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The Journal of Neuroscience, July 2, 2003, 23(13):5723-5731
Previous Article | Next Article
Caspase-Independent Photoreceptor Apoptosis in Mouse Models of Retinal Degeneration
Francesca Doonan, Maryanne Donovan, andThomas G. Cotter Tumour Biology Laboratory, Biochemistry Department, Bioscience Research Institute, University College Cork, Cork, Ireland
Abstract
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Abstract
Introduction
Apoptosis is the mode of cell death in retinitis pigmentosa, a group of retinal degenerative disorders primarily affecting rod photoreceptors. Although caspases have been demonstrated to play a central role in many incidences of apoptosis, accumulating evidence suggests that they may not be required for all forms of apoptotic cell death. The present study examined the mechanism of cell death in two in vivo models of photoreceptor apoptosis: the retinal degeneration (rd) mouse, a naturally occurring mutant model, and N-methyl-N-nitrosourea-induced retinal degeneration. Specifically, we examined the activation status of caspase-9, -8, -7, -3, and -2 and determined the caspase requirements for cytochrome c release, DNA fragmentation, and apoptosis-associated proteolysis of specific caspase substrates. We show that apoptosis in both in vivo models is independent of caspase-9, -8, -7, -3, and -2 activation. DNA fragmentation occurs in the absence of caspase-mediated ICAD (inhibitor of caspase-activated DNase) proteolysis, suggesting that an alternative endonuclease is responsible for DNA cleavage in these models. Importantly, we show that apoptosome activation is prevented because of an absence of mitochondrial cytochrome c release. Experiments performed using a cell-free system indicate that cytochrome c-dependent proteolysis and activation of caspase-9 can be restored in a neonatal cell-free system. However, we found that cytochrome c-dependent proteolysis and activation of caspase-9 could not be restored in an adult cell-free system because of an age-related decrease in the expression of Apaf-1 in the normal developing mouse retina. In the rd mouse, however, this age-related downregulation of apoptotic proteins was not observed, highlighting a critical feature of this model and the prevention of cytochrome c release as an apical event in caspase-independent apoptosis in this system.
Key words: photoreceptor; apoptosis; caspase; independent; cytochrome c; rd; MNU
Introduction
Apoptosis is a mechanism of programmed cell death in which the cell plays an active role in its own demise. This form of cell death, although crucial to development, has been implicated in several neurodegenerative diseases, including the heterogeneous group of inherited retinal degenerations referred to as retinitis pigmentosa (RP). RP is characterized by an initial disturbance of night vision associated with a loss of rod photoreceptors, followed by a loss of peripheral vision and visual acuity caused by cone photoreceptor degeneration. The genetics of RP is complex: at least 30 genes have been implicated, many of which encode photoreceptor-specific proteins. Examples include the structural proteins peripherin (Farrar et al., 1992) and rod outer segment membrane protein 1 (Bascom et al., 1992
In the retinal degeneration (rd) mouse, a mutation located in the gene encoding the
-subunit of rod cGMP phosphodiesterase (Bowes et al., 1990
The involvement of the caspase family of cysteine proteases in cell death is a topic of continuing debate. Initially, caspases were attributed a central role in the majority of apoptotic systems; however, increasing evidence suggests that certain features of apoptosis can be present in the absence of caspase activity (Borner and Monney, 1999
We show that apoptosis in both in vivo models is independent of caspase-9, -8, -7, -3, and -2 activation. We further show that caspase-9 activation is prevented because of an absence of mitochondrial cytochrome c release. Experiments performed using a cell-free system indicate that cytochrome c-dependent proteolysis and activation of caspase-9 can be restored in a neonatal cell-free system. However, we found that cytochrome c-dependent proteolysis and activation of caspase-9 could not be restored in an adult cell-free system because of an age-related decrease in the expression of Apaf-1 in the normal developing mouse retina. These studies highlight prevention of cytochrome c release as an apical event in caspase-independent activation in these in vivo models.
Materials and Methods
Animals. C57BL/6 (control), C3H/HEN (rd), and BALB/c animals were obtained from Harlan Olac (Bicester, UK). The animals were maintained in 12 hr light/dark cycles and killed by cervical dislocation.Intraperitoneal injections. Adult BALB/c mice were injected intraperitoneally with a single 75 mg/kg dose of MNU (Sigma, Dublin, Ireland) in Me2SO. Control animals received vehicle only. Animals were killed by cervical dislocation 14, 24, and 48 hr after treatment.
Cell lines. 32D cells were cultured in RPMI containing 10% FCS and 10% WEHI-conditioned media. Apoptosis was induced by exposure to UV irradiation for 8–10 min. Jurkat T-cells were cultured in RPMI containing 10% FCS. Apoptosis was induced using anti-human Fas (300 ng/ml) (Upstate Biotechnology, Lake Placid, NY).
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling. Briefly, enucleated eyes were fixed in 10% neutral buffered formalin overnight at 4°C, followed by cryoprotection in 30% sucrose overnight at 4°C. Frozen sections (5 µm) were incubated in 50 µl of reaction buffer containing 2.5 mM CoCl2, 0.1 U/ml terminal deoxynucleotidyl transferase in a 0.1 M Na cocadylate, pH 7.0, buffer, and 0.75 nM fluorescein-12-deoxyuridine triphosphate (Boehringer Mannheim, Mannheim, Germany). Sections were incubated at 37°C for 1 hr in a humidified chamber. After several washes in PBS, the sections were mounted in mowiol (Calbiochem, Nottingham, UK) and viewed under a fluorescence microscope (Nikon Eclipse E600) using an FITC filter.
