From the Tumour Biology Laboratory, Department of Biochemistry, Lee Maltings, University College Cork, Cork, Ireland
Received for publication, June 20, 2000, and in revised form, February 23, 2001
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ABSTRACT |
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Apoptosis is the mode of
photoreceptor cell death in inherited and induced retinal degeneration.
However, the molecular mechanisms of photoreceptor cell death in human
cases and animal models of retinal dystrophies remain undefined.
Exposure of Balb/c mice to excessive levels of white light
results in photoreceptor apoptosis. This study delineates the molecular
events occurring during and subsequent to the induction of retinal
degeneration by exposure to white light in Balb/c mice. We demonstrate
an early increase in intracellular calcium levels during photoreceptor
apoptosis, an event that is accompanied by significant superoxide
generation and mitochondrial membrane depolarization. Furthermore, we
show that inhibition of neuronal nitric-oxide synthase (nNOS) by
7-nitroindazole is sufficient to prevent retinal degeneration
implicating a key role for neuronal nitric oxide (NO) in this model. We
demonstrate that inhibition of guanylate cyclase, a downstream effector
of NO, also prevents photoreceptor apoptosis demonstrating that
guanylate cyclase too plays an essential role in this model. Finally,
our results demonstrate that caspase-3, frequently considered to be one
of the key executioners of apoptosis, is not activated during retinal
degeneration. In summary, the data presented here demonstrate that light-induced photoreceptor apoptosis in vivo is
mediated by the activation of nNOS and guanylate cyclase and is
caspase-3-independent.
Retinitis pigmentosa
(RP)1, refers to a group of
hereditary disorders of the retina characterized by a progressive loss
of rod and cone photoreceptors. Nyctalopia (night-blindness), an initial symptom of the disease is associated with rod photoreceptor degeneration. As the disease progresses, severe loss of peripheral and
central fields occur, due to the subsequent loss of cone
photoreceptors. The genetics of RP is complex and to date at least 30 genes have been implicated in the etiology of RP, the majority of which
encode photoreceptor-specific proteins that are either components of the phototransduction cascade or structural components of these cells
(see the Retinal Information Network available on the University of
Texas School of Public Health Web server). Despite the diverse genetics underlying the pathology of RP, one feature common to both
human cases and animal models is the apoptotic cell death of rod and
cone photoreceptors (1, 2). Apoptosis, therefore, represents a common
final pathway in the pathology of RP.
Exposure to excessive levels of white light induces photoreceptor
apoptosis and has previously been used as a model for the study of
retinal degeneration (3). Furthermore, several animal studies have
demonstrated that photoreceptors from retinal degeneration mutants are
more susceptible to the damaging effects of excessive light than normal
photoreceptors (4-7). The evidence from these animal models suggests
that excessive light may enhance the progression and severity of some
forms of human RP. The mechanism by which light induces retinal
degeneration is at present unclear. A recent study demonstrated that
rhodopsin is essential for light-induced retinal degeneration,
indicating that signal flow through the phototransduction cascade is
necessary to mediate the damaging effects of light (8). Indeed,
constitutive signal flow in phototransduction is thought to underlie
some forms of inherited retinal disorders (9). This is supported by the
significant number of mutated genes that encode components of the
phototransduction cascade and are implicated in the etiology of RP or
of related conditions. These include rhodopsin, cyclic GMP
phosphodiesterase, the rod cGMP-gated channel, and rhodopsin kinase
(see the Retinal Information Network available on the Web).
Although the exact mechanism of photoreceptor apoptosis resulting from
exposure to excessive levels of light remains undefined, a diverse
range of agents have been used to prevent photoreceptor apoptosis in
this model (10-12). These include calcium channel blockers (13) as
well as antioxidants (14, 15) implicating a role for both intracellular
calcium and reactive oxygen species in retinal apoptosis. However, the
molecular basis for the rescue of photoreceptors from apoptosis in
light-induced retinal degeneration by these agents has yet to be elucidated.
In this study, the molecular events that occur during light-induced
photoreceptor apoptosis are delineated. We demonstrate that inhibition
of NOS not only prevents photoreceptor cell death, but also additional
features of apoptosis, including superoxide generation, mitochondrial
membrane depolarization, and elevated intracellular calcium levels,
implicating a key role for NO in this model. In addition, the potential
involvement of cyclic guanosine monophosphate (cGMP), a central
component of the phototransduction cascade, is explored by examining
the effects of ODQ
(1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one), a potent
and selective inhibitor of guanylate cyclase. Inhibition of guanylate
cyclase in this study prevents light-induced retinal degeneration.
These results suggest that NO mediates cell death in this model through
activation of guanylate cyclase, resulting in increased levels of
intracellular calcium through cGMP-gated calcium channels. Finally, our
results demonstrate that caspase-3 is not activated during
photoreceptor apoptosis in this model.
Retinal Light Damage--
Adult male Balb-c mice were maintained
in the dark for 18 h before being exposed to constant light.
Immediately prior to light exposure their pupils were dilated with 5%
cyclopentolate. The mice were then exposed to 2 h of cool white
fluorescent light at a luminescence level of 5000 lux. The mice were
sacrificed after treatment by cervical dislocation at the following
time points: 30 min and 1 h after light onset, immediately after
light exposure (0 h) and after 6, 14, and 24 h of darkness that
followed the 2-h light exposure.
Intraperitoneal Injections--
Mice were injected
intraperitoneally with the following:
NG-nitro-L-arginine methyl ester
(L-NAME), 100 mg kg Cell Lines--
Jurkat T-cells were maintained in RPMI
containing 10% FCS. 32D cells were cultured in RPMI containing 10%
FCS and 10% WEHI-conditioned media. Agents used to induce
apoptosis were anti-human Fas (300 ng/ml) (Upstate Biotechnology Inc.,
Lake Placid, NY) and exposure to ultraviolet (UV) irradiation
(10 min).
Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End
Labeling of Fragmented DNA--
DNA strand breaks in photoreceptor
nuclei were detected by terminal deoxynucleotidyl transferase-mediated
dUTP nick end labeling (TUNEL). Briefly, enucleated eyes were fixed in
10% buffered formalin for 24 h, dehydrated, processed, and
embedded in paraffin. Sections (5 µm) were incubated in 50 µl of
reaction buffer containing 2.5 mM CoCl2, 0.1 unit/ml terminal deoxynucleotidyl transferase (TdT) in a 0.1 M sodium cacodylate (pH 7.0) buffer and 0.75 nM
fluorescein-12-dUTP (Roche Molecular Biochemicals, Germany). These
sections were incubated at 37 °C for 1 h in a humidified
chamber. Following several washes in PBS, the sections were mounted in
mowiol (Calbiochem) and viewed under a fluorescence microscope (Nikon
Eclipse E600) using a fluorescein isothiocyanate filter. Three animals
were used for each of the time points; 0, 6, 14, and 24 h after
light exposure.
Agarose Gel Electrophoresis--
Enucleated eyes were placed in
PBS, and retinal dissection was carried out using a watchmaker's
forceps. The choroid, sclera, and pigmented epithelium were removed,
and the retina was then separated from the vitreous and lens. Retinal
DNA was isolated following phenol-chloroform extraction. Briefly,
retinas were placed in 150 µl of lysis buffer (20 mM
EDTA, 100 mM Tris, and 0.8% sodium lauryl sarcosinate)
containing proteinase K (20 µg/ml) and incubated at 50 °C for
18 h, vortexing occasionally. The DNA was then extracted with
phenol chloroform and chloroform isoamyl alcohol (v/v, 24:1). DNA was
precipitated with ethanol, and the pellet was dissolved in Tris-EDTA.
Total RNase-treated DNA was visualized by including ethidium bromide
(0.5 µg/ml) in the agarose and observed by illuminating on a 302-nm
UV transilluminator.
Assessment of Active Caspase-3 Content in Light-induced and
Uninduced Balb/c Retina--
Retinal dissection was carried out as
above. Tissue dissociation was achieved in a 0.25% trypsin solution
(Life Technologies, Inc., Paisley, UK). The cells were washed in PBS
and fixed in 1% paraformaldehyde at 4 °C for 30 min. The cells were
then washed again with PBS and then with permeabilization buffer (PB:
10 mM HEPES, 150 mM NaCl, 4% FCS, 0.1% sodium
azide, and 0.1% Triton X-100). Cells were resuspended in PB containing
0.125 µg of anti-active caspase-3 antibody (PharMingen International,
San Diego, CA), or the same concentration of an isotype control (rabbit
IgG, Sigma, UK) and incubated for 1 h at 4 °C. Following two
washes in PB, cells were resuspended in 20 µg/ml fluorescein
isothiocyanate-conjugated secondary antibody (goat anti-rabbit, Sigma,
UK) and incubated for 1 h at 4 °C. After a further two washes
in PB, cells were resuspended in 0.5 ml of PBS for FACScan analysis on
a Becton-Dickinson FACScan flow cytometer.
Determination of Ac-DEVD-pNA Cleavage--
Retinas were
dissected and washed with cold PBS. Total protein was obtained by
homogenizing retinas in 50 µl of chilled lysis buffer containing 10 mM HEPES, pH 7.4, 2 mM MgCl2, 5 mM EGTA, 50 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 2 µg/ml
leupeptin. The cells were incubated on ice for 20 min and then lysed by
3-4 cycles of freezing and thawing. Insoluble material was pelleted by
centrifuging at 20,000 × g for 15 min at 4 °C. The
protein content of each sample was determined by the Bio-Rad protein
assay (Bio-Rad, Hemel Hempstead, UK) using bovine serum albumin as a
standard, and 80 µg of protein in 50 µl of lysis buffer was
dispensed into each well of a microtiter plate. An equal volume of 2×
reaction buffer (50 mM HEPES, pH 7.4, 0.2% CHAPS, 20%
glycerol, 2 mM EDTA, and 10 mM dithiothreitol) was added to each sample with 50 µM caspase-3
substrate-DEVD-pNA (Bahcem, Saffron Walden, UK; 1 mM stock in Me2SO). Reactions were incubated at
37 °C for 1 h and then cleavage of the peptide substrate DEVD-pNA was monitored by liberation of the chromogenic
pNA in a SpectraMax-340 plate reader (Molecular
Devices, CA) by measuring absorption at 405 nm.
Western Blot Analysis--
The retina was dissected, and total
protein was obtained by lysing in radioimmune precipitation buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-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
phenylmethylsulfonyl fluoride (0.1 mM). The amount of total
protein of each sample was determined by the Bio-Rad protein assay
(Bio-Rad, Hemel Hempstead, UK) using bovine serum albumin as a
standard. 60-80 µg of total protein from each sample was
electrophoresed on polyacrylamide gels followed by transfer to
nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) and
incubated overnight with the appropriate antibodies. Antibodies
reactive to caspase-3 (Upstate Biotechnology Inc.), ICAD (Oncogene),
PARP (PharMingen), nNOS (Transduction Laboratories), iNOS and eNOS
(Santa Cruz Biotechnology) were used in this study. Membrane
development was achieved using Enhanced Chemiluminescence (ECL)
(Amersham Pharmacia Biotech, Buckinghamshire, UK).
Analysis of Intracellular ROS Generation--
Superoxide anion
levels were measured using a modified version of the assay as
previously described (16). Briefly, cells (5 × 105)
were loaded with 10 µM hydroethidine (DHE) (Molecular
Probes), prepared from a 10 mM stock in Me2SO
for 15 min at 37 °C. Superoxide anion oxidizes DHE intracellularly
to produce ethidium bromide, which fluoresces upon interaction with
DNA. Superoxide anion levels were assessed by monitoring the
fluorescence due to ethidium bromide on a Becton-Dickinson FACScan flow
cytometer with excitation and emission settings of 488 and 590 nm, respectively.
