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(Investigative Ophthalmology and Visual Science. 2003;44:2774-2782.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-1061
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Cell-Type-Specific Opening of the Retinal Ganglion Cell Mitochondrial Permeability Transition Pore

Joshua P. Vrabec, Christopher J. Lieven, and Leonard A. Levin

From the University of Wisconsin Medical School, Department of Ophthalmology and Visual Sciences, Madison, Wisconsin.


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. To study the role of the mitochondrial permeability transition pore (PTP) in apoptosis of axotomized retinal ganglion cells (RGCs) in vitro.

METHODS. Primary rat retinal cultures containing DiI-labeled RGCs were treated with pharmacological agents that modulate the PTP. Ratiometric imaging of the mitochondrial membrane potential ({Delta}{Psi}m) were conducted on similarly treated cultures, with the dual-emission probe JC-1, and the correlation with the results of the viability experiments were determined.

RESULTS. The peripheral benzodiazepine receptor agonist PK11195 induced RGC death, but this was not inhibited by cyclosporin A (CsA), which normally maintains the PTP in the closed configuration. Paradoxically, the combination of CsA and PK11195 caused massive RGC death and decreased {Delta}{Psi}m, suggesting aberrant regulation of the PTP in these cells. Imaging of {Delta}{Psi}m revealed morphologic changes in the mitochondria after depolarization, characterized by formation of ringlike bodies, and similar to that with the potassium ionophore valinomycin. There were no such findings with other retinal neurons or neuronally differentiated PC-12 cells. The anomalous RGC death was independent of caspase activation or reactive oxygen species production.

CONCLUSIONS. These results suggest an aberrant opening of the RGC PTP and could be the result of structural differences in its components or its interaction with intracellular ligands. Unique RGC PTP behavior could underlie the pathophysiology of those mitochondrial diseases in which RGCs are specifically affected (e.g., Leber hereditary optic neuropathy).

Mitochondria are intimately involved with signaling of apoptosis. Opening of the mitochondrial outer membrane causes release of cytochrome c and other apoptosis-inducing factors.1 2 3 4 5 6 7 Loss of the mitochondrial transmembrane potential ({Delta}{Psi}m) results from opening of the mitochondrial permeability transition pore (PTP), which spans the mitochondrial inner membrane and allows passage of molecules smaller than 1500 Da between the mitochondrial matrix and the intermembrane space, and ultimately the cytoplasm.8 9 10 11 The PTP represents a putative association of the adenine nucleotide translocator (ANT),12 the voltage-dependent anion channel,13 14 proteins from the mitochondrial matrix (cyclophilin D15 ), and a peripheral benzodiazepine receptor (PBR).16 Under what circumstances the PTP opening is necessary for neuronal apoptosis induced by various injuries1 2 3 4 5 6 7 or whether mitochondrial depolarization can itself induce apoptosis is highly controversial.17

To elucidate the possible role of opening of PTPs in neuronal apoptosis, we studied axotomized retinal ganglion cells (RGCs) in vitro as a model system. RGCs are paradigm for central neurons, which undergo apoptosis after axonal injury.18 19 20 21 Death after axotomy partly depends on ROS signaling22 and we and others have shown that RGC survival depends on the cellular redox state.23 24 Although ROS generation can occur late in apoptosis, as a result of cytochrome c release,25 ROS regulate early stages of apoptosis in neurotrophin-deprived sympathetic neurons.26 27 There are vicinal thiol groups in one or more PTP proteins, which when oxidized result in opening of the pore and when reduced prevent its opening.28 29 30 31 We therefore hypothesized that oxidative changes of RGC PTP components could be one of the primary mechanisms by which apoptosis is signaled after axonal injury. To study this, we pharmacologically manipulated the PTP and measured {Delta}{Psi}m in RGCs after axotomy. In doing so, we not only found that RGC axotomy was not associated with early mitochondrial depolarization, but that modulation of the PTP with cyclosporin A (CsA) paradoxically decreased RGC viability.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Animals
All experiments were performed in accordance with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals, as well as the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and institutional guidelines regarding animal research.

Materials
Cell culture reagents were obtained from Gibco (Grand Island, NY). The retrograde fluorescent tracers 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) and 4'-6-diamidino-2-phenylindole (DAPI) and the fluorescent viability agent calcein-AM were obtained from Molecular Probes (Eugene, OR). 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was obtained from Molecular Probes and prepared as a 200-µM stock solution in dimethyl sulfoxide (DMSO). Papain was obtained from Worthington Biochemicals (Freehold, NJ). Unless noted, all other reagents were obtained from Sigma (St. Louis, MO).