Agarose gel electrophoresis. Enucleated eyes were placed in PBS, and retinal dissection was performed using a watchmaker's forceps. Retinal DNA was isolated using GenElute mammalian genomic DNA kit (Sigma). Extractions were performed according to the manufacturer's instructions except that retinas were initially incubated in 180 µl of tissue lysis buffer containing proteinase K (20 µl/ml) at 55°C for 8 hr. Electrophoresis was performed using 1.5% agarose gels and visualized under UV light after staining with ethidium bromide.
Western blot analysis. Retinas were dissected and lysed in radioimmunoprecipitation analysis (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM sodium fluoride) containing antipain (1 µg/ml), aprotinin (1 µg/ml), chymostatin (1 µg/ml), leupeptin (0.1 µg/ml), pepstatin (1 µg/ml), and PMSF (0.1 mM). Equivalent amounts of protein, as determined by the Bio-Rad (Hemel Hempstead, UK) protein assay, using bovine serum albumin as standard, were resolved using SDS-PAGE followed by transfer to nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). Membranes were blocked for 1 hr in 5% Blotto, followed by incubation overnight at 4°C with the appropriate antibodies. Antibodies reactive to caspase-9, caspase-3, caspase-7 (Cell Signaling Technology, Hertfordshire, UK), caspase-8, caspase-2, Apaf-1, (Santa Cruz Biotechnology, Santa Cruz, CA), poly(ADP)ribose polymerase (PARP) (PharMingen, San Diego, CA), inhibitor of caspase-activated DNase (ICAD) (Oncogene Research Products, Nottingham, UK), cytochrome c (Molecular Probes, Leiden, The Netherlands),
Determination of acetyl-Asp-Glu-Val-Asp-
-nitroanilide cleavage. Retinas were dissected and washed in cold PBS. The tissue was homogenized in 50 µl of lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 mM DTT, and 100 µM EDTA) on ice for 20 min followed by three to four cycles of freeze thawing. Insoluble material was pelleted by centrifugation at 20,000 x g for 15 min at 4°C. Lysates were prepared from 32D and Jurkat cells in the same manner. The protein content of each sample was determined by Bio-Rad protein assay using bovine serum albumin as standard. An equal quantity (80 µg) of retinal protein and positive control (32D or Jurkat) was loaded into each well of a microtiter plate. Lysates were incubated with an equal volume of 1x reaction buffer (as lysis buffer; plus 10% glycerol) and 50 µM caspase-3 substrate Asp-Glu-Val-Asp (DEVD)-
Subcellular fractionation. Separation of mitochondrial and cytosolic fractions was performed as follows: retinas were dissected in PBS and homogenized in lysis buffer (210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EGTA, 0.05% BSA, and 1 mM DTT) containing antipain (1 µg/ml), aprotinin (1 µg/ml), chymostatin (1 µg/ml), leupeptin (0.1 µg/ml), pepstatin (1 µg/ml), and PMSF (0.1 mM). Disruption of the cell membrane was achieved by passage through a Pasteur pipette, followed by incubation on ice for 10 min. After centrifugation at 1000 x g for 5 min (4°C), the supernatant was retained. Additional centrifugation at 10,000 x g for 20 min (4°C) yields a cytosolic fraction (supernatant) and a mitochondrial fraction (pellet). The pellet was resuspended in RIPA lysis buffer.
Preparation of cell-free extracts. Retinas were dissected in PBS and transferred to a 2 ml Dounce homogenizer, and 120 µl of cell extraction buffer (in mM: 20 HEPES, 10 KCl, 1.5 MgCl2, 1 EDTA, 1 EGTA, and 1 DTT) containing PMSF (0.1 mM), leupeptin (10 µg/ml), and aprotinin (2 µg/ml) was added. Cells were allowed to swell under hypotonic conditions for 15 min on ice. Cells were disrupted with 20 strokes of the pestle and incubated on ice for 15 min. The lysate was transferred to an Eppendorf tube and centrifuged at 20,000 x g for 15 min at 4°C. Cell-free extracts (CFEs) were incubated with cytochrome c and 2'-deoxyadenoside triphosphate (dATP) at 37°C for 2 hr.
Results
Detection of rod photoreceptor apoptosis in the rd mouse and MNU-treated BALB/c mice
To establish a time course for retinal degeneration in both of our in vivo models, apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) of frozen retinal sections. The number of TUNEL-positive cells in the rd mouse was comparable with that of wild-type (C57) mice until the second postnatal week when degeneration of rod photoreceptors in the outer nuclear layer (ONL) commenced. The level of apoptosis peaked at postnatal days 12–13 (P12–P13) (Fig. 1Ai), with the process virtually completed by P15. Consistent with these observations, Portera-Cailliau et al. (1994
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Figure 1. Detection of rod photoreceptor apoptosis in both the rd mouse and MNU-treated BALB/c mice. A, Detection of rod photoreceptor apoptosis in rd and MNU-treated BALB/c mice. Apoptotic cell death was assessed by detection of DNA strand breaks in photoreceptor nuclei by TUNEL. i, Retinas of rd mice at P9 show scattered labeling of the inner nuclear layer (INL) because of a developmentally associated reduction in cell number. This developmental apoptosis is essentially complete by P10. A similar pattern is seen in C57 mice at P9 and P10. Scattered photoreceptor apoptosis is visible at P11 in the ONL, with significant apoptosis observed at P12 and P13. By P14, the number of TUNEL-labeled cells has diminished, correlating with the loss of photoreceptor layers. ii, Retina of untreated control BALB/c mice did not exhibit any TUNEL-positive cells; however, 14 hr after MNU treatment, scattered apoptosis is observed in the ONL. Time points examined 24 and 48 hr after treatment reveal large numbers of apoptotic photoreceptor cells. B, Apoptosis was confirmed through detection of DNA fragmentation by agarose gel electrophoresis. The presence of a DNA ladder in MNU-treated mice 24 and 48 hr after exposure (lanes 4 and 5, respectively) confirms that apoptosis is the mode of cell death. Untd, Untreated.