Analysis of Mitochondrial Membrane
Depolarization--
Mitochondrial membrane depolarization was analyzed
using the probe
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolecarbocyanine iodide (JC-1, Molecular Probes). At high mitochondrial membrane potential JC-1 forms J-aggregates, which fluoresce strongly at 590 nm
(measured in FL-2). Cells were incubated with JC-1 (5 µg/ml) in
darkness for 15 min at 37 °C, and fluorescence was measured on a
Becton-Dickinson FACScan flow cytometer with excitation at 488 nm.
Intracellular Free Calcium Measurement--
Intracellular
calcium levels were determined using the intracellular calcium probe,
Fluo-3 AM (acetoxymethyl ester) (Molecular Probes, Leiden, The
Netherlands). Cells were incubated in darkness with Fluo-3 (250 nM), prepared from a 500 µM stock for 15 min at 37 °C, and fluorescence was measured in FL-1 (530 nm) on a Becton-Dickinson FACScan flow cytometer with excitation at 488 nm.
Detection of Photoreceptor Apoptosis in Light-induced Balb/c
Mice--
Exposure to excessive levels of light results in
photoreceptor apoptosis (3). In the present study, Balb/c mice were
exposed to 2 h of cool white fluorescent light at a luminescence
level of 5000 lux. DNA strand nicking in the outer nuclear layer was assessed by Terminal dUTP nick end labeling (TUNEL) over 24 h in
control animals and in animals exposed to 2 h of white fluorescent light. Retinas of control animals showed no TUNEL-positive labeling in
photoreceptor nuclei immediately after light exposure (Fig. 1A). At 6 h, positive
labeling increased, and at 14 and 24 h, abundant TUNEL-positive
cells were apparent in the ONL (Fig. 1, b-e). Apoptotic
cell death was confirmed by DNA agarose gel electrophoresis (Fig.
1B). Biochemically, apoptosis is characterized by
internucleosomal DNA cleavage-producing DNA fragments that are
multiples of 180-200 bp, which appear in agarose gel electrophoresis
as a ladder pattern. The presence of this typical ladder pattern at 14 and 24 h (lane 3 and 4) confirms apoptotic
cell death in this model and verifies the efficacy of the TUNEL assay
to detect apoptosis following light exposure.
Inhibition of NOS by L-NAME Prevents Light-induced
Photoreceptor Apoptosis--
To elucidate the molecular mechanism of
light-induced photoreceptor apoptosis we investigated the role of NOS,
previously shown to be involved in another model of retinal
degeneration where rats were exposed to light for 7 days at an
illuminescence of 90 footcandles (17). To investigate the role of NOS
in this model of light-induced photoreceptor apoptosis, animals
were treated with the NOS inhibitor L-NAME (100 mg
kg Elevation of Intracellular Calcium Is an Early Event in Retinal
Apoptosis and Is Inhibited by L-NAME--
A role for
calcium has previously been implicated in retinal cell death in
vivo. The ability of calcium channel blockers to prevent rod
photoreceptor apoptosis in two different models of retinal degeneration
has been demonstrated (13, 18). However, measurement of calcium levels
during photoreceptor apoptosis has not previously been conducted. In
this study, intracellular calcium levels were analyzed using the
fluorescent probe Fluo-3 AM (Fig. 3).
Increased levels of calcium were detected after 30 min of light
exposure, and the number of cells with elevated calcium continued to
increase up to 3 h. This was followed by a significant decrease in
intracellular calcium concentration. These data demonstrate that
calcium elevation is an early and rapid event in retinal degeneration.
This transient elevation of intracellular calcium is blocked by the NOS
inhibitor L-NAME, suggesting that elevated intracellular
calcium requires NOS activity.
Superoxide Anion Formation and the Collapse of Mitochondrial
Membrane Potential (
Mitochondria are the major site of cellular ROS production. The
increase in superoxide production observed here prompted the investigation of mitochondrial dysfunction as alterations in
mitochondrial membrane potential ( Light-induced Retinal Degeneration Is Accompanied by Increased
Expression of nNOS--
Although the NOS inhibitor L-NAME
is an established inhibitor of in vivo NO production (20),
L-NAME shows little or no selectivity for individual NOS
isoforms. As both constitutive and inducible isoforms of NOS have been
reported in the retina (see "Discussion"), it is unclear which of
the three isoforms; inducible nitric-oxide synthase (iNOS), endothelial
nitric synthase (eNOS), or neuronal nitric oxide (nNOS) is responsible
for the production of NO following light exposure. Indeed, elevated
levels of NO may be due to increased NOS protein expression or
activation by calcium entry into the cells. Immunoblot analysis
revealed no detectable increase in either iNOS or eNOS expression (Fig.
