucose or to low glucose medium which contained 11 mM glucose plus 34 mM mannitol. Mannitol was added to control for osmolar effects.

Animals

C57BL/6 mice (Harlan-Sprague Dawley) were used in these experiments. Type I, or insulin-dependent diabetes mellitus, was induced chemically in three week old mice following our published method [36]. Briefly, mice received an intraperitoneal injection of 75 mg kg−1 streptozotocin (STZ) dissolved in sodium citrate buffer (0.01 mol/l, pH 4.5) on three successive days. Mice were screened for diabetes beginning three days after the first dose of STZ by testing for the presence of glucose in urine using the Urine Strip Test. At the time of the experiment, the diabetic state of the animal was confirmed by measuring blood glucose levels via a glucometer. Diabetic C57BL/6 and age-matched control mice were maintained in our mouse colony as described [36]. Care and use of the animals adhered to the principles set forth in the DHEW Publication, NIH 80-23 “The Guiding Principles in the Care and Use of Animals.”

Semi-quantitative RT-PCR analysis of σR1 mRNA in rat ganglion cells cultured under high and low glucose

Using the TRIzol reagent, total RNA was prepared from RGC-5 cells cultured in the presence of 11 mM glucose plus 34 mM mannitol or 45 mM glucose for 48 h. RT-PCR was carried out using primer pairs specific for rat σR1 (sense, 5’-GTTTCTGACTATTGTGGCGGT GGTG-3’; antisense, 5’-CAAATGCCAGGGTAGACGGAATAAC-3’). These primers correspond to nucleotide positions 80–104 and 567–591, respectively, of the cloned rat σR1 cDNA (GenBank accession no. AF004218) [43]. RT-PCR conditions were as follows: denaturing phase 94°C, annealing phase of 30 sec at 60.5°C, and an extension of 2 min at 72°C. RT-PCR was carried out with cycles ranging from 12 to 32 to determine the linear range for the formation of the product. The PCR products (10 µl) were size fractionated on an agarose gel and subjected to Southern hybridization with 32P-cDNA probe specific for rat σR1 to quantitate the PCR product. Rat GAPDH was utilized as an internal control. The upstream primer 5’-TGGAGTCTACTGGCGTCTTC-3’(sense), and the downstream primer 5’-TCATGAGCCCTTCCACGATG-3’(antisense) correspond to nucleotide positions 1130–1149 and 1350–1369, respectively, in rat GAPDH cDNA (GenBank accession no. AF106860). Hybridization signals were quantified using STORM phosphorimaging system (Molecular Dynamics, Sunnyvale, CA) and processed using ImageQuaNT (Version 4.2a) software application. Intensities were analyzed using the linear range of the PCR cycle.

Semi-quantitative RT-PCR analysis of σR1 mRNA in retinas of control and streptozotocin-induced diabetic mice

The neural retina was dissected from the remainder of the eyecup of mice that had been diabetic for 3, 6 and 12 weeks. Retinas from age-matched C57Bl/6 mice were used as controls. Average blood glucose levels were 436 ± 22, 383 ± 20, 351 ± 34 mg/dl for animals that had been diabetic 3, 6 and 12 weeks, respectively. Control mice had blood glucose levels of 172 ± 14 mg/dl. Six eyes per group per experiment were pooled for analysis. Total RNA was isolated using the RNAWIZ reagent. RT-PCR was carried out using primer pairs specific for mouse σR1. The sense primer 5’-TATCGCAGTGCTGATCCA-3’ and the antisense primer 5’-TACTCCACCATCCACGTGTT-3’ correspond to nucleotide positions 75 to 92 and 520 to 539, respectively, in the cloned mouse σR1 cDNA (GenBank accession no. AF030198) [45]. As an internal control, RT-PCR was carried out with primer pairs specific for mouse GAPDH (sense primer 5’-ACCGGATTTGGCCGTATT-3’, antisense primer 5’-TCTGGGATGGAAATTGTG AG-3’. These primers correspond to positions 65 to 82 and 1132 to 1151, respectively in the cloned mouse GAPDH cDNA (GenBank accession no. M32599). Semiquantitative RT-PCR was carried out as described earlier.