Photoreceptor apoptosis is independent of caspase-3 in the rd mouse and in MNU-treated BALB/c mice
As a key executioner of apoptosis, the activity of caspase-3 in both model systems was analyzed by Western blot and cleavage of the colorimetric substrate acetyl (Ac)-DEVD-
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Figure 2. Caspase-3 is not activated during photoreceptor apoptosis in the rd mouse or in MNU-treated BALB/c mice. A, Analysis of caspase-3 activity by immunoblot. Retinal-cell lysates were taken from rd mice between P9 and P15 (i) and MNU-treated BALB/c mice 14, 24, and 48 hr after exposure (ii). Untreated (–) and UV-treated (+) 32D cells (apoptotic index, <10%) served as negative and positive controls, respectively, to confirm the ability of the antibody to detect the large 17–20 kDa active fragment of caspase-3. Bi, Bii, These cells demonstrated the processing of procaspase-3 (32 kDa) to its active fragment of 17–20 kDa. i, Although procaspase-3 was detectable in retinal-cell lysates from rd mice at all of the time points examined, the 17–20 kDa fragment was absent up to P15. A similar pattern was observed in C57 retinal-cell lysates. ii, Equally, procaspase-3 was detectable in retinal-cell lysates from MNU-treated BALB/c mice at all of the time points examined; however, the 17–20 kDa fragment was absent up to 48 hr. Each blot was reprobed with an antibody to
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Figure 6. Cytochrome c is not released from mitochondria during photoreceptor apoptosis in the rd mouse or MNU-treated BALB/c mice. A, Cells were fractionated at each time point from P9–P15 in the rd mouse (i) and 14, 24, and 48 hr after MNU treatment in BALB/c mice (ii). [Mitochondrial fractions were collected and analyzed for each time point (data not shown)]. The first lane was loaded with a representative mitochondrial fraction (M) as a positive control for cytochrome c. Cytochrome c was readily detectable in the mitochondrial fraction but was absent from the cytosol of rd mice at all of the time points examined. Cytochrome c was also absent from the cytosol of MNU-treated BALB/c mice up to 48 hr after treatment. Untreated and UV-treated 32D cells served as negative and positive controls, respectively, to demonstrate the release of cytochrome c from the mitochondria into the cytosol. B, Retinal-cell-free extracts were prepared from rd mice between P9 and P15 (i) and MNU-treated BALB/c mice 14, 24, and 48 hr after exposure (ii). Equivalent quantities of protein were incubated with cytochrome c and dATP for 2 hr and then resolved by SDS-PAGE, transferred onto nitrocellulose membrane, and probed with an antibody to caspase-9. Untreated (–) and 32D-cell-free extracts incubated with cytochrome c (+) were used as a positive control to demonstrate the processing of procaspase-9 to its large 37–39 kDa fragment. Caspase-9 was cleaved in the rd mouse at P10 and also at P15 after addition of cytochrome c. In C57 mice, however, caspase-9 could be cleaved at P10, but not at P15, after cytochrome c treatment. MNU-treated BALB/c mice retain caspase-9 in its inactive form; however, it was not cleaved on addition of cytochrome c. All of the blots were reprobed with an antibody to
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Figure 5. Neither caspase-8 nor caspase-9 is activated during photoreceptor apoptosis in the rd mouse or MNU-treated BALB/c mice. A, Analysis of caspase-8 activity by immunoblot. Retinal-cell lysates were taken from rd mice between P9 and P15 (i) and MNU-treated BALB/c mice 14, 24, and 48 hr after exposure (ii). Untreated (–) and anti-Fas IgM-treated (+) Jurkat cells were used as negative and positive controls, respectively, to demonstrate the processing of procaspase-8 (53 kDa) to its 42 kDa cleaved intermediate. The 53 kDa species was present at all of the time points analyzed in the rd mouse; however, the 42 kDa fragment was absent up to P15. Equally, procaspase-8 was detectable in retinal-cell lysates from MNU-treated BALB/c mice at all of the time points examined; however, the 42 kDa active fragment was absent up to 48 hr after treatment. *Nonspecific bands recognized by the antibody. B, Retinal-cell lysates were taken from rd mice between P9 and P15 (i) and MNU-treated BALB/c mice 14, 24, and 48 hr after exposure (ii). Untreated (–) and UV-treated (+) 32D cells (apoptotic index, <10%) served as negative and positive controls, respectively, to demonstrate the processing of procaspase-9 (46 kDa) to its large 37–39 kDa active fragment. Although procaspase-9 was detectable in retinal-cell lysates from rd mice at all of the time points examined, the 37–39 kDa large fragment was absent up to P15. A similar pattern was observed in C57 retinal-cell lysates. Equally, procaspase-9 was detectable in retinal-cell lysates from MNU-treated BALB/c mice at all of the time points examined; however, the 37–39 kDa fragment was absent even at 48 hr. All of the blots were reprobed with an antibody to
PARP and ICAD were not cleaved by caspase-3 in the rd mouse and in MNU-treated BALB/c mice
To verify further the absence of caspase-3 activation in these two models of retinal degeneration, cleavage of the caspase-3 substrate PARP was analyzed by Western blot. PARP is inactivated by cleavage from 116 to 85 and 25 kDa fragments (Tewari et al., 199540 kDa, which increased over time in MNU-treated mice and was observed in the rd mouse particularly at P11. It is possible that an alternative protease (e.g., calpain), which yields a 40 kDa product, may be cleaving and inactivating PARP (McGinnis et al., 1999