5A, part a) and
Fig. 5A (part b). However, a
time-dependent increase in nNOS expression was evident
(Fig. 5A, part c). Densitometric analysis
revealed a 2.5-fold increase in nNOS expression at 3 h, a 3-fold
increase at 6 h, a 3.5-fold increase at 14 h, and a 5-fold
increase at 24 h. The retinas taken from mice treated with 100 mg
kg 7-Nitroindazole Inhibits Light-induced Retinal
Degeneration--
To clarify the role of nNOS in light-induced retinal
degeneration, we investigated the effects of treatment with 7NI, a
selective inhibitor of neuronal nitric-oxide synthase that has been
employed in several in vivo studies to inhibit the activity
of neuronal NOS. 7 NI has high selectivity for nNOS (IC50
710 nm) over iNOS (IC50 20 µm). In addition, several
studies have demonstrated that 7NI had no effect on eNOS in the brain
in a number of animal species, including rodents (21-23). Animals were
treated with 100 mg kg Inhibition of Guanylate Cyclase by ODQ Blocks Photoreceptor
Apoptosis--
A major action of NO is to activate the soluble form of
the enzyme guanylate cyclase (24, 25). Binding of NO to guanylate cyclase increases the latter's activity leading to the formation of
cGMP, which may result in excess calcium influx through cGMP-gated channels. To explore the potential involvement of cGMP in photoreceptor apoptosis, we examined the effect of ODQ, a potent and selective inhibitor of NO-sensitive guanylate cyclase. Animals were treated with
50 mg kg Light-induced Photoreceptor Degeneration Occurs Independently of
Caspase-3 Activation--
Caspase-3 is recognized as one of the key
executioners of apoptosis. Recently, activation of caspase-3 was
reported during photoreceptor apoptosis in transgenic rats with the
rhodopsin mutation S334ter (26). In these mutants, pretreatment with
the irreversible caspase-3 inhibitor, z-DEVD-fmk prevents photoreceptor apoptosis. However, emerging evidence now suggests that not all cell
types require caspase-3 to undergo apoptosis, and it has been reported
that oxidative stress can inhibit caspase activation during
photoreceptor apoptosis in vitro (27). Because the free radical NO appears to play a key role in light-induced retinal degeneration, we determined the activation status of caspase-3 during
photoreceptor apoptosis in this model, using three different analytical
techniques (Fig. 7). Caspase-3 is
synthesized as a 32-kDa inactive proenzyme and is cleaved at
Asp28-Ser29 and
Asp175-Ser176 to generate active subunits of 17 and 12 kDa. Analysis of the levels of the 32- and 17-kDa caspase-3
species using Western blot demonstrates the absence of active, 17-kDa
caspase-3 in cell lysates taken from the retinas of light-induced
Balb/c mice 3, 6, and 14 h after light exposure (Fig.
7A). The murine hematopoietic 32D cell line is included as a
control to demonstrate the processing of pro-caspase-3 (32 kDa) to the
active 17-kDa fragment as these cells undergo apoptosis following
ultraviolet light irradiation.
The activity of caspase-3-like proteases was further assessed by
measuring the cleavage of the colorimetric substrate
AcDEVD-pNA (Fig. 7B). Again no evidence of
AcDEVD-pNA cleavage was obtained in light-induced Balb/c
mice 3, 6, or 14 h after light exposure. Untreated and anti-Fas
IgM-treated Jurkat cells served as negative and positive controls, respectively.
To further confirm the absence of active caspase-3 in the retina of
light-induced Balb/c mice, assessment of active caspase-3 content by
flow cytometry was carried out using an anti-active caspase-3 antibody
(Fig. 7C). This antibody is raised against the active
fragment and preferentially recognizes active caspase-3. Untreated and
anti-Fas IgM-treated Jurkat cells served as negative and positive
controls, respectively. There was no detectable active caspase-3 at 3, 6, or 14 h after light exposure.
Western blot analysis of two caspase-3 substrates, ICAD
(inhibitor of caspase-activated Dnase) and PARP (poly
(ADP-ribose)polymerase) was carried out to verify the lack of caspase-3
activity during light-induced retinal degeneration. ICAD is a caspase-3
substrate that exists as a complex with CAD and is cleaved at two sites by caspase-3 during apoptosis resulting in the release of CAD. PARP is
cleaved and inactivated by caspase-3 into 25- and 85-kDa fragments.
Analysis of the levels of the cleaved products of ICAD (12 kDa) and
PARP (85 kDa) by Western blot demonstrates the absence of these
fragments in cell lysates taken from the retinas of light-induced BALB/C mice 3, 6, and 14 h after light exposure (Fig. 7,
D and E). The murine hematopoietic 32D cell line
was employed to demonstrate the processing of both ICAD and PARP to 12- and 85-kDa fragments, respectively, as these cells undergo apoptosis
following etoposide treatment. These results taken together with the
lack of detection of caspase-3 activity suggest that light-induced
retinal degeneration is caspase-3-independent.
In this study, we delineate the molecular events occurring during
and subsequent to the induction of retinal degeneration by constant
exposure to white light in mice. It has been known for some time that
constant exposure to white light results in retinal degeneration (28)
and accumulated data to date has shown this to be a useful model for
the study of photoreceptor apoptosis in vivo. It has been
suggested that some of the molecular defects observed in inherited
retinal degenerations may produce signals equivalent to real light,
leading to retinal degeneration by a similar mechanism (9).
Elevated intracellular calcium has been suggested to be a key event in
apoptosis (29, 30). In the central nervous system, it has been
demonstrated to play an important role in degeneration during stroke or
trauma (31). Elevated calcium is thought to disrupt the membrane
potential and outer membrane of the mitochondria and induce the release
of several potent apoptosis-inducing factors (32). In this study,
intracellular calcium levels were monitored during and following light
exposure, and it was determined that elevated calcium is an early and
rapid event in photoreceptor apoptosis (Fig. 3). This is indirectly
supported by the potent anti-apoptotic effects of calcium channel
blockers in both the light-induced model (13) and in inherited retinal
degeneration in the rd mouse (18). The results presented here provide a
molecular basis for the inhibition of photoreceptor apoptosis by
calcium channel blockers.
Intraperitoneal administration of the NOS inhibitor L-NAME
inhibited the elevation of intracellular calcium observed, suggesting that NOS activity is required for increased intracellular calcium. To
determine the mechanism by which NO regulates intracellular calcium
levels in photoreceptors, we examined the potential role of guanylate
cyclase, a downstream effector of NO that converts GTP into cGMP. In
photoreceptors, cGMP binds cation specific channels on the
photoreceptor membrane, maintaining them in an open conformation. Excessive calcium influx could potentially occur, therefore, through cGMP-gated channels following activation of guanylate cyclase by NO. In
this study, intraperitoneal administration of ODQ a potent and
selective inhibitor of nitric oxide-sensitive guanylate cyclase
prevented photoreceptor apoptosis (Fig. 6), demonstrating that
NO-sensitive guanylate cyclase plays a key role in light-induced photoreceptor apoptosis. NO possibly acts in this model by activating guanylate cyclase and eliciting a cGMP increase. Indeed, there is
considerable evidence from other animal studies supporting a role for
increased intracellular cGMP in rod photoreceptors in retinal
degeneration. Increased levels of cGMP have been demonstrated to
precede photoreceptor degeneration in the rd mouse (33), and rod-cone
dysplasia in Irish Setter dogs (34). Accumulation of cGMP in the outer
segment would result in an increase in the opening of the cGMP-gated
channels and a subsequent increase in intracellular calcium. Such a
model would explain the ability of calcium channel blockers,
L-NAME and ODQ, to protect against light-induced
photoreceptor apoptosis.