In situ hybridization

To localize the mRNA transcript encoding σR1 in control and streptozotocin-induced diabetic mice, in situ hybridization was performed on mouse eyes. For the preparation of the mouse σR1-specific riboprobe, a 0.65 kbp fragment of the mouse σR1 cDNA, obtained by the digestion of the pSPORT mouse σR1 cDNA plasmid by SalI/SmaI, was subcloned into pBluescript vector. The orientation of the cDNA insert in the pBluescript vector was established by sequencing. Antisense and sense riboprobes were synthesized with T7 RNA polymerase or T3 RNA polymerase after linerization of the plasmid with appropriate restriction enzymes. The riboprobes were labeled using a digoxigenin-labeling kit. Eyes from C57BL/6 diabetic and control mice were enucleated, frozen immediately in Tissue-Tek OCT sectioned at 10 µm thickness, and fixed in 4% paraformaldehyde. Following our published protocol [38], sections were rinsed in ice-cold PBS and treated with active 1% diethylpyrocarbonate (DEPC) prepared in PBS to facilitate penetration of the labeled probes. Sections were permeabilized further with proteinase K (1 (g/ml) in PBS for 4 min. The proteinase K activity was stopped by rinsing the slides in glycine (2 mg/ml) in PBS. Sections were washed in PBS, equilibrated in 5% SSC, and were prehybridized for 2 h at 58°C in 50% (v/v) formamide, 5% SSC, 2% (w/v) blocking reagent (provided with the DIG Nucleic Acid Detection Kit), 0.1% w/v N-lauroylsarcosine, and 0.02% (w/v) sodium dodecyl sulfate. Sections were hybridized with the probes (1 µg/ml) and were incubated overnight at 58°C. They were washed twice in 2% SSC at room temperature, twice in 1% SSC at 55°C, and twice in 0.1% SSC at 37°C. For immunologic detection of the probe, sections were washed in a buffer containing 0.1 M maleic acid and 0.15 M NaCl (pH 7.5) and were blocked with the same buffer containing 1% blocking reagent. The anti-DIG-antibody conjugated to alkaline phosphatase was diluted 1:5000, and slides were incubated with this antibody for 2 h at room temperature. Sections were washed in the preceding wash buffer containing levamisol (200 (g/ml) twice for 10 min and were equilibrated with a buffer containing 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2. The color reaction was developed in NBT/BCIP. Slides were washed in distilled water and coverslipped, but not counterstained so that the purplish-red colored precipitate, indicative of a positive reaction, could be visualized in the sections.

Preparation of the antibody against σR1

An antipeptide antibody was raised against the peptide sequence SEVYYPGETVVHGPGEATD VEWG, which corresponds to residues 143–165 of rat σR1. An extra cysteine residue was added at the N-terminus of the peptide to permit conjugation with keyhole limpet hemocyanin. The peptide (2 mg) was conjugated to 2 mg of Imject maleimide activated mariculture keyhole limpet hemocyanin and purified by overnight dialysis. Approximately 300 µg of the peptide-conjugated hemocyanin in Freund’s complete adjuvant was administered intradermally to New Zealand white rabbits (approximately 50 µl each). The initial injection was followed by two boosters. Antiserum was obtained 10 days after the second booster and purified using affinity chromatography.

Western blot analysis of σR1 levels in retinas of diabetic mice compared to controls