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Figure 3. PARP and ICAD are not cleaved by caspase-3 in the rd mouse and in MNU-treated BALB/c mice. Western blot analysis was performed to detect cleavage of PARP and ICAD. Retinal-cell lysates were taken from rd mice between P9 and P15 (Ai, Bi) and MNU-treated BALB/c mice 14, 24, and 48 hr after exposure (Aii, Bii). Untreated (–) and UV-treated (+) 32D cells (apoptotic index, <10%) served as negative and positive controls, respectively, to demonstrate the processing of PARP (116 kDa) to its cleaved fragment (85 kDa) and to confirm the ability of the antibody to detect murine ICAD (45 kDa). Ai, PARP levels decreased in the rd mouse from P12 onward; however, the 85 kDa cleaved fragment was absent from P9 to P15. Levels of PARP remained constant in C57 mice from P9 to P15. Aii, Native PARP levels decreased in treated BALB/c mice, whereas the p85 fragment, present at basal levels in untreated BALB/c mice, did not increase. Bi, ICAD did not undergo cleavage in the rd mouse at any of the time points examined. Bii, Similarly, cleavage of ICAD was not observed in MNU-treated mice at any of the time points examined. All of the blots were reprobed with an antibody to
Finally, we examined the activity of ICAD, a second caspase-3 substrate (45 kDa). ICAD exists in complex with caspase-activated DNase (CAD) and is cleaved at two sites by caspase-3 during apoptosis, releasing CAD (12 kDa) (Sakahira et al., 1998
Caspase-7 and caspase-2 were not activated during photoreceptor apoptosis in the rd mouse or MNU-treated BALB/c mice
We next examined the activity of caspase-7, an alternative effector caspase, and caspase-2, which is reported to be involved in neuronal apoptosis through its action as an initiator or as an effector. Caspase-7 is synthesized as a 35 kDa inactive proenzyme that is cleaved to generate fragments of 20 and 12 kDa. Western blot analysis of cell lysates prepared from the retinas of rd mice from P9 to P15 and MNU-treated BALB/c mice 14, 24, and 48 hr after exposure demonstrated the absence of cleaved caspase-7 fragments (Fig. 4A). Induction of apoptosis by UV irradiation in the murine hematopoietic 32D cell line served as a positive control and demonstrated the processing of procaspase-7 (35 kDa) to 20 and 12 kDa fragments in a population of cells with an apoptotic index of 10%. Caspase-2 has been attributed an important role in neuronal apoptosis (Troy et al., 2000
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Figure 4. Caspase-7 and caspase-2 are not activated during photoreceptor apoptosis in the rd mouse or in MNU-treated BALB/c mice. A, Analysis of caspase-7 activity by immunoblot. Retinal-cell lysates were taken from rd mice between P9 and P15 (i) and MNU-treated BALB/c mice 14, 24, and 48 hr after exposure (ii). Untreated (–) and UV-treated (+) 32D cells (apoptotic index, <10%) served as negative and positive controls, respectively, to demonstrate the processing of procaspase-7 (35 kDa) to its large 20 kDa and small 12 kDa fragments. The 35 kDa species was present at all of the time points analyzed in the rd mouse; however, both cleaved fragments were absent up to P15. Procaspase-7 was detectable in retinal-cell lysates from MNU-treated BALB/c mice at all of the time points examined; however, both large and small cleaved fragments were absent even up to 48hr. A positive 32D control has been included in this blot to rule out any ambiguity regarding the nonspecific bands observed in the MNU-treated samples. B, Analysis of caspase-2 activity by immunoblot. Retinal-cell lysates were taken from rd mice between P9 and P15 (i) and MNU-treated BALB/c mice 14, 24, and 48 hr after exposure (ii). Untreated (–) and UV-treated (+) 32D cells (apoptotic index, <10%) served as negative and positive controls, respectively, to demonstrate the processing of procaspase-2 (48 kDa) to its 12 kDa product. The 48 kDa species was present at all of the time points analyzed in the rd mouse; however, both cleaved fragments were absent up to P15. Procaspase-2 was detectable in retinal-cell lysates from MNU-treated BALB/c mice at all of the time points examined; however, the large cleaved fragment was absent even up to 48 hr. All of the blots were reprobed with an antibody to
Neither caspase-8 nor caspase-9 was activated during photoreceptor apoptosis in the rd mouse or MNU-treated BALB/c mice
The activation status of both caspase-8, a mediator of receptor-activated apoptosis, and caspase-9, an important initiator caspase, were studied. Caspase-8 is synthesized as a 53 kDa inactive proenzyme that is processed into a cleaved 42 kDa intermediate and 18 and 12 kDa active fragments. We observed neither a decrease in the proenzyme nor the appearance of the 42 kDa cleaved fragment in cell lysates prepared from the retinas of rd mice from P9 to P15 (Fig. 5Ai) and MNU-treated BALB/c mice 14, 24, and 48 hr after exposure (Fig. 5Aii). The Jurkat cell line treated with anti-Fas IgM demonstrated the processing of the 53 kDa procaspase-8 to its 42 kDa fragment upon induction of apoptosis. Caspase-9, activated by release of cytochrome c from mitochondria, is essential for the initiation of a cascade of caspase activity, including caspase-2, -3, -6, and -7 (Slee et al., 1999
Cytochrome c was not released from the mitochondria in the rd mouse or after MNU treatment
Cytochrome c is central to the initiation of a cascade of caspase activity through caspase-9. Release of cytochrome c from the mitochondrial intermembrane space allows formation of the apoptosome, consisting of Apaf-1, caspase-9, dATP, and cytochrome c, which subsequently leads to the proteolytic cleavage and activation of caspase-9. The lack of caspase-9 cleavage during photoreceptor apoptosis led us to investigate the cellular localization of cytochrome c. Subcellular fractions were prepared from the retinas of rd mice from P9 to P15 and MNU-treated BALB/c mice 14, 24, and 48 hr after exposure. Analysis by Western blot revealed that, in both cases, cytochrome c was localized entirely in the mitochondria and did not translocate to the cytosol during photoreceptor apoptosis (Fig. 6A). UV irradiation of the murine hematopoietic 32D cell line demonstrated the release of cytochrome c from mitochondria into the cytosol. We also examined activation of caspase-12, because it has been implicated in cytochrome c-independent activation of caspase-9 (Morishima et al., 2002Addition of cytochrome c to cell-free extracts initiated activation of caspase-9 in the rd mouse, but not in MNU-treated BALB/c mice