RGC Labeling and Culture
RGCs were labeled and cultured using previously described methods.22 Briefly, ganglion cells were labeled by stereotactic injection of the fluorescent tracers DiI or DAPI dissolved in dimethylformamide into the superior colliculi of anesthetized postnatal day-2 to -4 Long-Evans rats. During a period of 3 to 4 days, the tracers flowed through retrograde transport from RGC projection sites in the superior colliculi to the RGC somas in the retina, where they associate with either cell membrane (DiI) or nucleus (DAPI). DiI was used for studies of cell death, whereas DAPI was used for imaging experiments measuring {Delta}{Psi}m, because its excitation and emission spectra do not overlap with those of JC-1. At postnatal days 7 to 9 the animals were killed by decapitation, the eyes enucleated, and the retinas dissected in Hanks’ balanced salt solution (HBSS). After two incubations in HBSS-containing papain (12.5 U/mL), each for 7 minutes at 37°, the retinas were gently triturated with a Pasteur pipette and plated on poly-L-lysine-coated plates at a density of approximately 2000 cells/mm2. For the counting experiments, 96-well flat-bottomed tissue culture plates were used, whereas an 8-well chambered coverglass (Nalgene; NalgeNunc, Rochester, NY) was used for the cell-imaging experiments. The cells were cultured for 24 hours in Eagle’s minimum essential medium (MEM) with methylcellulose (0.7%), glutamine (2 mM), gentamicin (1 µg/mL), glucose (22.5 mM final concentration), and prescreened fetal calf serum (5%). For fluorescent imaging of {Delta}{Psi}m, cells were cultured in serum-free medium (Neurobasal-A with 2% B27 defined serum-free supplement; Gibco).

Ganglion Cell Identification and Counting
RGCs were identified by the presence of retrogradely transported cytoplasmic dye, either DiI, which appeared red-orange when viewed with dye-specific filters under epifluorescence, or DAPI, which stains RGC nuclei blue (Fig. 1) . Cell viability was determined by metabolism of calcein-AM, producing green fluorescence when viewed with fluorescein filters. Briefly, cells were incubated in a 1-µM solution of calcein-AM in PBS for 20 minutes, after which the medium was replaced with fresh PBS. Survival of RGCs was determined by identifying the percentage of DiI-positive cells that were also calcein positive in five low-power fields per well, totaling a minimum of 50 to 100 RGCs in each well. Wells were counted in duplicate for each condition. Survival of non-RGCs was determined by identifying the percentage of phase-visible cells that were also calcein positive in five high-power fields per well, totaling a minimum of 200 to 300 non-RGCs in each well. Wells were counted in duplicate. Although using phase positivity to identify non-RGCs cells would also include some cells that were DiI positive, the percentage of RGCs in the retina is so low (approximately 1%) that this would not significantly affect counting. Results are expressed as the mean ± SEM (normalized to mean survival in control wells).



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  		FIGURE 1. RGC labeling and identification. Mixed retinal cultures from rats injected with DAPI were stained with the mitochondrial probe JC-1. Cultures were viewed under epifluorescence with either JC-1 or DAPI-specific filters using a 100x oil-immersion objective. Shown are images of the same field, obtained with different fluorescent emission filters. (A) The cellular uptake of the monomer form of JC-1 ({lambda} = 535 nm), independent of mitochondrial {Delta}{Psi}m. Bright green cells had low mitochondrial {Delta}{Psi}m and were probably apoptotic. (B) JC-1 J-aggregate formation ({lambda} = 580 nm) in areas of high mitochondrial {Delta}{Psi}m. (C) DAPI-stained nucleus of a retrograde labeled RGC ({lambda} = 450 nm). Arrows: RGCs in each panel. None of the other cells in this field were DAPI positive and therefore not RGCs.

 
Measurement of Mitochondrial Membrane Potential
The mitochondrial membrane potential ({Delta}{Psi}m) was measured using the voltage-sensitive dye JC-1,32 which translocates to the mitochondrial inner membrane. JC-1 exists in two forms. In its base state and when {Delta}{Psi}m is low, it associates with the mitochondrial inner membrane as a monomer, and fluoresces green (535 nm emission; Fig. 1A ). When {Delta}{Psi}m is high, it forms J-aggregates near areas of high membrane potential, and fluoresces red (580 nm emission; Fig. 1B ). Because the total mitochondrial mass varies from cell to cell, these absolute fluorescence values also are quite variable. Therefore, the ratio of red to green fluorescence F{lambda}580/F{lambda}535 is a more accurate method for comparing mitochondrial {Delta}{Psi}m status in different cells. Typically, cells with a healthy population of mitochondria with a high {Delta}{Psi}m would have a high ratio of F{lambda}580/F{lambda}535, whereas cells whose mitochondria are in the process of losing their {Delta}{Psi}m (depolarization) would have a low F{lambda}580/F{lambda}535 ratio.