Several studies have implicated the importance of oxidative stress in
animal models of retinal degeneration in which antioxidants retard or
inhibit the degenerative pathology. In the light-induced model, a
number of compounds possessing anti-oxidant properties have
successfully prevented photoreceptor degeneration, these include:
methylprednisolone (15), dithiomethyleurea (14), and ascorbic acid
(10). However, to date, little is known about the oxidative
pathways involved in such retinal death, nor have ROS measurements been
conducted in vivo. This laboratory recently reported ROS as
mediators of photoreceptor apoptosis in vitro and has
demonstrated the early and rapid generation of ROS while also
demonstrating inhibition of photoreceptor apoptosis with antioxidants
(19). In this study, for the first time we undertake the analysis of
the superoxide anion in an in vivo model of retinal degeneration. Our data demonstrate that superoxide production is an
early event in light-induced retinal degeneration (Fig. 4), occurring
alongside increased intracellular calcium levels. These results, taken
together with the ability of antioxidants to prevent apoptosis in
vivo, establish a critical role for ROS in photoreceptor apoptosis.
Emerging evidence now suggests that mitochondria play a central role in
the execution of the apoptotic program. Alterations in
Inhibition of NOS by L-NAME in this study was sufficient to
prevent all features of apoptosis monitored, implicating a key role for
NOS in light-induced photoreceptor apoptosis. However, because all
three isoforms of this enzyme have been reported in the retina
(43-45), it was unclear which of the three isoforms was responsible
for mediating light-induced photoreceptor apoptosis. Western blot
analysis demonstrates the expression of all three isoforms (Fig. 2) in
the retina consistent with previous reports. A
time-dependent increase in nNOS expression was demonstrated 3 h after the light insult, but there was no detectable increase in expression of either eNOS or iNOS. Pre-treatment of the mice with
7NI, a selective inhibitor of nNOS, was found to prevent photoreceptor
apoptosis, demonstrating a prominent role for neuronal derived NO in
this model.
Activation of caspases, a family of cysteine proteases, has been
demonstrated to be a central event in apoptosis in several studies
(46). Caspase-3 is considered to be one of the major downstream
caspases of the apoptotic machinery and has been shown to play a
critical role in apoptosis induced by a range of stimuli in a wide
variety of cell types. However, emerging evidence now suggests that not
all cell types require caspase activity to undergo apoptosis (47-50).
Indeed, caspase-3-independent apoptosis is well documented in neurons
(51-54). Analysis of caspase-3 activity in this study of light-induced
retinal degeneration using a range of different analytical techniques,
demonstrated the absence of caspase-3 activation during apoptosis.
Furthermore, Western blot analysis of ICAD and PARP, both caspase-3
substrates, revealed that these are not cleaved during photoreceptor
apoptosis (Fig. 7). These results suggest that light-induced retinal
degeneration is caspase-3-independent. This also strongly implies that
this cell death is caspase-independent, because caspase-3 activation has been shown to be dependent on prior activation of upstream caspases
(caspase-2, 8, 9, 10) (55-57). This laboratory has previously provided
evidence for a caspase-independent apoptotic pathway in photoreceptors
in vitro (27). This study demonstrated oxidative inactivation of caspases. The presence of an essential cysteine residue
in the active site of the caspases renders the activity of
this family of proteases susceptible to redox modification (58). The
key role of NO in photoreceptor apoptosis in this study may provide an
explanation for the failure of caspase activation by a similar
mechanism. Inhibition of caspase activity by
S-nitrosylation, whereby nitric oxide is transferred to the
cysteine sulfydryl group, has been reported in other apoptotic models,
including neuronal apoptosis (59).
Despite the lack of evidence for caspase-3 involvement in this study,
the photoreceptor apoptosis described retains one of the key
characteristics of apoptosis attributed to caspase-3 activation; DNA
fragmentation. CAD (a caspase-activated DNase that requires cleavage of
its endogenous inhibitor (ICAD) in a caspase-3-dependent manner) is at present the only apoptosis-specific endonuclease identified (60). However, there is much evidence to support a role for
Ca2+-dependent endonucleases during DNA
fragmentation and death in apoptotic cells (61). Given the evidence for
a role of calcium in this model, it is possible that this apoptotic
pathway culminates in the activation of calcium-dependent endonucleases.
The results of this and previous in vitro studies (27) are
in contrast to the recent reports of activation of caspases during photoreceptor apoptosis in rhodopsin S334ter rats (26) and RCS rats
(62) and during photo-oxidative stress in vitro (63). The
experiments described by Krishnamoorthy and colleagues (63) were
carried out on cells derived from SV40-transformed mouse retina. This
cell line is continuously dividing, non-differentiated, and, therefore,
may not be directly comparable to the study presented in this paper,
which is conducted on fully differentiated, mature, post mitotic
photoreceptor cells in vivo. Furthermore, it is likely that
the type and the severity of the initiating insult may determine whether the apoptosis is caspase-dependent or
caspase-independent. It was recently reported that neurons exposed to a
relatively mild nitrosative insult died by
caspase-dependent apoptosis while those that were exposed
to a high nitrosative insult still died by apoptosis, but caspase
inhibitors were no longer protective (64). This is further
evidence that the severity of the insult confers the shape of neuronal
demise. Indeed, a caspase-independent pathway for apoptosis may be
essential for neuronal programmed cell death, because the inherent
biochemical and physiological characteristics of neuronal cells,
including high lipid concentrations and energy requirements, make them
particularly susceptible to free radical mediated insult.