To confirm that the absence of cytochrome c release directly prevented processing of caspase-9, we attempted to overcome this barrier by addition of cytochrome c and dATP to cell-free systems. Cell-free extracts were prepared from the retinas of rd mice at P10 and P15. We discovered that caspase-9 was cleaved on addition of cytochrome c to cell-free extracts prepared from 10- and 15-d-old rd retinas (Fig. 6Bi). However, analysis of cell-free extracts, prepared from retinas of C57 mice at the same ages and treated with cytochrome c, indicated that, although caspase-9 was susceptible to proteolysis at P10, it was not susceptible to cytochrome c-mediated proteolysis at P15 (Fig. 6Bi). This indicated the existence of an additional barrier to caspase activation in a normal adult retina. Cell-free extracts were also prepared from the retinas of untreated and MNU-treated BALB/c mice 24 hr after exposure and incubated with cytochrome c. Similarly, Western blot analysis demonstrated that caspase-9 could not be processed to its active subunits in untreated adult BALB/c mice or 24 hr after MNU treatment (Fig. 6Bii). These cell-free studies demonstrated that activation of the downstream caspase cascade is dependent only on cytochrome c release in the rd mouse.Apaf-1 and caspase-3 were downregulated at P15 in C57 mice, but not in the rd mouse
Release of mitochondrial cytochrome c is a central event in caspase-dependent apoptosis. However, in our cell-free systems, the addition of cytochrome c did not initiate the proteolytic cleavage and activation of caspase-9 in C57 mice at P15 in untreated adult BALB/c mice, or adult BALB/c mice treated with MNU. This laboratory recently showed an age-related downregulation of apoptotic proteins, including Apaf-1, in adult BALB/c and C57 mice (from P10 to P60) and demonstrated that this decrease in Apaf-1 correlates with an age-related inability to activate caspase-9 in the presence of cytochrome c (Donovan and Cotter, 2002
Discussion
The purpose of this study was to analyze the activity of the caspase family of cysteine proteases in two models of in vivo photoreceptor apoptosis with differing severity of insult. It appears that there is increasing disparity between studies performed in cell systems compared with the actual disease state, highlighting the importance of in vivo models. In this regard, our results show that photoreceptor apoptosis in the rd mouse and in MNU-treated BALB/c mice occurs independently of both cytochrome c release and caspase activation (Figs. 2, 3, 4, 5, 6).In particular, we studied the effectors caspase-3 and -7 as well as the initiators caspase-8 and -9. We also examined the activity of caspase-2, which can function either downstream of caspase-3 as an effector caspase, or upstream of the mitochondria as an initiator. It is important to note that, although caspase-2 was not cleaved in either of our in vivo models, a very recent report has demonstrated caspase-2 activation without proteolysis in vitro (Read et al., 2002b
The results shown here are in direct contrast to a report of caspase activation during retinal degeneration in the rd mouse (Jomary et al., 2001
Despite the apparent lack of caspase activity in either of our models, both exhibit DNA fragmentation, one of the key characteristics of apoptosis. Although CAD, which is activated by caspase-3, is the primary endonuclease identified, evidence exists that DNA fragmentation can occur independently of caspase activity. It has been shown that granzyme B can cleave ICAD in a caspase-independent manner, resulting in DNA fragmentation (Thomas et al., 2000
To establish the point at which activation of the caspase cascade is prevented, we examined the intrinsic or mitochondrial pathway of cell death. This pathway requires release of cytochrome c into the cytosol where it forms a complex with caspase-9 and Apaf-1. Subcellular fractionation studies revealed that cytochrome c is not released from mitochondria in the rd mouse or in response to MNU treatment in BALB/c mice (Fig. 6A). Addition of cytochrome c to rd cell-free extracts confirmed that the absence of cytochrome c release was the only barrier to the activation of caspase-9 in rd mice at both P10 and P15. It has been demonstrated that cGMP, through activation of protein kinase G, can prevent mitochondrial cytochrome c release (Kim et al., 1999
Similarly, the absence of cytochrome c release in MNU-treated BALB/c mice prevents activation of the caspase cascade; however, a supplementary mechanism prevents caspase-9 activation. A possible explanation is provided by the observation that caspase-9 may be activated by cytochrome c in vitro in C57 cell-free extracts at P10 but is precluded from activation at P15, correlating with a significant age-related decrease in Apaf-1. Therefore, at some point between P10 (essentially the conclusion of developmental cell death) and P15, the ability to activate the caspase pathway in the retina of C57 mice is removed. Downregulation of Apaf-1 and caspase-3 has been demonstrated in adult BALB/c and C57 mice (Donovan and Cotter, 2002
This study describes photoreceptor apoptosis in two different in vivo models via a pathway that is independent of a number of key caspases. Despite the absence of caspase activation, characteristic features of apoptosis are retained, including DNA fragmentation. Caspases of the post-mitochondrial pathway are precluded from activation during photoreceptor apoptosis by the absence of cytochrome c release. The key components of the apoptotic pathway are present in the rd mouse but cannot be activated because of a block on cytochrome c release. In MNU-treated adult BALB/c mice, caspase activation is blocked primarily by the absence of cytochrome c release from the mitochondria and secondarily by what appears to be a more general phenomenon of the adult mouse retina, i.e., the decrease in expression of two key components of the caspase-dependent apoptotic pathway. In conclusion, from our studies, it is evident that photoreceptors use a caspase-independent apoptotic pathway; therefore, targeting caspases with the aim of retarding or preventing photoreceptor apoptosis may not be sufficient, and alternative targets for potential therapeutic intervention must be identified.