Cells plated on poly-L-lysine-treated chambered coverslips, as described earlier, were treated with JC-1 (1 µg/mL) in serum-free medium (Neurobasal-A; Gibco) for 15 minutes at 37°C, washed with medium without JC-1, and imaged in serum-free medium, 2% B27, 21 mM HEPES, and 1 µg/mL gentamicin. RGCs were located in each well with a DAPI filter set (330 nm excitation, 450 nm emission). Filters used to detect JC-1 were 480 nm for excitation, 505 nm dichroic, and dual 535- and 580-nm emission filters, mounted in a computer-controlled filter wheel (Sutter Instruments, Novato, CA). A cooled charge-coupled device camera (Roper Scientific, Trenton, NJ) mounted on an inverted microscope (Axiovert 135; Carl Zeiss, Oberkochen, Germany) collected the images, with image processing performed in real-time (binning of 2, exposure time 100 ms, 2x gain; MetaFluor software; Universal Imaging Corp., West Chester, PA).

After 20 minutes of acquisition to establish baseline {Delta}{Psi}m, cells were treated by adding reagents directly to the well. Background fluorescence was subtracted from the initial fluorescent measurements, and the ratio of fluorescence at 580 and 535 nm (F{lambda}580/F{lambda}535) was calculated. The point of maximal depolarization was chosen as that time after treatment when the ratio reached a minimum, and data from this time point were used for subsequent analyses. Because of changes in fluorescence due to photobleaching and dye leakage, the effect of pharmacological agents on maximal {Delta}{Psi}m depolarization from baseline was determined compared with that of media alone.

Pharmacological Treatments
Various components of the pore were perturbed as follows: dithiothreitol (DTT) and tris-(2-carboxyethyl) phosphine (TCEP) were used to reduce vicinal thiols, and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was used to oxidize them, 1-(2-chlorophenyl)-N-methyl-N-(1-methylprolyl)-3-isoquinoline carboxamide (PK11195) and protoporphyrin IX were used to induce pore opening by binding the PBR found on the outer membrane, atractyloside was used to inhibit the adenine nucleotide transporter, and CsA was used to close the pore. Valinomycin, a mitochondrial inner membrane-specific K+ ionophore, was used as a positive control in all PTP imaging experiments, for baseline depolarization events. All concentrations were chosen based on results of initial dose-response experiments.

Statistical Analysis
Mean results were compared with Student’s unpaired t-test.


   Results
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 Abstract
 Methods
 Results
Discussion
 References
 
Effect of CsA on RGC Death at 24 or 72 Hours In Vitro
In other neuronal systems, the cyclophilin D-binding agent CsA has been shown to inhibit opening of PTPs and prevent apoptosis.33 We assessed the potential neuroprotective aspects of CsA in primary rat retinal cultures. Mixed retinal cultures containing RGCs retrogradely labeled with DiI were incubated for 24 and 72 hours with CsA (1, 2, 5, and 10 µM). The viability of DiI-positive RGCs treated with CsA was then assessed by staining with calcein-AM and compared with the control. Under control conditions, RGC survival at 24 hours was typically between 50% and 75%. By 72 hours, 90% to 95% of cells were dead as a result of developmental and dissociation-induced cell death. We found that at 24 hours, there was no significant difference in RGC survival in cultures containing 0.2, 2, and 20 µM CsA compared with control medium (respectively, 52.5% ± 0.9%, 56.3% ± 2.5%, 50.8% ± 7.6% vs. control 55.2% ± 3.8%). Similar results were recorded at 72 hours. The failure of CsA to protect axotomized RGCs suggests that either PTP opening is not required for death under these conditions or that CsA does not modulate pore closure in these cells at this developmental age. To differentiate these possibilities, we perturbed the RGC PTP, in the presence or absence of CsA.