It has been demonstrated in some models that inhibition of apoptosis is
sufficient for the prevention of retinal degeneration (65). This is
supported by recent studies on congenital stationary night blindness, a
disease that involves loss of night vision due to blockage of the
phototransduction cascade (66, 67). However, daylight vision is
maintained as the rod and cone photoreceptors do not undergo apoptosis.
Therefore, by blocking the death of rod photoreceptors and the
subsequent death of the cones, visual function may be retained. The
data presented here not only provides a possible mechanism by which
nitric oxide mediates light-induced retinal degeneration but also
demonstrates that oxidative stress and elevated calcium contribute
significantly to an apoptotic process, which does not involve
caspase-3. These findings suggest that therapeutic strategies, which
target initiating signals of apoptosis such as calcium channel blockers
and antioxidants may be of more therapeutic benefit, than caspase inhibitors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (Sigma Chemical Co., UK)
in PBS or the inactive isomer
NG-nitro-D-arginine methyl ester
(D-NAME), 100 mg kg
1 (Sigma, UK) in PBS as a
control, 7-nitroindazole (7NI), 100 mg kg
1 (Sigma, UK) in
peanut oil or peanut alone as a control, and ODQ, 50 mg
kg
1 (Calbiochem) in Me2SO or
Me2SO alone as a control. All intraperitoneal injections were administered 1 h prior to light exposure.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Detection of rod photoreceptor apoptosis in
light-induced Balb/c mice. Apoptotic cell death was
assessed by two methods over 24 h. A, detection of DNA
strand breaks in photoreceptor nuclei by terminal dUTP nick end-
labeling (TUNEL). Retina of dark-adapted control mouse (a)
does not show labeling. Photoreceptors of mice sacrificed immediately
after 2-h light exposure remain negative (b). 6 h in
darkness following light exposure reveals scattered labeling in the ONL
(c). Mice sacrificed 14 h (d) and 24 h
(e) after light exposure show significant labeling of
photoreceptors. ONL, outer nuclear layer; INL,
inner nuclear layer. B, detection of DNA fragmentation by
DNA gel electrophoresis. Total retinal DNA was prepared from Balb/c
mice at each of the following time points: 0 h (untreated)
(lane 1), 6, 14, and 24 h after the light insult
(lanes 2-4, respectively). Untreated mice do not have
internucleosomal fragmentation. Internucleosomal DNA fragments that are
multiples of ~180 bp are evident in mice at 14 and 24 h.
1), 1 h prior to light exposure. In treated
animals no TUNEL-positive photoreceptor cells were detected immediately
following light exposure or following a further incubation of 6, 14, and 24 h in darkness (Fig. 2,
e-h). Animals that were injected with D-NAME, the inactive isomer of L-NAME, showed similar TUNEL
labeling to untreated light-induced animals (Fig. 2, a-d).
These results suggest a key role for NO in this model of light-induced
retinal degeneration.
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Fig. 2.
Effect of intraperitoneal administration of
L-NAME on light-induced photoreceptor apoptosis. Mice
were treated with 100 mg kg 1 (intraperitoneal) of the NOS
inhibitor L-NAME or its inactive isomer D-NAME,
1 h prior to light exposure. Retinas of mice treated with
D-NAME (a-d). Retina of dark-adapted control
mouse (a) does not show labeling. Photoreceptors of mice
sacrificed 6 h in darkness following light exposure reveals
scattered labeling in the ONL (b). Mice sacrificed 14 h
(c) and 24 h (d) after light exposure show
significant labeling of photoreceptors. Retinas of mice treated with
100 mg kg
1 of the NOS inhibitor L-NAME,
1 h prior to light exposure (e-h). Photoreceptors of
dark-adapted control mice (e) or those sacrificed or 6 (f), 14 (g), and 24 (h) hours of
darkness following the light exposure show no labeling. ONL,
outer nuclear layer; INL, inner nuclear layer.
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Fig. 3.
Analysis of intracellular calcium levels in
the retinas of untreated light-induced Balb/c mice and in those treated
with 100 mg kg 1 of the NOS inhibitor
L-NAME. Intracellular calcium levels were monitored
using the fluorescent probe Fluo-3 prior to light exposure (0 h),
during light exposure (0.5 h, 1 h), immediately after light
exposure (2 h) and 3 and 6 h of darkness following light exposure.
Increased fluorescence in the FL-1 channel indicates increased levels
of calcium. The percentage of cells displaying increased levels of
calcium is shown at each time point. Results are representative of
three independent experiments.
m)--
A key role for ROS has
been demonstrated in an in vitro model of retinal
degeneration, where antioxidants prevent photoreceptor cell death (19).
In vivo, a number of compounds possessing antioxidant properties have prevented photoreceptor apoptosis (see
"Discussion"). However, to date, little is known about the
oxidative pathways involved in such retinal death, nor have ROS
measurements been conducted in an in vivo model of retinal
degeneration. In this study, superoxide anion formation was monitored
using the probe DHE in control light-induced Balb/c mice and in those
treated with L-NAME (Fig.
4a). Increased levels of
superoxide were detected following 30 min of light exposure, and the
number of cells with elevated superoxide levels increased up to 3 h after the light insult. This was followed by a decrease in
intracellular levels. This data demonstrates, therefore, that
superoxide production is an early event in light-induced retinal
degeneration occurring alongside increased calcium levels. Furthermore,
our results show that superoxide generation is inhibited by
L-NAME, suggesting that NOS activity is also required for
the generation of superoxide.
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Fig. 4.