Footnotes
Received Aug. 12, 2002; revised Feb. 25, 2003; accepted Apr. 8, 2003.This work was supported by prize money from the Royal Dublin Society Irish Times Boyle Medal, the Health Research Board of Ireland, and Enterprise Ireland. In particular, we acknowledge Fighting Blindness Ireland for their continued support. We also acknowledge Dr. Justin McCarthy for his critical reading of this manuscript.
Correspondence should be addressed to Dr. Thomas G. Cotter at the above address. E-mail: t.cotter{at}ucc.ie.
Copyright © 2003 Society for Neuroscience 0270-6474/03/235723-09$15.00/0
References
Azarian SM, Williams DS (1995) Calpain activity in the retinas of normal and RCS rats. Curr Eye Res 14: 731–735.[Medline]
Bascom RA, Manara S, Collins L, Molday RS, Kalnins VI, McInnes RR (1992) Cloning of the cDNA for a novel photoreceptor membrane protein (rom-1) identifies a disk rim protein family implicated in human retinopathies. Neuron 8: 1171–1184.[Web of Science][Medline]
Borner C, Monney L (1999) Apoptosis without caspases: an inefficient molecular guillotine? Cell Death Differ 6: 497–507.[Web of Science][Medline]
Boulares AH, Zoltoski AJ, Contreras FJ, Yakovlev AG, Yoshihara K, Smulson ME (2002) Regulation of DNAS1L3 endonuclease activity by poly(ADP-ribosyl)ation during etoposide-induced apoptosis. Role of poly(ADP-ribose) polymerase-1 cleavage in endonuclease activation. J Biol Chem 277: 372–378.
[Abstract/Free Full Text] Bowes C, Li T, Danciger M, Baxter LC, Applebury ML, Farber DB (1990) Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase. Nature 347: 677–680.[Medline]
Caffe AR, Soderpalm AK, Holmqvist I, van Veen T (2001) A combination of CNTF and BDNF rescues rd photoreceptors but changes rod differentiation in the presence of RPE in retinal explants. Invest Ophthalmol Vis Sci 42: 275–282.
[Abstract/Free Full Text] Carmody RJ, Cotter TG (2000) Oxidative stress induces caspase-independent retinal apoptosis in vitro. Cell Death Differ 7: 282–291.[Web of Science][Medline]
Chang GQ, Hao Y, Wong F (1993) Apoptosis: final common pathway of photoreceptor death in rd, rds, and rhodopsin mutant mice. Neuron 11: 595–605.[Web of Science][Medline]
Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME (1997) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91: 231–241.[Web of Science][Medline]
de Bilbao F, Guarin E, Nef P, Vallet P, Giannakopoulos P, Dubois-Dauphin M (1999) Postnatal distribution of cpp32/caspase 3 mRNA in the mouse central nervous system: an in situ hybridization study. J Comp Neurol 409: 339–357.[Web of Science][Medline]
Donovan M, Cotter TG (2002) Caspase-independent photoreceptor apoptosis in vivo and differential expression of apoptotic protease activating factor-1 and caspase-3 during retinal development. Cell Death Differ 9: 1220–1231.[Web of Science][Medline]
Farrar GJ, McWilliam P, Bradley DG, Kenna P, Lawler M, Sharp EM, Humphries MM, Eiberg H, Conneally PM, Trofatter JA, Humphries P (1990) Autosomal dominant retinitis pigmentosa: linkage to rhodopsin and evidence for genetic heterogeneity. Genomics 8: 35–40.[Web of Science][Medline]
Farrar GJ, Kenna P, Jordan SA, Kumar-Singh R, Humphries MM, Sharp EM, Sheils D, Humphries P (1992) Autosomal dominant retinitis pigmentosa: a novel mutation at the peripherin/RDS locus in the original 6p-linked pedigree. Genomics 14: 805–807.[Web of Science][Medline]
Harvey NL, Trapani JA, Fernandes-Alnemri T, Litwack G, Alnemri ES, Kumar S (1996) Processing of the Nedd2 precursor by ICE-like proteases and granzyme B. Genes Cells 1: 673–685.[Abstract]
He L, Poblenz AT, Medrano CJ, Fox DA (2000) Lead and calcium produce rod photoreceptor cell apoptosis by opening the mitochondrial permeability transition pore. J Biol Chem 275: 12175–12184.
