Serum Deprivation Induces Apoptotic Cell Death of Transformed Rat Retinal Ganglion Cells via Mitochondrial Signaling Pathways

Abstract

purpose. Apoptosis-related signaling pathways were investigated in a cultured rat retinal ganglion cell (RGC-5) line deprived of growth factors after serum withdrawal from the culture medium.

methods. RGC-5 cells were subjected to serum deprivation for 2 to 6 days and compared with RGC-5 cells cultured in growth medium containing 10% fetal calf serum. Cell viability was determined by a neutral red dye uptake assay. Apoptosis of RGC-5 cells was established by DNA laddering. The expression of various apoptosis-related genes was investigated by immunoblot analysis, and or reverse transcription polymerase chain reaction (RT-PCR) analysis. The redox state of the cell was determined by biochemical methods, including NF-κB binding activity by electrophoretic mobility gel shift assays (EMSA) and mitochondrial damage by JC-1 (5,5′, 6,6′-tetrachloro 1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide) staining, using live cell confocal microscopy and cytosolic release of cytochrome c.

results. Fifty percent cell loss was evident after 2 days of serum deprivation, as demonstrated by neutral red dye uptake assay. This cell loss was due to apoptotic cell death, as established by DNA laddering. The oxidative state of serum-deprived RGC-5 cells was perturbed as suggested by the increase in malonyldialdehyde (MDA) and a decrease in reduced glutathione (GSH) levels in cell lysates. The apoptosis of the RGC-5 cells was associated with the activation of caspase-3, -8, and -9, and increased levels of Bax with corresponding decreases in Bcl-2 levels and NF-κB (NF-κB) binding activity. Serum deprivation was also associated with a loss of mitochondrial function, as revealed by cytosolic release of cytochrome c and JC-1 staining of mitochondria of dying RGC-5 cells.

conclusions. Taken together, these results indicate that serum withdrawal induces apoptotic cell death in RGC-5 cells via mitochondrial pathways. These studies lead to the speculation that growth factor deprivation arising from blockade of retrograde transport of neurotrophins may involve similar mechanism(s) of retinal ganglion cell death in glaucoma.

Glaucoma is currently the second leading cause of blindness in the United States, and its incidence is on the rise every year. ^{1 2} It is a progressive optic neuropathy that is characterized by an excavated appearance of the optic nerve head and loss of retinal ganglion cells (RGCs) due to apoptosis. ³ Glaucoma was originally characterized by an elevation in intraocular pressure (IOP), which remains one of the highest risk factors for the development of glaucoma. Although several other risk factors also contribute, the cause of vision loss in all cases is ultimately through apoptosis of RGCs, ^{4 5 6 7} which was first shown by transection of the optic nerve, the axons of the RGCs, in the rat. ^{8 9 10}  

Other investigators found apoptosis of RGCs in a monkey model of experimental glaucoma. ^{4 11} Studies in human primary open angle glaucoma (POAG) showed TUNEL-positive labeling in the RGC layer in 50% of the patients compared with <10% in the control group. ¹² One of the hypotheses to explain apoptotic cell death of RGCs is that the elevated IOP results in obstruction of axonal transport within the optic nerve head, leading to blockade of retrograde transport of neurotrophins. ^{3 13 14 15 16 17 18 19}  

RGCs play a key role in integrating visual information and relaying it to the visual centers of the brain through the optic tract. The RGCs are sustained by neurotrophic factors that are retrogradely transported to the ganglion cells. ³ When the axons are damaged, as occurs in glaucoma, retrograde transport is disrupted, and the ganglion cells die by apoptosis. ³ Because trophic withdrawal has been hypothesized as a primary cause of RGC death in glaucoma, ²⁰ we attempted to elucidate the pathways that RGCs use when they undergo apoptosis during serum deprivation. The in vitro model described herein mimics the “blocked axonal transport of neurotrophins” paradigm and therefore may shed some light on the prospective mechanisms involved in RGC degeneration in glaucoma. 