Analysis of intracellular superoxide levels
and mitochondrial transmembrane potential
( m) in the retinas
of untreated light-induced Balb/c mice and mice treated with 100 mg
kg
1 of the NOS inhibitor L-NAME.
a, analysis of intracellular superoxide levels. Superoxide
levels were detected with DHE prior to light exposure (0 h), during
light exposure (0.5 h, 1 h), immediately after light exposure (2 h) and 3 and 6 h of darkness following light exposure. Increased
fluorescence in the FL-2 channel indicates increased levels of
superoxide. The percentage of cells displaying increased levels of
superoxide is shown at each time point. Results are representative of
three independent experiments. b, analysis of mitochondrial
transmembrane potential.
m was assessed by flow
cytometry using the fluorescent probe JC-1. At high
m, JC-1 fluoresces strongly, therefore, a reduction
in fluorescence due to JC-1 indicates a reduction of
m. The percentage of cells displaying reduced
m is shown at each time point; prior to light
exposure (0 h), during light exposure (0.5 h, 1 h), immediately
after light exposure (2 h), and 3 h in darkness following light
exposure. Results are representative of three independent
experiments.
m) can result in
increased ROS generation. The lipophilic probe JC-1 was used to
analyze
m. In the presence of an intact
m, JC-1 forms J-aggregates, which are associated with
a shift in fluorescence emission (590 nm). Thus, a reduction in
fluorescence emission at 590 nm can be interpreted as a reduction in
m. As illustrated in Fig. 4b, a reduction
in
m is evident in a significant number of cells
after 30 min of light exposure and 3 h after light exposure 75%
of cells have reduced
m. This reduction in
m is blocked by L-NAME.
1 of L-NAME 1 h prior to light
exposure showed no increase in nNOS expression (Fig. 5A,
part d). These results suggest that initial NOS activity is
required for up-regulation of nNOS.
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Fig. 5.
Determination of the NOS isoform responsible
for production of NO following light exposure. A,
immunoblot analysis of the three isoforms of nitric-oxide synthase;
iNOS, eNOS, and nNOS. Western blot analyses were performed to detect
expression of iNOS, eNOS, and nNOS using polyclonal antibodies against
the three different isoforms. Equivalent quantities of total protein
from retinal cell lysates taken prior to light exposure (0 h), during
light exposure (0.5 h and 1 h), immediately after light exposure
(2 h), and 3, 6, 14 and 24 h of darkness following light exposure
were resolved using SDS-PAGE and transferred to a nitrocellulose
membrane. There was no detectable increase in expression of either iNOS
(A, part a) or eNOS (A, part
b). A time-dependent increase in nNOS expression was
evident (A, part c) with a 2.5-fold increase in
nNOS expression at 3 h, a 3-fold increase at 6 h, a 3.5-fold
increase at 14 h, and a 5-fold increase at 24 h. The retinas
taken from mice treated with 100 mg kg 1
L-NAME, 1 h prior to light exposure, showed no
increase in nNOS expression (A, part d).
B, effect of intraperitoneal administration of 7NI on
light-induced retinal degeneration. 1 h prior to light exposure,
mice were injected with 7NI, a selective inhibitor of nNOS (100 mg
kg
1) in peanut oil, or peanut alone as a control. Retinas
of control mice (a-d). Retina of dark-adapted control mouse
(a) does not show labeling. Photoreceptors of mice
sacrificed 6 h in darkness following light exposure reveals
scattered labeling in the ONL (b). Mice sacrificed 12 h
(c) and 24 h (d) after light exposure show
significant labeling of photoreceptors. Retinas of mice treated with
100 mg kg
1 of the nNOS inhibitor 7NI, 1 h prior to
light exposure (e-h). Photoreceptors of dark-adapted
control mice (e) or those sacrificed or 6 (f), 14 (g), and 24 (h) hours of darkness following light
exposure show no labeling. ONL, outer nuclear layer;
INL, inner nuclear layer.
1 of 7NI or peanut oil 1 h
prior to light exposure. Retinas of control animals (injected with
peanut oil) had scattered TUNEL labeling at 6 h and abundant TUNEL
labeling at 14 and 24 h (Fig. 5B, a-d)
similar to untreated light-induced animals (Fig. 1A). No
TUNEL-positive photoreceptor cells were detected at 6, 14, and 24 h in the retina of animals treated with 7NI (Fig. 5B,
e-h). 7 NI, therefore, prevents light-induced photoreceptor
apoptosis thus establishing the role of neuronal NOS in promoting
light-induced retinal degeneration.
1 of ODQ or Me2SO 1 h prior to
light exposure. In the Me2SO control animals, positive
TUNEL labeling was apparent at 6, 14, and 24 h (Fig.
6, a-d), similar to untreated
animals (Fig. 1A). In animals treated with ODQ, no
TUNEL-positive photoreceptor cells were detected at 6, 14, and 24 h (Fig. 6, e-h). These results demonstrate that guanylate
cyclase activity is required for light-induced retinal degeneration.
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Fig. 6.
Effect of ODQ, an inhibitor of guanylate
cyclase on photoreceptor apoptosis. Mice were treated with ODQ
(intraperitoneal 50 mg/kg) in Me2SO or Me2SO
alone as a control, 1 h prior to light exposure. Retinas of mice
treated with Me2SO alone (a-d). Retina of
dark-adapted control mouse (a) does not show labeling.
Photoreceptors of mice sacrificed 6 h in darkness following light
exposure reveals scattered labeling in the ONL (b). Mice
sacrificed 14 h (c) and 24 h (d) after
light exposure show significant labeling of photoreceptors. Retinas of
mice treated with 50 mg kg 1 of the guanylate cyclase
inhibitor, ODQ, 1 h prior to light exposure (e-h).
Photoreceptors of dark-adapted control mice (e) or those
sacrificed or 6 (f), 14 (g), and 24 (h) hours of darkness following the light exposure show no
labeling. ONL, outer nuclear layer; INL, inner
nuclear layer.