[Abstract/Free Full Text] Jomary C, Neal MJ, Jones SE (2001) Characterization of cell death pathways in murine retinal neurodegeneration implicates cytochrome c release, caspase activation, and bid cleavage. Mol Cell Neurosci 18: 335–346.[Web of Science][Medline]
Katai N, Kikuchi T, Shibuki H, Kuroiwa S, Arai J, Kurokawa T, Yoshimura N (1999) Caspaselike proteases activated in apoptotic photoreceptors of Royal College of Surgeons rats. Invest Ophthalmol Vis Sci 40: 1802–1807.
[Abstract/Free Full Text] Kennedy SG, Kandel ES, Cross TK, Hay N (1999) Akt/protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria. Mol Cell Biol 19: 5800–5810.
[Abstract/Free Full Text] Kim YM, Chung HT, Kim SS, Han JA, Yoo YM, Kim KM, Lee GH, Yun HY, Green A, Li J, Simmons RL, Billiar TR (1999) Nitric oxide protects PC12 cells from serum deprivation-induced apoptosis by cGMP-dependent inhibition of caspase signaling. J Neurosci 19: 6740–6747.
[Abstract/Free Full Text] Liu C, Li Y, Peng M, Laties AM, Wen R (1999) Activation of caspase-3 in the retina of transgenic rats with the rhodopsin mutation s334ter during photoreceptor degeneration. J Neurosci 19: 4778–4785.
[Abstract/Free Full Text] Lolley RN, Rong H, Craft CM (1994) Linkage of photoreceptor degeneration by apoptosis with inherited defect in phototransduction. Invest Ophthalmol Vis Sci 35: 358–362.
[Abstract/Free Full Text] Mathiasen IS, Sergeev IN, Bastholm L, Elling F, Norman AW, Jaattela M (2002) Calcium and calpain as key mediators of apoptosis-like death induced by vitamin D compounds in breast cancer cells. J Biol Chem 277: 30738–30745.
[Abstract/Free Full Text] McGinnis KM, Gnegy ME, Park YH, Mukerjee N, Wang KK (1999) Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates. Biochem Biophys Res Commun 263: 94–99.[Web of Science][Medline]
McLaughlin ME, Sandberg MA, Berson EL, Dryja TP (1993) Recessive mutations in the gene encoding the beta-subunit of rod phosphodiesterase in patients with retinitis pigmentosa. Nat Genet 4: 130–134.[Web of Science][Medline]
Mooney SM, Miller MW (2000) Expression of bcl-2, bax, and caspase-3 in the brain of the developing rat. Brain Res Dev Brain Res 123: 103–117.[Medline]
Morishima N, Nakanishi K, Takenouchi H, Shibata T, Yasuhiko Y (2002) An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12. J Biol Chem 277: 34287–34294.
[Abstract/Free Full Text] Mouatt-Prigent A, Karlsson JO, Agid Y, Hirsch EC (1996) Increased M-calpain expression in the mesencephalon of patients with Parkinson's disease but not in other neurodegenerative disorders involving the mesencephalon: a role in nerve cell death? Neuroscience 73: 979–987.[Web of Science][Medline]
Nakajima M, Nambu H, Shikata N, Senzaki H, Miki H, Tsubura A (1996) Pigmentary degeneration induced by N-methyl-N-nitrosourea and the fate of pigment epithelial cells in the rat retina. Pathol Int 46: 874–882.[Medline]
Nambu H, Yuge K, Nakajima M, Shikata N, Takahashi K, Miki H, Uyama M, Tsubura A (1997) Morphologic characteristics of N-methyl-N-nitrosourea-induced retinal degeneration in C57BL mice. Pathol Int 47: 377–383.[Web of Science][Medline]
Nicotera P (2002) Apoptosis and age-related disorders: role of caspase-dependent and caspase-independent pathways. Toxicol Lett 127: 189–195.[Web of Science][Medline]
Nylandsted J, Rohde M, Brand K, Bastholm L, Elling F, Jaattela M (2000) Selective depletion of heat shock protein 70 (Hsp70) activates a tumor-specific death program that is independent of caspases and bypasses Bcl-2. Proc Natl Acad Sci USA 97: 7871–7876.
[Abstract/Free Full Text] Ogino H, Ito M, Matsumoto K, Yagyu S, Tsuda H, Hirono I, Wild CP, Montesano R (1993) Retinal degeneration induced by N-methyl-N-nitrosourea and detection of 7-methyldeoxyguanosine in the rat retina. Toxicol Pathol 21: 21–25.
[Abstract/Free Full Text] Okuno S, Shimizu S, Ito T, Nomura M, Hamada E, Tsujimoto Y, Matsuda H (1998) Bcl-2 prevents caspase-independent cell death. J Biol Chem 273: 34272–34277.
[Abstract/Free Full Text] Portera-Cailliau C, Sung CH, Nathans J, Adler R (1994) Apoptotic photoreceptor cell death in mouse models of retinitis pigmentosa. Proc Natl Acad Sci USA 91: 974–978.
[Abstract/Free Full Text] Read DS, McCall MA, Gregg RG (2002a) Absence of voltage-dependent calcium channels delays photoreceptor degeneration in rd mice. Exp Eye Res 75: 415.[Web of Science][Medline]
Read SH, Baliga BC, Ekert PG, Vaux DL, Kumar S (2002b) A novel Apaf-1-independent putative caspase-2 activation complex. J Cell Biol 159: 739–745.
[Abstract/Free Full Text] Robertson JD, Enoksson M, Suomela M, Zhivotovsky B, Orrenius S (2002) Caspase-2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis. J Biol Chem 277: 29803–29809.