A transformed rat retinal culture of RGCs, RGC-5 cells, ²¹ recently established in the laboratory, were used in the present studies. Our data show that serum deprivation of RGC-5 cells for various durations results in the activation of caspase-3 and -9. Increased Bax levels, decreased Bcl-2 levels, NF-κB binding activity, and cellular GSH levels were also observed in the serum-deprived RGC-5 cells. Moreover, serum deprivation also resulted in cytochrome c release from the mitochondria with associated loss of mitochondrial membrane potential. Taken together, these data suggest that serum deprivation promotes apoptotic cell death of RGC-5 cells primarily via a mitochondrial pathway. 

Materials and Methods

Culture of RGC-5 Cells

Cultures of RGC-5 cells were maintained in growth medium containing low-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO) in a humidified atmosphere of 95% air and 5% CO₂ at 37°C, as described by Krishnamoorthy et al. ²¹ The cells were passaged every 3 to 4 days, with a doubling time of 18 to 20 hours. 

Serum Deprivation of RGC-5 Cells

The RGC-5 cells were seeded in the tissue culture dishes at the desired density. After 2 to 3 hours, when the cells attached to the dish, the dishes were rinsed with serum-free medium three to five times. The dishes were then incubated with DMEM containing 0% FCS or 10% FCS for 2 to 6 days under the conditions described for cell culture. 

Cell-Viability Assays

The effects of serum deprivation on the survival of RGC-5 cells were evaluated by using the neutral red uptake viability assay. ²² Neutral red dye (Invitrogen-Gibco, Grand Island, NY) was added to the cells to a final concentration of 0.033% in HEPES buffer (in mM; 125 NaCl, 5 KCl, 1.8 CaCl₂, 2 MgCl₂, 0.5 NaH₂PO₄, 5 NaHCO₃, 10 mM d-glucose, and 10 HEPES [pH 7.2]) after exposure to various conditions of serum deprivation. After 2 hours, cells were gently washed with 2 volumes of HEPES buffer to wash off the dye not taken up by the live cells. The cells were air dried for 20 minutes and treated with 500 μL of ice-cold solubilization buffer (1% acetic acid and 50% ethanol). Twenty minutes later, 100-μL aliquots were transferred to wells of flat-bottomed 96-well plates, and optical densities of samples were read at 570 nm. ²¹  

Reverse-Transcription–Polymerase Chain Reaction Analysis

RGC-5 cells exposed to growth medium containing 0% or 10% serum were used for isolation of total RNA (RNAzol B reagent; Tel-Test Inc., Friendswood, TX) and subjected to cDNA synthesis with avian myeloblastosis virus [AMV] reverse transcriptase. ²³ The PCR primers for Bcl-2, Bax, and β-actin were purchased either commercially (Continental Laboratory Products, Inc., San Diego, CA) or were designed from published sequences by using Primer 3 (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi/results_from_primer3 Massachusetts Institute of Technology, Cambridge, MA). The PCR primers for Bcl-2 were: TGC ACC TGA CGC CCT TCA C (S) and AGA CAG CCA GGA GAA ATC AAA CAG (A); for Bax: ACC AAG AAG CTG AGC GAG TGT C (S) and ACA AAG ATG GTC ACG GTC TGC C (A); and for β-actin: TGT GAT GGT GGG AAT GGG TCA G (S) and TTT GAT GTC ACG CAC GAT TTC (A), where S represents the forward or sense primer and A represents the backward or reverse primer. PCR reactions were run with the hot start method, which utilizes a monoclonal antibody to Taq polymerase. Programmable temperature cycling (Perkin Elmer, Wellesley, MA) was performed as follows: 1 initial denaturing cycle for 5 minutes at 94°C and 5 minutes at 60°C, followed by 30 to 40 amplification cycles for 2 minutes at 72°C, 1 minute at 94°C, and 1 minute at 60°C, and a final extension of one cycle of 10 minutes at 72°C. The authenticity of the RT-PCR product was established either by Southern blot hybridization or sequencing. All the test samples were amplified simultaneously with a particular primer pair by using a master mix containing all the components in the PCR reaction, except the target cDNA, or in the case of the control, water. ²³ β-Actin primers served as the internal control for the amount of cDNA for each condition and to confirm proper cDNA synthesis. For quantification, band densities of Bcl-2 and Bax PCR products were determined by densitometry using NIH Image (available by ftp at zippy.nimh.nih.gov/or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). 