Effect on RGCs of Agents that Open the Mitochondrial PTP
The opening of the PTP has been identified as a possible triggering event in the apoptotic cascade. To assess whether modulation of the PTP could induce cell death in RGCs, we treated RGC cultures with agents that purportedly open the PTP. Retinal cultures containing retrogradely labeled RGCs were treated with various concentrations of two PBR agonists, PK11195 and protoporphyrin IX, the adenine nucleotide transporter inhibitor atractyloside, or the nonspecific pore oxidizing agent tert-butyl-hydroperoxide (tBHP). All these agents have been shown to induce opening of the PTP.16 34 35 RGC viability was assessed at 24 hours with calcein-AM staining. Even though protoporphyrin IX-treated cells appeared light red when viewed under epifluorescence, because of the fluorescent properties of the compound itself, it did not interfere with the ability to distinguish RGCs, because DiI-positive RGCs fluoresced much more intensely than protoporphyrin IX-stained cells alone and thus were easily discernible. In these experiments, the pore-opening compounds PK11195 and protoporphyrin IX significantly decreased RGC survival above control, as did the oxidant tBHP, which is thought to open the pore indirectly by oxidizing vicinal sulfhydryls (Fig. 2) .36 However, atractyloside did not decrease RGC survival, suggesting that pore opening itself was not sufficient for RGC death.



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  		FIGURE 2. Dose-response curves of permeability transition pore openers. RGCs in culture were exposed to varying concentrations of compounds known to open the mitochondrial permeability transition pore in vitro. Treatments were made at the time of plating and were present in the following concentrations for 24 hours in culture before survival was assessed: (A) tert-Butyl-hydroperoxide (0.5, 5, and 50 µM); (B) PK11195 (1, 10, 100, and 1000 µM); (C) protoporphyrin IX (0.01, 0.1, 1, 10, and 100 µM); (D) Atractyloside (0.02, 0.2, and 2 mM). With the exception of atractyloside, all pore-opening agents increased RGC death when compared with control, although only at relatively high concentrations. Drug concentrations for subsequent experiments were chosen based on these results.

 
Paradoxical Effect of CsA on RGC Survival in the Presence of PK11195
To determine whether PTP opening was involved in RGC death after axotomy, we examined whether CsA could protect against RGC death induced by chemically opening the pore. RGC survival in mixed cultures was assessed for each pore-opening compound (PK11195, protoporphyrin IX, and tBHP), alone and with CsA. There was decreased survival with 100 µM PK11195 treatment compared with the control (45.5% ± 4.6% vs. 71.5% ± 3.3%; P = 0.044), but unexpectedly, there was even less survival when cultured with PK11195 in combination with 2 µM CsA (9.76% ± 0.2% vs. 71.5% ± 3.3%; P = 0.003). When CsA was used in combination with protoporphyrin IX and tBHP, survival was unchanged compared with the control (data not shown). A dose-response characterization of the observed CsA+PK11195 toxicity revealed that as little as 0.6 µM CsA potentiated the toxicity of PK11195 (Fig. 3A) . These results imply that attempting to maintain the pore in a closed state with CsA paradoxically increases RGC death in the presence of the pore opener PK11195, suggesting that cyclophilin-D binding may not inhibit pore opening in axotomized RGCs, as has been described in other cells.9 37 38



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  		FIGURE 3. Dose-response and time-course of CsA+PK11195 toxicity. (A) Retinal cultures were plated in the presence of control medium or PK11195 (100 µM) with various concentrations of CsA. After 24 hours, RGCs were counted, with calcein-AM used to assess viability. (B) Retinal cultures were plated in the presence of control medium, CsA (2 µM), PK11195 (100 µM), or CsA+PK11195. RGC viability was assessed at 3, 6, 10, or 24 hours. Unlike observations in other experiments, cell survival for each condition at t = 3 hours was considered baseline, and survival at subsequent time-points was compared with survival at 3 hours RGC survival remained relatively constant over all conditions from 3 to 10 hours, then significantly decreased at 24 hours in the CsA+PK11195 condition compared with the control (P = 0.018).

 
Time Course of Aberrant PTP-Related RGC Death
To understand the timing of the paradoxical RGC death induced by CsA in the presence of PK11195, we studied the viability of RGCs treated for various lengths of time. Retrogradely labeled RGCs were cultured as described earlier, and viability was assessed by calcein-AM staining at 3, 6, 10, and 24 hours after dissociation. Although RGC cell death has occurred at 3, 6, and 10 hours, there was no significant decrease in survival with combined CsA (2 µM) and PK11195 (100 µM), compared with the control at these times (Fig. 3B , Table 1 ). However, RGC death at 24 hours was significantly greater with CsA PK11195 compared with the control (37.2% ± 4.8% vs. 81.8% ± 3.7; P = 0.018). Survival data were normalized to percentage survival at the 3-hour time point. Similar to previous assays, the difference in percentage of RGCs surviving between the CsA+ PK11195 condition and control medium remained significant at 24 hours. These results suggest that aberrant RGC death was not immediate, but required several hours to develop.