The neural retina was dissected from the remainder of the eyecup of mice that had been diabetic for 3, 6 and 12 weeks. Eyes from age-matched C57Bl/6 were used as controls. Ten eyes per group per experiment were pooled for analysis. Tissues were homogenized using a glass-glass homogenizer 20–30 times in 50 mM Tris-HCl buffer pH 7.4, containing 1 mM PMSF and a protease inhibitor cocktail. The sample was then centrifuged at 21000×g for 30 min. The pellet was collected and dissolved in lysis buffer (50 mM Tris-HCl, pH 7.4, containing 1% Triton-X-100, 0.1% SDS, 10 mM EDTA, 2 mM Na3VO4, 10 mM sodium pyrophosphate and 50 mM NaF). The sample was sonicated for 10–15 sec and then passed through a syringe containing a 26 g needle 10 times. The sample was then centrifuged at 21,000×g for 30 min at 4°C. The supernatant was collected and the protein concentrations was estimated according to the method of Lowry et al [31]. Equivalent amounts of protein from the tissue lysates were boiled in Laemmli’s buffer for 5 min and analyzed by 10% SDS-PAGE. After transferring the separated proteins onto nitrocellulose membranes, the membranes were blocked for 1.5 h at room temperature with Tris-buffered saline-0.1% Tween-20 containing 5% non-fat milk. The membranes were incubated for 3 h at room temperature with the polyclonal antibody against σR1 (1:1000). The membranes were probed with a secondary HRP-conjugated goat anti-rabbit IgG antibody (1:3000) for 1.5 h, washed and the proteins visualized using the ECL Western detection system. The membranes were washed and reprobed with an antibody against β–actin. Following immunoblotting, films were placed on a white light box vertically and image capture was performed using the AlphaImager 2200 digital imaging system (Alpha Innotech Corporation, San Leandro, CA). A drag-and-drop rectangular grid of identical size was placed on the band of interest and captured for processing. Background density was automatically subtracted. Data represent an average of all the pixel values enclosed in the grid.

The specificity of the polyclonal antibody against σR1 that was generated for these studies was determined in the following manner. MCF-7 cells, which do not contain endogenous σR1, were transfected using the vaccinia virus expression system as described previously [43]. The cDNA encoding σR1 was cloned into the plasmid pSPORT in such an orientation that the sense transcription of the cDNA was under the control of T7 promoter in the plasmid. MCF-7 cells were grown in DMEM medium supplemented with 5% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Subconfluent MCF-7 cells were first infected with the recombinant vaccinia virus (VTF7-3) encoding T7 RNA polymerase. This was followed by Lipofectin-mediated transfection of the plasmid cDNA into the cells. Cells transfected with empty vector served as control. After transfection, cells were re-cultured in OPTI-MEMI medium for 12 h before collecting the protein for western blot analysis. As shown in Fig. 1, the proteins extracted from the cells transfected with the σR1 cDNA showed the expected band with a molecular mass of ~27 kDa, while this band was not detected in the cells transfected with the empty vector. Reprobing of the membrane with an antibody specific for β-actin showed the expected band of ~45 kDa in both the transfected and non-transfected cells. These data suggest that the antipeptide antibody used in these studies is specific for σR1.

Figure 1. Western blot analysis to determine the specificity of the polyclonal σR1 antibody.

Figure 1

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MCF-7 cells were transfected with cDNA encoding σR1 using vaccinia virus expression system; cells transfected with empty vector served as control. After transfection, proteins were extracted from the cells and were processed for western blot analysis using our polyclonal antibody against σR1 followed by an antibody against β-actin. Lanes (C) and (T) shown protein from control cells and cells transfected with cDNA of σR1, respectively.

Immunohistochemistry

Immunohistochemical methods were used to localize σR1 in retina of intact control and diabetic mouse eyes. Cryosections of mouse eyes were fixed with ice-cold acetone, blocked with 10% normal goat serum and were incubated for 3 h at room temperature with the antibody against σR1 at a dilution of 1:50 followed by an overnight incubation at 4°C. Incubation with 0.1% normal rabbit serum or with buffer only served as negative controls. After rinsing, all sections were incubated overnight at 4°C with a fluorescein thiocyanate (FITC)-conjugated AffiniPure goat anti-rabbit IgG at a dilution of 1:100. Sections were examined using a Zeiss Axioplan 2 flourescent microscope (Carl Zeiss Inc., Germany) equipped with a Spot Camera and Spot Software version 2.2 (Diagnostic Instruments, Inc., Sterling Heights, MI).