JC-1 Mitochondrial Staining in Live RGC-5 Cells: Confocal Microscopy

RGC-5 cells were seeded in 35-mm glass-bottomed dishes (World Precision Instruments, Inc., Sarasota, FL) with DMEM supplemented with or without 10% FBS as described earlier. In healthy cells due to higher mitochondrial membrane potentials, JC-1 forms red fluorescent “J aggregates” that exhibit a broad excitation spectrum and an emission maximum at ∼590 nm and appear red. However, JC-1 exists as a monomer at low mitochondrial membrane potential, as seen in apoptotic cells, and appears green. Thus, the emission of this cyanin dye can be used as a sensitive measure of mitochondrial membrane potential ²⁴ (Molecular Probes, Eugene, OR). A stock solution of JC-1 (Molecular Probes) was prepared at 4 mg/mL in dimethylsulfoxide (DMSO). The stock JC-1 solution was added drop-wise, while vortexing, to an appropriate volume of control medium to a final concentration of 10 μg/mL. The diluted JC-1 solution was then passed through a 0.2-μM syringe filter (GeneMate, Kaysville, UT). An appropriate volume was added to the RGC-5 culture dishes and incubated for 15 minutes at 37°C. 

After incubation, the staining solutions were decanted, each dish was washed three times with Ringer’s buffer (in mM; 130 NaCl, 5 KCl, 2 CaCl₂.2H₂O, 1 MgSO₄, 8 NaOH, 1 NaPO₄, 5.5 d-glucose [pH 7.4]) at 37°C, and an appropriate volume of Ringer’s buffer was added to each dish. Live cell images were then acquired with JC-1 mitochondrial staining, using the Argon laser (488 nm/568 nm) on a confocal microscope (model LSM410; Carl Zeiss Meditec, Dublin, CA). The JC-1 was excited at 488 nm and nonconjugated light emissions were collected at 530 nm (green) and conjugated at 590 nm (red). To quantitate the effect of serum deprivation on the mitochondrial membrane potential of RGC-5 cells, red to green fluorescence image ratios of individual cells for JC-1 were then calculated on computer (MetaMorph ver. 6.1; Universal Imaging Corp., Downingtown, PA). 

Immunoblot Analysis

The RGC-5 cells were seeded in 100-mm tissue culture dishes and serum deprived as described earlier. After various periods of serum deprivation, the cells were collected, pelleted, and washed three times in equal volumes of phosphate-buffered saline (PBS). They were then resuspended in 200 μL of lysis buffer (50 mM Tris, 150 mM NaCl, 10% glycerol, 1 mM EGTA, 1 mM Na orthovanadate, 5 μM ZnCl₂, 100 mM NaF, 10 μg/mL aprotinin, 1 μg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride [PMSF], 0.4 μg/mL okadaic acid, and 1% Triton X-100), sonicated, and centrifuged. The protein concentrations of the resultant supernatants were determined by the Bradford-Lowry method, and the samples were stored at −80°C until used for immunoblot analysis. 

Lysates from control and serum-deprived RGC-5 cells were subjected to SDS-PAGE and immunoblot analyses with enhanced chemiluminescence (ECL) detection (Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD) to compare the levels of the various caspases, Bcl-2/Bax. ^{23 25} The antibodies against caspases 3 and -9 and Bcl-2 and Bax were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA) and used at a 0.2-μg/mL concentration. To ensure the equal loading of protein in each lane, the blots were reprobed with an antibody against β-actin. For cytochrome c-release assays in the cytosol, the mitochondria-free cytosolic extracts and heavy mitochondrial fractions from control and serum-deprived RGC-5 cells were prepared ^{23 25} and subjected to immunoblot analysis with a commercially available antibody against cytochrome c (Santa Cruz Biotechnologies) as described. 

Preparation of Cytoplasmic and Nuclear Extracts

The RGC-5 cells were exposed to serum-deprived conditions, and nuclear and cytoplasmic extracts were prepared as described earlier. ^{23 25} Briefly, the cells were suspended in 100 μL of buffer C (10 mM HEPES [pH 7.9], 1.5 mM MgCl₂, 10 mM KCl, 10% glycerol, 1 mM dithiothreitol [DTT], and 0.5 mM PMSF) and incubated on ice for 15 minutes. To this, 3 μL of 10% NP-40 was added and then briefly vortexed. The nuclei were pelleted by centrifugation at low speed. The supernatant (cytoplasmic extract) was collected and stored at −80°C. The nuclear pellet was resuspended in 70 μL of buffer D (20 mM HEPES [pH 7.9], 400 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, and 0.5 mM PMSF) and incubated for 20 minutes at 4°C before being centrifuged at 8000g for 5 minutes. The resultant supernatant containing extracted nuclear proteins was transferred to a fresh microfuge tube and stored at −80°C. Protein concentrations of the cytoplasmic and the nuclear extracts were measured with a DC protein assay kit (Bio-Rad, Hercules, CA). 