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  		TABLE 1. Time-Course Relationship of CsA+PK11195-Related Toxicity

 
Mechanism of PTP-Related RGC Death
RGCs underwent massive death when treated with CsA+PK11195, more than that induced by opening the PTP with PK11195 alone. For better characterization of the factors involved in this anomalous response, we attempted to rescue RGCs using agents that scavenge or decrease reactive oxygen species (ROS) or that block effector caspases (Fig. 4) . Mixed retinal cultures treated with a combination of CsA (2 µM) and PK11195 (100 µM) were coincubated with the cell-permeable thiol reducing agents tris-(2-carboxyethyl) phosphine (TCEP; 200 µM) or DTT (300 µM), the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 5 mM), the superoxide dismutase mimetic Mn(III)-tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride (MnTMPyP; 50 µM), reduced glutathione (GSH; 2 µM), or the caspase 3 inhibitor DEVD-CHO (1 µM). None of these rescued RGCs from the effects of CsA+PK11195. This suggests that the mechanism of RGC death under these conditions probably does not involve thiol oxidation, generation of ROS, or activation of caspases.



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  		FIGURE 4. CsA+PK11195 toxicity was not rescued by a variety of agents that interfere with RGC apoptosis. (A) Retinal cells were cultured in the presence or absence of the combination of CsA (2 µM) + PK11195 (100 µM), along with one of the following: TCEP (200 µM); DEVD-CHO (1 µM); DTT (300 µM); L-NAME (5 mM); MnTMPyP (50 µM); and GSH (2 µM). RGC survival for each condition was compared with the control at 24 hours using calcein-AM to assess viability. None of the treatments inhibited RGC death due to CsA+PK11195. (B) Potential pathways transducing RGC injury and apoptosis. The exact composition and ordering of these events is controversial, and the pathways presented are organized in one of several possible configurations. To assess the possibility of rescuing RGCs from CsA+PK11195-induced cell death, we selected pharmacological agents affecting multiple points in the proposed RGC apoptotic cascade. Nitric oxide synthase is inhibited by L-NAME; superoxide radical is scavenged by MnTMPyP (a superoxide dismutase mimetic) to hydrogen peroxide, which can be further metabolized to water by GSH; DEVD-CHO is a nonspecific caspase inhibitor, functioning downstream of mitochondrial depolarization and/or release of cytochrome c. (C) Proposed structure of PTP in the RGC. Basic pore components in most animal models include a voltage-dependent anion channel (VDAC); the ANT; a PBR, to which PK11195 binds; and cyclophilin D (Cyp-D), bound by CsA. Essential to the opening of the PTP is the oxidative state of several sulfhydryl residues located on the inner membrane surface of the ANT, which can be modified by the reducing agents DTT and TCEP.

 
Calcineurin Inhibition and Paradoxical CsA Toxicity
CsA binds to cyclophilin-D to prevent the PTP from opening.39 However, it also binds (in the presence of other cyclophilins) to the calmodulin-dependent phosphatase calcineurin. It therefore is possible that CsA amplification of PK11195 toxicity is mediated though an interaction with calcineurin. To assess this, we coincubated PK11195-treated RGCs with either FK-506, which also binds calcineurin in the presence of its immunophilin or the calmodulin inhibitor trifluoperazine (TFP). Although incubation with CsA (2 µM) and PK11195 (100 µM) resulted in low RGC survival (12.9% ± 4.8%), equipotent concentrations of FK-506 (2 µM) and TFP (1 µM) in combination with PK11195 (100 µM) did not decrease RGC survival compared with the control (52.7% ± 7.3% and 56.4% ± 9.2%, respectively, vs. 74.7% ± 2.1%; P = NS). These results suggest that the paradoxical toxicity observed with CsA is not due to direct or indirect inhibition of calcineurin.