Results

Semiquantitative RT-PCR analysis of σR1 mRNA in RGC-5 cells cultured in high and low glucose

The influence of high glucose on the steady-state levels of mRNA transcripts specific for σR1 in cultured retinal ganglion cells was investigated using semiquantitative RT-PCR. Cells were cultured in the presence of 45 mM glucose or 11 mM plus 34 mM mannitol (osmolar control). The steady-state levels of mRNA encoding σR1 were normalized against GAPDH for glucose-treated and control cells. Figs. 2A and 2B show Southern hybridization signals in the linear cycles for both σR1 and GAPDH. Fig. 2C shows representative RT-PCR products for analysis of σR1 in cells grown in 11 mM glucose compared to those grown in 45 mM glucose. The band intensities obtained were nearly identical. Similarly, steady-state levels of GAPDH mRNA did not appear to differ between the cells grown in 11 mM glucose versus those grown in 45 mM glucose. The phosphorimage analysis of the data gave a ratio of σR1 mRNA bands to GAPDH bands in cells grown in 45 mM glucose that was quite similar to that of cells grown in 11 mM glucose (Fig 2D). These results suggest that hyperglycemic conditions do not alter the steady-state levels of σR1 mRNA in RGC-5 cells.

Figure 2. Analysis of steady-state levels of mRNA for σR1 and GAPDH in rat retinal ganglion cells exposed to high levels of glucose.

Figure 2

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Total RNA was isolated from RGC cells treated with 45 mM glucose or 11 mM glucose plus 34 mM mannitol (control) for 48 h at 37°C. Primer pairs specific for rat σR1 and GAPDH were used. RT-PCR was performed over a wide range of PCR cycles (9–32) for both σR1 and GAPDH. The resultant PCR products were run on agarose gels and stained with ethidium bromide solution. The number of cycles, which were not visualized with UV exposure, were considered to be in the linear range. The linear range was analyzed by performing RT-PCR in the lower range of PCR cycles (15–24) for σR1 and (9–18) for GAPDH. The resultant products were run on a gel and then subjected to Southern hybridization with 32P-labelled cDNA probes specific for σR1 and GAPDH. The hybridization signals were quantified by phosphorescence imaging and a graph was plotted of band intensities versus number of cycles. (A) Southern hybridization signal in linear cycles (18–24) for σR1. (B) Southern hybridization signal in linear cycles (9–15) for GAPDH. (C) Representative Southern hybridization signal in 45 mM and 11 mM glucose. (D) The relative band intensity (σR1/GAPDH) in cells treated with high glucose relative to that in control cells.

Analysis of steady-state levels of σR1 mRNA in retinas of diabetic and control mice

The analysis of σR1 gene expression in the cultured ganglion cells described in Fig.2 provided evidence that short term exposure to high glucose levels did not alter σR1 mRNA levels. Since our interest is in determining whether σR1 ligands may be beneficial for prevention of ganglion cell loss in diabetes, we analyzed σR1 mRNA expression in neural retinas of control and diabetic mice. Neural retinas were dissected free from the remainder of the eyecup. The retinas of control mice and mice that had been diabetic for 3, 6 or 12 weeks were used for semiquantitative RT-PCR for the determination of levels of mRNA transcripts encoding σR1. As an internal control, the steady-state levels of GAPDH mRNA in the samples were determined in parallel. Representative gels from these experiments are shown in Fig. 3. The σR1 mRNA levels in retinas of control mice were similar to those of diabetic mice. This was true not only at 3 weeks, but also at the 6 and 12 week time points. The phosphorimage analysis of the data (Fig. 3B) gave a ratio of σR1 mRNA bands to GAPDH bands in diabetic retinas that was quite similar to that of control retinas, although at six weeks there appeared to be a slight upregulation of the σR1 mRNA expression. This shows that the steady state levels of σR1 mRNA are not changed by long-term hyperglycemia in the intact diabetic retina.

Figure 3. Analysis of steady-state levels of mRNA for σR1 and GAPDH in retina of control (C) and diabetic (D) mice.

Figure 3

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Total RNA was isolated from neural retinas of mice at 3, 6, 12 weeks post-onset of diabetes and in age-matched control mice. Primers specific for mouse σR1 and mouse GAPDH mRNA were used for RT-PCR with appropriate PCR cycle numbers so that the product formation was within the linear range (24 cycles for σR1 and 12 cycles for GAPDH). (A) Representative Southern hybridization signal. (B) The relative band intensity (σR1/GAPDH) in diabetic retina relative to control retina. The σR1/GAPDH ratio in control retina was taken as 1.