Electrophoretic Mobility Shift Assays

For electrophoretic mobility shift assays (EMSAs), a double-stranded oligonucleotide containing the NF-κB DNA-binding consensus sequence (denoted in bold type) 5′-AGT TGA GGG GAC TTT CCC AGG C-3′-and a double stranded mutant oligonucleotide, 5′-AGT TGA GGC GAC TTT CCC AGG C-3′ (Santa Cruz Biotechnology) were used to study the DNA-binding activity of NF-κB by EMSA, as described elsewhere. ^{23 25} Briefly, the double-stranded NF-κB oligos (50 ng) were end labeled with (γ-³²P)-ATP (NEN, Boston, MA) using T4 polynucleotide kinase. This labeled probe was then purified by ethanol precipitation. A DNA-binding reaction mixture containing 10 μg cytoplasmic or nuclear extract, 10 mM Tris (pH 7.6), 60 mM NaCl, 1 mM DTT, 4 mM MgCl₂, 1 mM EDTA, 6 femtomoles ³²P-labeled oligonucleotide (approximately 20,000 cpm) and 5% glycerol, in a total volume of 20 μL, was incubated and the binding reaction proceeded for 20 minutes at 37°C. Subsequently, the samples were subjected to electrophoresis on a 4% native polyacrylamide gel using 0.25× TBE. The gel was dried and autoradiographed. Similar to this, a cold competitive binding assay with molar excess of consensus and mutant oligos of NF-κB were performed to validate the authenticity of the NF-κB band, as described earlier. ²⁵  

Measurements of Reduced and Oxidized Glutathione Levels

The membrane lipid peroxidation of serum-deprived RGC-5 cells was studied by measuring the malonyldialdehyde (MDA) levels by a colorimetric method involving thiobarbituric acid (TBA) adduct formation. ²⁶ The reduced glutathione (GSH) levels in serum-deprived RGC-5 cells was studied by using the 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) reagent. ²⁷  

Statistical Analysis

An unpaired t-test with Welch’s correction was used for statistical analysis (Prism 4 for McIntosh; Graph Pad Software, Inc., San Diego, CA). P <0.05 was considered statistically significant. 

Results

Effect of Serum Deprivation on RGC-5 Cells

RGC-5 cell viability was determined by neutral red dye uptake assay. Cells deprived of serum for 48 hours showed a 50% to 60% loss relative to the nondeprived cells (Fig. 1A) . To determine whether the cell loss is due to apoptosis, serum-deprived RGC-5 cells were subjected to DNA laddering (Fig. 1B)and showed the classic ladder formation, a hallmark of apoptosis, of genomic DNA in serum-deprived cells but not in the control cells (Fig. 1B , arrowheads). These results suggest that serum deprivation results in apoptosis of the RGC-5 cells. 

Serum Deprivation and the Release of Mitochondrial Cytochrome c and Oxidative Damage

It is becoming increasingly evident that neurotrophin deprivation of neurons activates oxidative processes, such as leakage of cytochrome c from mitochondria. The data indicate (Fig. 2A)a considerable release of cytochrome c in the cytosolic fraction from serum-deprived RGC-5 cells (Fig. 2A , SF) compared with the nondeprived cells (Fig. 2A , S+). After the cytosolic fraction was collected from the cells, the remaining heavy mitochondrial fraction (Mitos) was included as a positive control. As expected, there was no leakage of cytochrome c observed in the cytosolic fraction of control RGC-5 cells grown in 10% fetal calf serum containing medium compared with the serum-deprived cells (Fig. 2A) . To explore further whether serum deprivation results in oxidative damage to RGC-5 cells, MDA and GSH levels were measured. Serum deprivation resulted in increased levels of MDA and decreased levels of GSH in RGC-5 cells compared with the control RGC-5 cells (Fig. 2B) . 