Nonspecific Benzodiazepine Receptor Activity of PK11195 and Paradoxical CsA Toxicity
Agents that bind to the PBR induce PTP opening, and PK11195 is presumed to kill cells by that mechanism. To test whether the PTP-related RGC death is due to PK11195 binding to central benzodiazepine receptors, we added the central agonists clonazepam or flunitrazepam to mixed retinal cultures in the presence of CsA and compared survival with that in cultures containing PK11195 and CsA. Combining the central benzodiazepine receptor agonists clonazepam (10 µM) or flunitrazepam (100 nM) with CsA (2 µM) did not significantly reduce RGC survival when compared with the control (46.2% ± 2.5% or 43.8% ± 1.9%, respectively, vs. 54.2% ± 3.3%; P = NS). These results showed that the paradoxical RGC death was related to PK11195’s activity at the PBR and not central benzodiazepine receptors.

Association of Opening of the Mitochondrial PTP with Aberrant PTP-Related RGC Death
All experiments up to this point involved indirect measurements of pore transition, based on treatment with pharmacological agents and observation of cell death. To directly determine whether the toxicity observed when RGCs were exposed to the combination of CsA and PK11195 was related to opening of the PTP, we imaged {Delta}{Psi}m in cultured RGCs by using the dual-emission probe JC-1. After baseline acquisition, pharmacological treatments were applied and measurements made for 100 minutes (Fig. 5) . As would be expected, for cells containing mitochondria of varying size and number, there were variable differences in the baseline fluorescence at {lambda} = 580 and {lambda} = 535 nm for treatments with media alone, PK11195 (100 µM), CsA (2 µM), or PK11195+CsA. However, there was a significant decrease from baseline in the more significant F{lambda}580/F{lambda}535 ratio with the combination of PK11195+CsA (39.2% ± 4.5%) compared with the control (58.6% ± 5.9%; P = 0.012), but not with CsA alone (59.6% ± 7.4%; P = 0.46) or PK11195 alone (55.8% ± 6.0%; P = 0.37).



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  		FIGURE 5. Change in mitochondrial membrane potential over time. Cells preincubated with JC-1 were imaged for 20 minutes to establish baseline red ({lambda}580) and green ({lambda}535) fluorescence intensities and then were treated with (A) medium; (B) PK11195 (100 µM); (C) CsA (2 µM); or (D) CsA (2 µM) + PK11195 (100 µM). Left: tracings showing fluorescence measured at {lambda} = 580 nm (solid line) and {lambda} = 535 nm (dashed line) over the course of the treatment, normalized to readings at t = 0. Right: tracings showing the ratio of F{lambda}580/F{lambda}535. Ratios vary between experiments because of differential dye loading and the mitochondrial membrane potential at the beginning of imaging. (E) Mitochondrial depolarization in RGCs treated with PTP modulators. Medium alone, CsA (2 µM), PK11195 (100 µM), or CsA+PK11195 was added to cultures 20 minutes after acquisition of baseline. The point of maximum depolarization was set at the point where the fluorescence ratio F{lambda}580/F{lambda}535 was at a minimum and compared with the baseline fluorescence ratio. The decrease in F{lambda}580/F{lambda}535 was significantly larger with CsA+PK11195 compared with all other conditions (P = 0.012).

 
We also observed a morphologic change in the appearance of the mitochondria after treatment with 100 µM PK11195 and 2 µM CsA, but not with either drug alone (Fig. 6) . The mitochondria occasionally took on a ring-like appearance consistent with pore opening.40 A similar effect was noted with valinomycin (10 µM), which depolarizes mitochondria because of its activity as a potassium ionophore. Together, these results indicate that the paradoxical RGC death observed in mixed retinal cultures treated with CsA and PK11195 is intimately related to mitochondrial depolarization, most likely from PTP opening.



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  		FIGURE 6. Morphologic and membrane potential changes in mitochondria after depolarization. Cells preincubated with JC-1 were imaged for 20 minutes to establish baseline fluorescence intensities, then treated with either (A) CsA (2 µM) + PK11195 (100 µM), or (B) valinomycin (10 µM), and imaged for 100 minutes. The tracings below each micrograph demonstrate the change in fluorescence at {lambda} = 580 nm (solid line) and {lambda} = 535 nm (dashed line) over the course of the treatment. Images were captured (A) 100 and (B) 20 minutes after treatment, once depolarization had occurred and morphologic changes had become apparent.