In situ hybridization analysis of σR1 in retinas of diabetic and control mice

The semiquantitative RT-PCR analysis of σR1 mRNA expression shown in figure 3 provided an analysis of the entire neural retinas of diabetic and control mice. The dissection eliminates the RPE from the analysis as well as other non-retinal ocular tissues, but it does not permit isolation of specific cell types, such as ganglion cells, which are at risk in diabetic retinopathy. To determine whether the cells that were expressing σR1 mRNA included the retinal ganglion cells, in situ hybridization was performed with mRNA probes specific for mouse σR1. In situ hybridization analysis was performed on cryosections of eyes of mice that had been diabetic 2, 6 and 12 weeks and age-matched control C57BL/6 mice using a digoxigenin-labeled riboprobe. As shown in figure 4, σR1 mRNA transcripts were expressed in the cells of the ganglion cell layer at all ages studied in both control (A, C, E) and diabetic (B, D, F) mice. At 2 weeks post-onset of diabetes (Fig. 4B), there was intense expression of mRNA in nearly all cells of the ganglion cell layer as it was in age-matched controls (Fig. 4A). At 6 weeks post-onset of diabetes, the σR1 was expressed in ganglion cells (Fig. 4D) in a manner similar to age-matched controls (Fig. 4C). Even at 12 weeks post-onset of diabetes, a time when there are fewer ganglion cells present, σR1 is expressed in the ganglion cells remaining (Fig. 4F) just as it is in control retinas (Fig 4E). Other cells of the retina were also positive for σR1 expression, including cells of the inner nuclear layer, which contains amacrine, bipolar, horizontal and Müller cells. Hybridization of the sections with the sense probe showed no positive staining in any cells (data not shown).

Figure 4. Distribution of σR1-specific mRNA transcript in control and diabetic mouse retina as assessed by in situ hybridization.

Figure 4

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Cryosections of mouse retina from control age-matched to diabetic mice (2, 6, 12 weeks post-onset of diabetes) were probed with an antisense digoxigenin-labeled σR1 riboprobe. The analysis showed a positive reaction (deep purple stain) in the retinal ganglion cells (arrow). A positive reaction was observed in the ganglion cells of diabetic mice at all ages examined. (A) 2 week control, (B) 2 week diabetic, (C) 6 week control, (D) 6 week diabetic, (E)12 week control, (F) 12 week diabetic. gcl = ganglion cell layer, inl = inner nuclear layer. (Magnification: 400X).

Western blot analysis of σR1 in retinas of diabetic and control mice

While the semi-quantitative RT-PCR analysis suggested that mRNA levels encoding σR1 were similar between retinas of diabetic and control mice, it was not certain that the σR1 protein levels were comparable. To analyze this, western blot was used. Neural retinas of mice that had been diabetic for either 3, 6 or 12 weeks were dissected from the remainder of the eyecup and prepared for SDS-PAGE and subsequent western blotting using a polyclonal antibody against σR1. Figure 5A shows the data from a membrane probed initially with the antibody against σR1 (Mr ~27 kDa) and then stripped and reprobed with the antibody against β-actin (Mr ~45 kDa). The bands corresponding to σR1 were quite similar for retinas of diabetic mice compared with those of control mice. The bands corresponding to β-actin were similar at 3, 6 and 12 weeks. Quantitative analysis of the band density of the σR1 compared with the β-actin (Fig. 5B) showed very similar ratios of these two proteins in the diabetic and control retinas at 3, 6 and 12 weeks. These data are important as they provide evidence that the σR1 is present in the diabetic mouse retina and may be a useful target for intervention using the ligands for σR1.

Figure 5. Western blot analysis of σR1 in retinas of diabetic and control mice.

Figure 5

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Neural retinas from normal and diabetic mice were processed for western blot analysis using a polyclonal antibody against σR1 followed by an antibody against β-actin. The density of the bands was quantified by densitometry. (A) Immunoblot; the molecular sizes of the σR1 and β-actin bands are indicated. (B) Data from densitometric scans of blots (σR1/β-actin). The σR1/βactin ratio in control retinas was taken as 1.