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Fig. 7.
Light-induced retinal degeneration is
caspase-3 independent. A, analysis of caspase-3
activity by immunoblotting analysis. Equivalent quantities of protein
from cell lysates taken prior to light exposure (0 h), and 3, 6, and14
h of darkness following light exposure were resolved using SDS-PAGE and
transferred to a nitrocellulose membrane. The presence of pro-caspase-3
(32 kDa) and the proteolytically active 17-kDa fragment were determined
using an anti-caspase-3 antibody. A second Western blot was carried out
with untreated and UV-treated 32D cells, which served as negative and
positive controls, respectively, to determine the ability of this
antibody to detect active murine caspase-3. The 32D protein lysates
were extracted 16 h after a 10-min exposure to ultraviolet
irradiation. These cells demonstrate the processing of pro-caspase-3
(32 kDa) to the active 17-kDa fragment as these cells undergo
apoptosis. Two separate Western blots were necessary, because the
Balb/c samples required longer exposure time to enable detection of
pro-caspase-3. The 32-kDa pro-caspase species is present at all time
points analyzed; however, the active 17-kDa fragment is absent up to
14 h. A representative result of three independent experiments is
shown. B, analysis of caspase-3-like activity by detection
of DEVD-pNA cleavage. The measurement of DEVD-pNA
cleavage was performed in a spectrophotometric assay by monitoring the
liberation of pNA due to caspase activity prior to light
exposure (0 h), and 3, 6, and 14 h of darkness following light
exposure. Untreated and anti-Fas IgM-treated Jurkat cells served as
negative and positive controls, respectively. Jurkat cells were
suspended at 5 × 105 cells/ml and treated with
apoptosis-inducing anti-Fas IgM antibody (300 ng ml 1) for
5 h. Data are expressed as the mean ± S.E. of three
independent experiments. The slight increase in activity at 3 h of
light exposure is not statistically significant as determined by the
student t test (p < 0.5). C,
assessment of active caspase-3 content in light-induced and uninduced
Balb/c retina. Active caspase-3 was assessed by FACS scan analysis
using an anti-active caspase-3 antibody. Increased fluorescence
indicates increased levels of active caspase-3. Untreated and
Fas-treated Jurkat cells served as negative and positive controls,
respectively. A representative result of three independent experiments
is shown. D and E, Western blot analysis was
carried out to detect the cleaved products of ICAD (D) and
PARP (E). Equivalent quantities of protein from cell lysates
taken prior to light exposure (0 h), and 3, 6, and14 h of darkness
following light exposure were resolved using SDS-PAGE and transferred
to a nitrocellulose membrane. A second Western blot was carried out
with untreated and VP-16-treated 32D cells, which served as negative
and positive controls, respectively, to determine the ability of the
antibodies employed to detect the native and cleaved products of ICAD
and PARP. The 32D protein lysates were extracted 16 h after
treatment with VP-16 (5 µg/ml). These cells demonstrate the
processing of PARP (116 kDa) and ICAD (45 kDa) to the 85- and 12-kDa
fragments, respectively, as these cells undergo apoptosis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m have been reported as early events during
apoptosis induced by a diverse range of stimuli (35). Consistent
with these reports, an early reduction in
m was
observed during photoreceptor apoptosis in this model (Fig. 5). Loss of
m can result in the release of several potent
apoptosis-inducing factors whose presence have been reported in the
intermembrane space, these include cytochrome c (36),
apoptosis inducing factor (37), procaspase-3 (38), pro-caspase-2
(39), and pro-caspase-9 (39, 40). The loss of
m
observed here may occur as a result of elevations in intracellular calcium, because alterations in intracellular calcium can disrupt mitochondrial potential (32, 41). Indeed, calcium-induced mitochondrial
depolarization has previously been described in rod photoreceptor
apoptosis in vitro (42). This study revealed that
mitochondria are the initial target site in Ca2+-induced
apoptotic rod cell death and demonstrated that Ca2+ enters
the rod photoreceptor outer segment via cGMP-gated channels and
subsequently enters the mitochondria, ultimately resulting in
mitochondrial depolarization.
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ACKNOWLEDGEMENTS |
---|
We wish to acknowledge Maria Kenny for technical assistance and Professor Peter Humphries for useful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by Fighting Blindness (Ireland), The Health Research Board of Ireland, and Bausch and Lomb, Co. Waterford Ireland.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Dept. of Molecular and Cellular Engineering,
Institute of Human Gene Therapy, University of Pennsylvania, School of
Medicine, Philadelphia, PA 19104.
§ To whom correspondence should be addressed: Tel.: 353-21-4904068; Fax: 353-21-4904259; E-mail: t.cotter@ucc.ie.
Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M005359200
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ABBREVIATIONS |
---|
The abbreviations used are:
RP, retinitis
pigmentosa;
NO, nitric oxide;
NOS, nitric-oxide synthase;
L-NAME, NG-nitro-L-arginine methyl ester;
D-NAME, NG-nitro-D-arginine methyl ester;
PBS, phosphate-buffered saline;
7NI, 7-nitroindazole;
ODQ, 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one;
TUNEL, terminal dUTP nick end labeling;
cGMP, cyclic guanosine monophosphate;
TdT, terminal deoxynucleotidyl transferase;
DHE, hydroethidine;
JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolecarbocyanine
iodide;
ROS, reactive oxygen species;
m, mitochondrial membrane potential;
ONL, outer nuclear layer;
INL, inner
nuclear layer;
Ac-DEVD-pNA, Acetyl-Asp-Glu-Val-Asp-p-nitroanilide;
DEVD-fmk, Asp-Glu-Val-Asp-fluoromethylketone;
CAD, caspase-activated DNase;
PARP, poly(ADP-ribose)polymerase;
FCS, fetal calf serum;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PB, permeabilization buffer;
PAGE, polyacrylamide gel
electrophoresis.
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