Effect of Serum Deprivation of RGC-5 Cells on Levels of Bcl-2 and Bax

Bcl-2 is an antiapoptotic protein, whereas Bax is a proapoptotic protein belonging to the Bcl-2 gene family. Together, the levels of these anti-and proapoptotic proteins determine a cell’s fate for survival or cell death. The mRNA levels of Bcl-2 and Bax were measured in serum-deprived and control RGC-5 cells using the semiquantitative RT-PCR analysis. The results showed a decrease in the level of Bcl-2 with a concomitant increase in the level of Bax mRNA in serum deprived cells (Fig. 3A) . Densitometry of the RT-PCR products showed a significant decrease in the Bcl-2/Bax ratio in serum-deprived RGC-5 cells (Fig. 3B) . These results were further confirmed by immunoblot analysis with specific antibodies against Bcl-2 and Bax. These blots demonstrated a time-dependent decrease in Bcl-2 protein with a concomitant increase in Bax protein levels after serum deprivation (Fig. 3C) , whereas β-actin, used as an internal control for loading, did not change (Fig. 3C) . 

Effect of Serum Deprivation on Levels of Caspases in RGC-5 Cells

Levels of activated caspase-3 and -9 proteins were determined by immunoblot analysis. There were time-dependent increases in the levels of activated caspases in serum-deprived RGC-5 cells compared with the control RGC-5 cells (Fig. 4) . β-Actin, used as an internal control for loading of the caspases, did not change (Fig. 4) . 

Effect of Serum Deprivation on NF-κB-Binding Activity in RGC-5 Cells

After serum deprivation, nuclear and cytoplasmic extracts were prepared and subjected to EMSAs with end-labeled double-stranded oligos having the consensus binding sequence for NF-κB. A time course of serum deprivation of RGC-5 cells showed a decrease in NF-κB activity in both the cytoplasm (Fig. 5A , lanes 4, 6) and nucleus (Fig. 5A , lanes 8, 10, 12) compared with control cells grown in complete growth medium (Fig. 5A , lanes 1, 3, 5 for cytoplasm; lanes 7, 9, and 11, for nucleus). These results indicate that the RGC-5 cells express NF-κB constitutively and that the expression of NF-κB decrease after serum deprivation. The specificity of the binding of NF-κB was shown by the competition with cold NF-κB consensus and mutant NF-κB oligo ^{23 25} (Fig. 5B) . 

Serum Deprivation–Induced Loss of Mitochondrial Membrane Potential in RGC-5 Cells

To determine whether the serum deprivation resulted in loss of mitochondrial membrane potential, JC-1 staining was used after serum deprivation in RGC-5 cells. Live cell fluorescence confocal microscopy revealed that there was minimal red fluorescence labeling in serum-deprived RGC-5 cells (Fig. 6A , arrowheads) compared with the control RGC-5 cells cultured in serum-containing growth medium (Fig. 6B , arrows). Furthermore, the quantitation of the red/green fluorescence ratio, showed a significant lowering of the ratio in serum-deprived RGC-5 cells compared with the control cells (Fig. 6C) . These results indicate that serum deprivation results in the disruption of the mitochondrial membrane potential of RGC-5 cells. 

Discussion

In the present study, the prospective signaling pathways involved in RGC apoptosis were evaluated in a serum deprivation paradigm in cultured RGC5 cells. We hypothesized that serum provides sustenance for cells in culture because of the presence of trophic factors. Thus, serum deprivation would induce apoptosis of RGCs representing a blockade of retrograde trophic factor receptor–mediated signaling mechanism of RGC death in glaucoma. Serum deprivation of RGC-5 cells for various durations resulted in characteristics typical of apoptosis, such as DNA laddering, activation of caspase-3 and -9, increased levels of Bax, decreased levels of Bcl-2, NF-κB binding activity, as well as cellular GSH levels. Furthermore, serum deprivation resulted in cytochrome c release from the mitochondria with a loss of mitochondrial membrane potential. Thus, these findings support that serum deprivation results in apoptotic cell death of RGC-5 cells through mitochondrial pathways. 