Immunohistochemical analysis of σR1 protein in diabetic and control mouse eyes

The western blot analysis indicated that σR1 protein is present in neural retinas of diabetic mice, however it did not indicate which cells of the neural retina contained the protein. To determine whether σR1 was present in ganglion cells of diabetic mice, immunohistochemical analysis was performed. Eyes from mice that had been diabetic 2, 6 and 12 weeks and age-matched controls were cryosectioned and incubated with the antibody specific for σR1. Figure 6 shows representative immunohistochemical data of retinas from these mice. Examination of the photomicrographs shows that σR1 is present in ganglion cells of control mice (Fig. 6A,C and E) at all ages studied. The σR1 is present also in ganglion cells of diabetic mice (Fig. 6B, D, F). This is particularly evident at 2 and 6 weeks post-onset of diabetes (Fig. 6B and D). At 12 weeks after the onset of diabetes (Fig. 6 F), the immunolabeling of ganglion cells was not as uniform as in the retinas of the control mice. This is likely due to the apoptotic cell death that occurs in the ganglion cells of diabetic retina. Nevertheless, many of the ganglion cells were positive.

Figure 6. Fluorescent microscopic immunolocalization of σR1 in intact eyes of diabetic and control mice.

Figure 6

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Cryosections of mouse eyes from control and age-matched diabetic mice that were incubated with antibody against σR1 followed by incubation with FITC-labeled secondary antibody. (A) 2 week control, (B) 2 week diabetic, (C) 6 week control, (D) 6 week diabetic, (E)12 week control, (F) 12 week diabetic. gcl = ganglion cell layer, inl = inner nuclear layer. (Magnification: 400X).

Discussion

The present paper describes experiments designed to determine whether σR1 is expressed in retinal ganglion cells under hyperglycemic conditions. Previously, we reported that in the normal retina several cell types express σR1. These cells include retinal pigment epithelial cells, Müller cells and ganglion cells [38]. We were particularly interested in the finding that ganglion cells, the second order neurons of the visual pathway, expressed σR1. It has been reported that ganglion cells die by apoptosis in human patients with diabetes and in experimental models of diabetes [3, 27]. The ganglion cell death is thought to be due to overstimulation of the NMDA receptor, thus compounds that could interfere with this stimulation may prevent the apoptosis. Agonists for the σR1 receptor may offer neuroprotection to cells provided those cells continue to express σR1 during diabetes. Our analysis was conducted initially in vitro using a well-differentiated rat retinal ganglion cell line, RGC-5. These cells express several ganglion cell specific markers, including Thy1 and Brn-3C, and are considered a useful model to study aspects of retinal ganglion cell biology [24]. We exposed these cells to very high levels of glucose (45 mM) and found that σR1 mRNA levels were similar between hyperglycemic cells and those treated with 11 mM glucose. The observation that the σR1 expression was not altered significantly during a short term exposure to high glucose levels encouraged us to pursue studies in an animal model of diabetes. The remainder of the analyses was carried out with retinas of diabetic and age-matched control mice. Mice, made diabetic using streptozotocin, consistently had blood glucose levels greater than 300 mg/dl. We did not administer insulin to these animals as we did not wish to confound the interpretation of the data. The neural retinas expressed σR1 mRNA at levels comparable to age-matched controls as assessed by semiquantitative RT-PCR. This provided a crude estimate of the σR1 expression in ganglion cells, however the analysis actually represents σR1 mRNA for all cells in the neural retina. We know from previous in situ hybridization studies [38] that many retinal cells are positive for σR1 mRNA, not just ganglion cells. In subsequent experiments with diabetic mice we used in situ hybridization and found that σR1 was expressed in the ganglion cells of diabetic mice. We then examined σR1 protein levels and found both by western analysis and immunohistochemistry that σR1 protein is present in ganglion cells of diabetic mice.