 
Specific Vulnerability of RGCs to CsA+PK11195 Toxicity
The PTP-related toxicity that we observed in RGCs has not been described in other cell types. To test whether this was a nonspecific neuronal phenomenon, we studied two other neuronal populations. First, we neuronally differentiated PC-12 pheochromocytoma cells with nerve growth factor (NGF) and exposed them for 24 hours to the same treatments as were used for RGCs. There were no differences in PC-12 cell survival after treatment with 2 µM CsA (71.0% ± 2.3%), 100 µM PK11195 (70.7% ± 5.7%), the combination of these two agents (71.5% ± 1.6%), or control medium (77.9% ± 0.4%). We then examined the effects of CsA and PK11195 on non-RGC retinal cells. These could be identified by the absence of retrogradely transported DiI staining in mixed retinal cultures. Because the baseline survival of these cells over 24 hours is low, we included the reducing agent TCEP (200 µM) in the culture medium for these experiments. In contrast to RGCs, the combination of CsA and PK11195 was not toxic to non-RGCs from the same well (Fig. 7) . Furthermore, there was a significant difference between RGCs and non-RGC retinal cells in the fluorescence ratio F{lambda}580/F{lambda}535 decrease from baseline after treatment with CsA+PK11195 (39.2% ± 4.5% for RGCs vs. 62.1% ± 9.9% for non-RGCs; P = 0.031). Taken together, these experiments demonstrate that RGCs are selectively vulnerable to the mitochondrial depolarization and apoptosis caused by the combination of CsA and PK11195, compared with other neuronal cells in vitro.



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  		FIGURE 7. RGCs are selectively affected by CsA+PK11195 toxicity. Mixed retinal cultures were plated in the presence of control medium, CsA (2 µM), PK11195 (100 µM), or CsA+PK11195 for 24 hours and viability assessed with calcein-AM. The reducing agent TCEP (200 µM) was added to the culture medium for all conditions to increase baseline survival of retinal neurons other than RGCs, which otherwise die rapidly. In contrast to RGCs, other retinal neurons (non-RGCs) were significantly less susceptible to the toxicity of CsA+PK11195 (P = 0.019).

 

   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
For these experiments, we chose pore-modifying agents (CsA, PK11195, PPIX, tBHP) for which there is a consensus on the mechanism of action, but obtained results in RGCs that conflict with the supposed action of these agents. Although the PBR ligands PK11195 and protoporphyrin IX-induced variable RGC death, consistent with their known action on opening the PTP,16 34 this only occurred at concentrations (>10 and >1 µM, respectively) exceeding the nanomolar receptor binding affinity known for the PBR and was not associated with mitochondrial depolarization. Furthermore, CsA did not prevent mitochondrial depolarization or apoptosis, in contrast to results from several studies in non-neuronal cells.41 42 43 Even more unexpectedly, attempting to rescue RGCs from PK11195-induced cell death using CsA resulted instead in extensive neuronal death associated with rapidly progressive mitochondrial depolarization. This paradoxical effect was independent of the calcineurin inhibitory activity of CsA, because death was not seen with calcineurin inhibitors (FK-506, trifluoperazine) that do not close the pore. It was also probably related to activity at the PBR, because similar toxicity was not seen when CsA was combined with the central benzodiazepine agonists clonazepam and flunitrazepam. Together, these results suggest differential regulation of the PTP in RGCs, compared with other retinal neurons and neuronally differentiated PC-12 cells, analogous to the differences between PTP functioning in non-neuronal and neuronal cells.7

What mechanism could account for the paradoxical mitochondrial depolarization associated with exposure to CsA? A possible answer comes from studies examining the mechanism by which CsA causes nephrotoxicity in patients treated to prevent rejection of transplants. One group of proposed mediators of renal vasoconstriction after administration of CsA are ROS. Several studies have documented measurable increases in multiple ROS after administration of CsA in vitro. Exposing rat aortic smooth muscle cells to as little as 1 µM CsA generates significant amounts of nonspecific ROS, independent of P450 enzyme activity.44 Similarly, cultured human mesangial cells incubated with CsA demonstrate elevated hydrogen peroxide levels.45 If ROS are generated as a result of RGC exposure to CsA, it could help explain the combined toxicity of CsA and PK11195. There are other examples in which PK11195 itself is not sufficient to cause apoptosis without the presence of another mediator.46 We have shown that RGCs are differentially susceptible to certain ROS47 and that RGC survival is exquisitely sensitive to redox modulation,24 presumably by ROS. It is therefore possible that idiosyncratic responses of RGCs to ROS may explain the unique response of these cells to CsA in vitro. Intrinsic properties of PK11195 may provide additional clues to the mechanism of idiosyncratic RGC death from opening of the PTP. Although it binds to the PBR in nanomolar concentrations, much higher levels must be present to cause opening of the PTP.48 Similar to CsA, PK11195 also increases production of ROS, and the latter may be involved in the compound’s pore-modifying properties.34

However, although it is tempting to attribute the aberrant PTP opening to an ROS burst, attempts to rescue RGCs from toxicity by using a variety of ROS scavengers (catalase, glutathione, and MnTMPyP) or a non-thiol-containing reducing agent (TCEP) were unsuccessful. Furthermore, opening of the PTP occurring merely as a result of initiation of apoptosis by CsA or PK11195 is unlikely, because we found that blocking caspase activation with a broad-spectrum caspase inhibitor did not block RGC death under these circumstances. Instead, these findings suggest that some other explanation underlies the aberrant opening or structural differences of the PTP in RGCs in vitro.