Oxidative stress plays an important role in the pathophysiology of glaucoma. ^{6 28 29 30 31 32 33} Ischemia, excitotoxicity, and/or trophic insufficiency have all been suggested to play important roles in RGC death in glaucoma. ²⁰ These stimuli cause cellular damage, usually in the form of cleavage of structural and repair proteins and early degradation of DNA, ³⁴ leading to apoptosis. In addition to DNA and protein damage, reactive oxygen species (ROS) can be generated and released from the mitochondria. ³⁵ Because damage to mitochondria occurs in the apoptotic pathway, it is conceivable that ROS are released in the process, causing oxidation of proteins and membrane damage. It is clear from these studies that oxidative damage may play an important role in the pathophysiology of glaucoma in the RGCs. 

In this study, oxidative damage was shown to have a potential role in serum deprivation–induced cell death. For example, the levels of MDA were increased with a concomitant lowering of GSH levels during serum deprivation of RGC-5 cells. Recent reports suggest that NF-κB is also activated during oxidative signaling. ^{36 37 38 39} It has been suggested in many of these studies that reactive oxygen intermediates (ROIs) may be involved in the activation of NF-κB. The NF-κB signaling is also implicated in the regulation of apoptosis. One of the earliest significant observations in this direction was made by Beg et al., ⁴⁰ who demonstrated extensive apoptosis of liver cells leading to embryonic death of mice lacking the RelA subunit. Subsequent work by Beg and Baltimore ⁴¹ demonstrated that treatment of RelA-deficient (RelA^−/−) mouse fibroblasts and macrophages with TNF-α results in a significant reduction in cell viability. Along similar lines, NF-κB has been shown to result in suppression of TNF-α-induced apoptosis. ^{42 43 44} There is also evidence of proapoptotic aspects of RelA activity. In the retina, TNF- α and TNF-α receptor-1 have both been implicated in ganglion cell death, albeit in different experimental paradigms than those used in the present study. ^{45 46 47}  

It is also known that serum starvation causes cell death of human embryonic kidney 293 cells accompanied by the activation of RelA-containing NF-κB. ⁴⁸ Redox changes in RGC-5 cells due to serum deprivation resulted in lowering of the NF-κB binding activity along with a lowering of the p65 subunit of NF-κB protein in the nuclear fraction and apoptosis of RGC-5 cells. Similar to these results, lowering of NF-κB binding activity has been shown to be associated with apoptosis in the photo-oxidative induced insult of photoreceptor cells. ^{23 25} In those studies, it was proposed that caspase(s) might use NF-κB as a preferred substrate, resulting in lower levels and thus lower binding activity of NF-κB. It is thought that activation or induction of NF-κB is usually associated with cell survival signals. ^{49 50} Therefore, reduced NF-κB binding activity with the death of serum-deprived RGC-5 cells is a consistent finding. Relevant to these studies, failure to activate NF-κB promotes apoptosis of RGCs in vivo after optic nerve transection. ²⁸ Furthermore, because serum deprivation resulted in caspase activation, it is conceivable that the caspase(s) might use NF-κB as their substrate in this paradigm as well. ^{23 25} Further studies are needed, using caspase-specific tetrapeptide inhibitors to answer this question. 

The execution of apoptosis consists of a proteolytic cascade involving a family of proteases called caspases. ⁵¹ This phase is usually associated with an upregulation of Bax, which is a proapoptotic gene. The increased levels of Bax in the cell then combine with the Bcl-2 already present in the cell. This lowers the amount of free Bcl-2 in the cell, driving the cell toward apoptosis. Bax and Bcl-2 proteins then closely interact with mitochondria and affect the permeability of mitochondrial membranes. Bax binds to the outer membrane of the mitochondria. This binding results in opening of the permeability transition pore, which is a multiprotein complex that forms at points where the inner and outer mitochondrial membranes make contact. ⁵² This also causes the mitochondria to release cytochrome c to cytosol. ⁵³ Cytochrome c then activates a series of caspases. In the current studies, serum deprivation of RGC-5 cells resulted in loss of mitochondrial membrane potential with a release of cytochrome c, activation of caspase-3 and -9, and an increase in Bax with a decrease in Bcl-2. These results are consistent with previous studies demonstrating that a release of cytochrome c activates caspase-9, which then activates caspase-3 leading to DNA fragmentation. ^{54 55 56} Caspase-9 activation is increased in the RGC layer in a rat model of experimental glaucoma. ⁷ This pathway represents the classic mitochondrial or intrinsic pathway to apoptosis. 