These studies are very encouraging because they provide a rationale for using σR1 agonists to block ganglion cell death characteristic of diabetic retinopathy. σR1 agonists have been proposed as a neuroprotectant in other conditions. For example, (+) pentazocine, a potent σR1 agonist, afforded significant neuroprotection in a rat model of transient focal ischemia [47]. Similarly, the σR1 agonist PPBP ((4-phenylbutyl)-piperidine) protects against focal ischemia [16]. In those studies, it was shown that the PPP modulates NMDA evoked NO production in vivo. This observation is noteworthy as NO is a regulator of cerebral blood flow [18]. If present in abnormally high concentrations NO may exert neurotoxic effects [50]. NO levels are increased in diabetes [2, 21], including in patients with diabetic retinopathy [40] and in retinal cells cultured under high glucose conditions [22]. Senda and co-workers [41] demonstrated the protective effects of pentazocine and SA4503, two σR1 agonists on glutamate-induced damage of cultured retinal (amacrine) neurons. There have been no studies reported to date analyzing the neuroprotective effects of σR1 agonists on ganglion cell death either in vitro or in vivo. Nor is it known whether interfering with the early ganglion cell death characteristic of diabetes would alter the subsequent vasculopathy characteristic of diabetic retinopathy.

σR1 is an enigmatic molecule. While details about the gene encoding σR1 and the size of the protein have been determined (it has a molecular weight in the range of ~25 – 28 kD and contains 223 amino acids [12, 15, 43]), there are many questions remaining about how it functions. The endogenous ligand for σR1 is not known, although there has been speculation that progesterone is an endogenous ligand for σR1 [46]. σR1 contains a putative transmembrane region at the N-terminus and two stretches of hydrophobic amino acids. Some investigators have localized σR1 to the endoplasmic reticulum [17] while others have localized it to the nuclear membrane [19]. Precisely how σR1 ligands confer neuroprotection is not known. Largent and coworkers used pharmacological and autoradiographic analyses and found that the benzomorphan opioid, SKF 10,047, which is a prototypical agonist for the σR1, had not only affinity for σR, but also a lower affinity for the phencyclidine (PCP) site of the NMDA receptor channel complex [25]. Nishikawa and colleagues reported that σR1 ligands that bind to both the σR1 and the NMDA receptor channel complex prevented glutamate induced toxicity in their cultured cortical neurons, while the compounds that bound only the σR1 site were not neuroprotective [37]. They suggested that σR1 ligands interact directly with the NMDA/PCP receptor channels to protect against glutamate neurotoxicity. Others have suggested that the neuroprotective effects of σR1 ligands are due to modulation of NMDA receptors as well as muscarinic receptors [8, 35]. Lobner and Lipton [28] showed that glutamate release from the hippocampus during ischemic insult was attenuated by σR1 ligands suggesting additional mechanisms for the σR ligand-mediated neuroprotection against glutamate toxicity. Hayashi and Su [17] found that σR1 control the function of cytoskeletal proteins, such as ankyrin which in turn regulates Ca2+ signaling. Thus, though there have been numerous reports on the pharmacology of several σR1 ligands, the exact role of the receptor in neuroprotection remains to be determined. It will be very informative to determine first whether σR1 ligands are protective in ganglion cell death during diabetes and, if so, to determine the mechanism of that neuroprotection. While numerous beneficial effects have been ascribed to σR1 agonists, Bowen [6] has reviewed the literature associated with cyototoxic effects of sigma receptor ligands. He cites a number of studies in which ligands specific for the type 2 σR were cytotoxic. The σR-2 selective compounds CB-64D and ibogaine caused severe morphological changes in C6 glioma and other neuronal cells. Continued exposure of the cells to these compounds ultimately resulted in their death. Thus, as with any pharmacologic intervention, the enthusiasm about beneficial effects must be tempered with careful consideration of possible toxic side effects.

In summary, the present study has established that σR1 mRNA is expressed and the protein is present in ganglion cells cultured under hyperglycemic conditions and in ganglion cells of retinas of diabetic mice. Future studies will examine the efficacy of σR1 ligands in protecting against ganglion cell death in vitro and in vivo.

Acknowledgements

This work was supported by National Institutes of Health Grants EY12830, EY13089, and DA10065, Fight for Sight-Prevent Blindness America, an unrestricted award from Research to Prevent Blindness, Inc. to the Department of Ophthalmology, Medical College of Georgia, the Medical College of Georgia Research Institute and the American Health Assistance Foundation – National Glaucoma Foundation.

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