Another reason for this unusual observation could be related to the cell-cell interactions between RGCs and the other retinal cells that are cultured along with the RGCs. Although the dissociation process disrupts the physical connections between these cells, there may be cell-cell signaling that triggers apoptosis within the dissociated cells. We cannot exclude the possibility that other retinal neurons or glia could be mediating the toxicity while they themselves remain unharmed.

An alternate explanation for the absence of cytoprotective effect with CsA is that PTP-associated cyclophilin D behaves differently or is absent in RGCs, compared with other cells. For example, there is evidence that different PTPs may display different biochemical and functional behavior in the hexokinase complex.49 Given that the hexokinase complex associated with the PTP differs across cell types, it is also possible that there are differences in the cyclophilin D moiety. Because there are multiple pore components, it is likely that modulatory mechanisms of its conductance state are complex and highly regulated and may not be the same in all cell types or even among neuronal subtypes.

The underlying pathophysiology in RGC-selective optic neuropathies such as Leber hereditary optic neuropathy (LHON)50 may be related to unique properties of RGCs that make them especially susceptible to various noxious stimuli. RGCs differ from other retinal neurons in several fundamental ways. As relay cells in the inner retina, RGC somas must be small and transparent, yet they have extremely long axons projecting to the lateral geniculate nucleus, superior colliculus, and other targets. They rapidly undergo apoptosis after axonal injury.18 They are anatomically located in a hypoxic environment, yet are bathed in a high concentration of ascorbate.51 They are exquisitely sensitive to N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity.52 Most relevant to the present study, ganglion cell axons posterior to the lamina cribrosa of the optic nerve head undergo a transition from unmyelinated to myelinated that leads to impedance mismatch, which increases energy requirements and hence mitochondrial activity. These and other53 54 aspects of RGC pathophysiology suggest that the mechanisms by which their cell death program is activated and/or executed may diverge from that of other retinal neurons, perhaps with respect to mitochondrial signaling of apoptosis through PTP opening. In LHON, for example, the mitochondrial mutations may place the RGC axons at high risk of injury if energy requirements cannot be met. Similar findings in studies of methanol toxicity of the eye support a role for mitochondrial toxicity in optic neuropathy.55

In summary, the mechanism behind the paradoxical RGC-specific vulnerability to the combination of CsA and PK11195 remains unclear. However, it is a finding that suggests that the RGC PTP may differ from other neuronal cell types in structure and/or function. Furthermore, this finding indicates that mitochondrial signaling of apoptosis may be heterogeneous, even among neuronal populations in the same tissue. Although excitotoxicity, neurotrophin deprivation, ischemia, and axotomy have all been shown to cause opening of PTPs in neurons, it is still unclear whether this holds true for RGCs.33 37 56 57 Complicating the matter further are studies showing that apoptosis can occur in rat hippocampal neurons, even in the absence of changes in the mitochondrial membrane potential.7 Understanding the mechanisms by which opening of the PTP occurs in RGCs and how it leads to activation of apoptosis could potentially link these two processes. More important, dissecting the process of mitochondrial permeability transition in RGCs may suggest mechanisms to explain the particular susceptibility of these cells in certain diseases—for example, LHON.


   Footnotes
 
Supported by National Eye Institute Grant R01 EY12492, the Glaucoma Foundation, the Retina Research Foundation, and an unrestricted departmental grant from Research to Prevent Blindness, Inc. LAL is a Research to Prevent Blindness Dolly Green Scholar.

Submitted for publication October 16, 2002; revised December 26, 2002; accepted January 30, 2003.

Disclosure: J.P. Vrabec, None; C.J. Lieven, None; L.A. Levin, 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.

Corresponding author: Leonard A. Levin, University of Wisconsin Medical School, 600 Highland Avenue, Madison, WI 53792-4673; lalevin{at}facstaff.wisc.edu.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

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