ROS have also been shown to result in activation of caspase-8 and -3, which represent the key players in most extrinsic (death receptor) mediated pathways of apoptosis. ⁵⁷ Serum deprivation is also associated with activation of caspase-8 along with DR-3 and -4 (Charles I, unpublished observations, 2004), which are members of the tumor necrosis factor (TNF) receptor family consisting of a superfamily of receptors including Fas, TNFR1, TNF-α-related apoptosis inducing ligands (TRAIL)R1, TRAILR2, and DR-3/4. All these members contain a homologous intracellular region called the death domain (DD). ⁵⁸ The DDs are capable of initiating the death-inducing signaling complex (DISC) that catalyzes caspase activation and apoptosis. ⁵⁸ A number of ligands (membrane bound as well as secreted) of DR-3 have been suggested in the literature. ^{59 60 61 62} Other studies have implicated TNF-α and TNFR-1 in the signaling pathway for ganglion cell death in rat and mouse models of glaucoma. ^{45 46 47} Although these factors have not yet been investigated in RGC-5 cells, serum deprivation of RGC-5 cells could also result in secretion of a certain unknown factor(s), which in turn binds to DR-3/4, resulting in activation of a receptor-mediated apoptotic pathway. Activation of caspase-8 along with DR-3 and -4 has been shown to represent an extrinsic or receptor-mediated apoptotic pathway. ^{57 63} Further studies are needed to delineate the exact mechanism of the extrinsic pathway for apoptosis in the serum deprivation–induced cell death paradigm. 

The results in these studies suggest that maintenance of the redox state of the cell is an extremely important factor in considering neuroprotection strategies for RGCs. Therapeutics pertaining to NF-κB may also be valuable. Taken together, the results presented in this report suggest that serum deprivation–induced apoptosis of RGC-5 cells involves mitochondrial signaling pathways. A schematic representation of the molecular events occurring in the course of the serum deprivation–induced apoptotic pathway are shown in Figure 7 . Additional in vivo studies are needed, to extrapolate these results to the glaucomatous condition. 

²  Contributed equally to the work and therefore should be considered equivalent authors.

 Supported by the National Glaucoma Program of American Health Assistance Foundation (NA) and National Institutes of Health Grant P20 RR16481 (NC).

 Submitted for publication April 1, 2004; revised September 3 and October 11, 2004; accepted November 12, 2004.

 Disclosure: I. Charles, None; A. Khalyfa, None; D.M. Kumar, None; R.R. Krishnamoorthy, None; R.S. Roque, None; N. Cooper, None; N. Agarwal, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Effect of serum deprivation on the viability of RGC-5 cells. RGC-5 cells were deprived of serum for various times after which they were subjected to cell viability assays along with the control cells. (A) Cell survival neutral red dye uptake assay after 2 days of serum deprivation; (B) RGC-5 cells showing genomic DNA laddering after 2, 4, and 6 days of serum deprivation. There was a 50% cell loss after 2 days of serum deprivation (A) with a classic time-dependent apoptotic ladder formation of genomic DNA (B). * P < 0.05.

Figure 2.

 

Effect of serum deprivation on the oxidative state of RGC-5 cells. The cells were deprived of serum for 48 hours, after which the release of cytochrome c in the cytosol and mitochondria (Mitos) was determined by immunoblot analysis (A) or measurements of MDA and GSH were made by biochemical methods along with levels in the control cells (B). Release of cytochrome c was observed in the cytosol of serum-deprived RGC-5 cells (A, SF) compared with the cytosol of RGC-5 cells grown in full medium (A, S+). Mitos (A) were included as a control. As expected, cytochrome c was present in the mitochondrial fractions of both the serum-deprived and control cells, and it was released to cytosol only in the serum-deprived cells. The levels of GSH were significantly reduced and the levels of MDA significantly increased in serum-deprived RGC-5 cells (B). *P <0.05.

Figure 3.