[Abstract/Free Full Text] Sakahira H, Enari M, Nagata S (1998) Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391: 96–99.[Medline]
Sakamoto YR, Nakajima TR, Fukiage CR, Sakai OR, Yoshida YR, Azuma MR, Shearer TR (2000) Involvement of calpain isoforms in ischemia-reperfusion injury in rat retina. Curr Eye Res 21: 571–580.[Web of Science][Medline]
Selznick LA, Zheng TS, Flavell RA, Rakic P, Roth KA (2000) Amyloid beta-induced neuronal death is bax-dependent but caspase-independent. J Neuropathol Exp Neurol 59: 271–279.[Web of Science][Medline]
Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ (1999) Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol 144: 281–292.
[Abstract/Free Full Text] Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, Kroemer G (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397: 441–446.[Medline]
Tamada Y, Fukiage C, Daibo S, Yoshida Y, Azuma M, Shearer TR (2002) Involvement of calpain in hypoxia-induced damage in rat retina in vitro. Comp Biochem Physiol B Biochem Mol Biol 131: 221–225.[Medline]
Taomoto M, Nambu H, Senzaki H, Shikata N, Oishi Y, Fujii T, Miki H, Uyama M, Tsubura A (1998) Retinal degeneration induced by N-methyl-N-nitrosourea in Syrian golden hamsters. Graefes Arch Clin Exp Ophthalmol 236: 688–695.[Web of Science][Medline]
Tewari M, Quan LT, O'Rourke K, Desnoyers S, Zeng Z, Beidler DR, Poirier GG, Salvesen GS, Dixit VM (1995) Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81: 801–809.[Web of Science][Medline]
Thomas DA, Du C, Xu M, Wang X, Ley TJ (2000) DFF45/ICAD can be directly processed by granzyme B during the induction of apoptosis. Immunity 12: 621–632.[Web of Science][Medline]
Troy CM, Rabacchi SA, Friedman WJ, Frappier TF, Brown K, Shelanski ML (2000) Caspase-2 mediates neuronal cell death induced by beta-amyloid. J Neurosci 20: 1386–1392.
[Abstract/Free Full Text] Troy CM, Rabacchi SA, Hohl JB, Angelastro JM, Greene LA, Shelanski ML (2001) Death in the balance: alternative participation of the caspase-2 and -9 pathways in neuronal death induced by nerve growth factor deprivation. J Neurosci 21: 5007–5016.
[Abstract/Free Full Text] Tsuji T, Shimohama S, Kimura J, Shimizu K (1998) m-Calpain (calcium-activated neutral proteinase) in Alzheimer's disease brains. Neurosci Lett 248: 109–112.[Medline]
Ueyama H, Kumamoto T, Fujimoto S, Murakami T, Tsuda T (1998) Expression of three calpain isoform genes in human skeletal muscles. J Neurol Sci 155: 163–169.[Medline]
van Loo G, Schotte P, van Gurp M, Demol H, Hoorelbeke B, Gevaert K, Rodriguez I, Ruiz-Carrillo A, Vandekerckhove J, Declercq W, Beyaert R, Vandenabeele P (2001) Endonuclease G: a mitochondrial protein released in apoptosis and involved in caspase-independent DNA degradation. Cell Death Differ 8: 1136–1142.[Web of Science][Medline]
Yakovlev AG, Di X, Movsesyan V, Mullins PG, Wang G, Boulares H, Zhang J, Xu M, Faden AI (2001a) Presence of DNA fragmentation and lack of neuroprotective effect in DFF45 knockout mice subjected to traumatic brain injury. Mol Med 7: 205–216.[Web of Science][Medline]
Yakovlev AG, Ota K, Wang G, Movsesyan V, Bao WL, Yoshihara K, Faden AI (2001b) Differential expression of apoptotic protease-activating factor-1 and caspase-3 genes and susceptibility to apoptosis during brain development and after traumatic brain injury. J Neurosci 21: 7439–7446.
[Abstract/Free Full Text] Yoshizawa K, Nambu H, Yang J, Oishi Y, Senzaki H, Shikata N, Miki H, Tsubura A (1999) Mechanisms of photoreceptor cell apoptosis induced by N-methyl-N-nitrosourea in Sprague-Dawley rats. Lab Invest 79: 1359–1367.[Web of Science][Medline]
Yoshizawa K, Yang J, Senzaki H, Uemura Y, Kiyozuka Y, Shikata N, Oishi Y, Miki H, Tsubura A (2000) Caspase-3 inhibitor rescues N-methyl-N-nitrosourea-induced retinal degeneration in Sprague-Dawley rats. Exp Eye Res 71: 629–635.[Web of Science][Medline]
Yoshizawa K, Kiuchi K, Nambu H, Yang J, Senzaki H, Kiyozuka Y, Shikata N, Tsubura A (2002) Caspase-3 inhibitor transiently delays inherited retinal degeneration in C3H mice carrying the rd gene. Graefes Arch Clin Exp Ophthalmol 240: 214–219.[Web of Science][Medline]
Yuge K, Nambu H, Senzaki H, Nakao I, Miki H, Uyama M, Tsubura A (1996) N-methyl-N-nitrosourea-induced photoreceptor apoptosis in the mouse retina. In Vivo 10: 483–488.[Web of Science][Medline]
Zhang X, Chen J, Graham SH, Du L, Kochanek PM, Draviam R, Guo F, Nathaniel PD, Szabo C, Watkins SC, Clark RS (2002) Intranuclear localization of apoptosis-inducing factor (AIF) and large scale DNA fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite. J Neurochem 82: 181–191.[Web of Science][Medline]
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