 

Effect of serum deprivation of RGC-5 cells on Bcl-2 and Bax mRNA expression and protein levels. RGC-5 cells were deprived of serum for various durations of time, after which they were subjected either to RT-PCR analysis of Bcl-2 and Bax mRNA expression (A) or immunoblot analysis of the protein levels (C). After 3 days of serum deprivation, the Bcl-2 message was downmodulated compared with the control RGC-5 cells maintained in complete growth medium (A). In contrast, serum deprivation resulted in an increase in Bax mRNA expression compared with the control cells (A). β-Actin was included as a control, to compare the levels of cDNA synthesis in both treatments (A). The band density of Bcl-2 and Bax was determined by densitometry, with NIH Image, and the ratio of Bcl-2/Bax was plotted (B). There was a significant decrease (*P < 0.01) in the ratio of Bcl-2/Bax in serum-deprived RGC-5 cells (B). Immunoblot analysis of control and serum-deprived RGC-5 cells showed a time-dependent increase of Bax protein levels with a concomitant decrease in Bcl-2 protein levels (C). β-Actin was included as a control to compare the equal loading of proteins in all treatments.

Figure 4.

 

Effect of serum deprivation of RGC-5 cells on activation of caspase-9 and -3. RGC-5 cells were deprived of serum for various periods, after which they were subjected to immunoblot analysis for caspases. The representative blot from Figure 3was stripped of Bcl-2 and Bax antibodies and reprobed with specific caspase antibodies. β-Actin was included as a control to compare the equal loading of proteins in all treatments. A time-dependent increase in cleaved caspases was observed in serum-deprived RGC-5 cells for caspase-3 and -9 (arrowheads) compared with the 0-day control cells. The upper arrow represents the uncleaved caspase band; the lower arrow represents the β-actin band.

Figure 5.

 

Effect of serum deprivation on NF-κB binding activity in RGC-5 cells. RGC-5 cells were deprived of serum for various periods, after which they were subjected to EMSA for NF-κB binding activity. RGC-5 cells expressed NF-κB constitutively (A, lanes 1 and 7, for cytoplasmic and nuclear fractions, respectively). Lanes 2, 4, 6, 8, 10, and 12: NF-κB binding activity in cytoplasm and nucleus after 1, 2, and 3 days of serum deprivation of the RGCs. Clearly, in serum-deprived (−) RGC-5 cells, NF-κB binding activity in the cytoplasm as well as the nucleus was reduced compared with control samples (+), at all stages of serum deprivation. The authenticity of the band in (A) was confirmed by a competitive binding assay with 100 to 200 M excess of cold consensus NF-κB oligo and with a mutant oligo (B). As expected, competition with cold consensus oligo resulted in the total disappearance of the band, whereas mutant oligo did not affect it (B).

Figure 6.

 

Effect of serum deprivation of RGC-5 cells on mitochondrial membrane potential by live cell confocal microscopy, using JC-1 mitochondrial dye. RGC-5 cells were deprived of serum for 3 days (A) and control cells maintained in 10% serum (B) were subjected to JC-1 labeling and visualized under a laser confocal microscope. The serum-deprived cells were devoid of red fluorescence (A, arrowheads) indicating loss of mitochondrial membrane potential, compared with the control cells, which showed several cells with red fluorescence (B, arrows). (C) The control cells showed an increase in the red/green ratio, indicating intact mitochondria (*P < 0.0002) compared with the serum-deprived RGC-5 cells. Scale bar, 10 μm.

Figure 7.

 

Summary of the proposed signaling mechanism of serum deprivation–induced apoptosis of RGC-5 cells. Serum deprivation of RGC-5 cells results in redox perturbations, with lowering of Bcl-2 and upregulation of Bax mRNA and protein levels, resulting in mitochondrial damage with loss of mitochondrial membrane potential and cytochrome c release and activation of caspase-9 and -3 and apoptosis. Mitochondrial damage due to ROS may decrease NF-κB binding and thus its transcriptional activity. Lowering of NF-κB binding activity may also be a result of caspase activation, which may use NF-κB as their substrate, as proposed earlier. ²⁵

The authors thank Jamboor K. Vishwanathan for a critical reading of the manuscript, Shaoyou Chu and I-fen Chang for help with confocal microscopy, and David F. Cummings for help with the analysis of the data pertaining to red/green fluorescence ratios of JC-1-stained cells from confocal microscopy.