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The Journal of Neuroscience, June 15, 1999, 19(12):4778-4785
Activation of Caspase-3 in the Retina of Transgenic Rats with the
Rhodopsin Mutation S334ter during Photoreceptor Degeneration
Changdong
Liu1,
Yiwen
Li2,
Min
Peng1,
Alan M.
Laties1, and
Rong
Wen1, 3
Departments of 1 Ophthalmology,
2 Neurology, and 3 Cell and Developmental
Biology, University of Pennsylvania, School of Medicine,
Philadelphia, Pennsylvania 19104
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ABSTRACT |
The role of caspase-3 in photoreceptor degeneration was examined in
a line of transgenic rats that carry a rhodopsin mutation S334ter.
Photoreceptor degeneration in these animals is rapid. It is detected as
early as postnatal day (PD) 8, and by PD 20, only one of the original
12 rows of nuclei remain in the outer nuclear layer. At PD 11 and 12, the number of photoreceptors dying per day reaches a peak of ~30% of
the total photoreceptors in the retina. Coincident with this rapid
degeneration is an increase in caspase-3-like activity as assessed by
the cleavage of a fluorescent substrate
N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin and an increase in activated caspase-3 as determined by Western blot analysis
for its 12 kDa subunit. Intraocular injection of an irreversible caspase-3 inhibitor
N-benzyloxycarbonal-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethyketone partially protected photoreceptors from degeneration. These findings indicate that a caspase-3-dependent mechanism is operative in photoreceptor death in the transgenic rats under investigation.
Key words:
caspase-3; photoreceptors; z-DEVD-fmk; rhodopsin
mutation; degeneration; retina; transgenic rat
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INTRODUCTION |
Inherited retinal degenerations,
collectively known as retinitis pigmentosa, are characterized by
progressive death of photoreceptors. Mutations in any of a number of
photoreceptor-specific proteins, including rhodopsin (Dryja et al.,
1990a ,b; Farrar et al., 1990), peripherin (Travis et al., 1989; Farrar
et al., 1991; Kajiwara et al., 1991), the  subunit of the cGMP
phosphodiesterase (Bowes et al., 1990; McLaughlin et al., 1993, 1995),
and a rod outer segment protein, ROM1 (Kajiwara et al., 1994), can lead
to photoreceptor degeneration. Moreover, introduction of mutant
rhodopsin genes into the genomes of mouse (Olsson et al., 1992), pig
(Petters et al., 1997), or rat (Steinberg et al., 1996, 1997) has
successfully mimicked the phenotypes of retinitis pigmentosa as
observed in humans.
Photoreceptor cell death in animal models is considered an apoptotic
process, because DNA fragmentation has been demonstrated in
degenerating photoreceptors (Chang et al., 1993; Portera-Cailliau et
al., 1994). Since apoptosis, or programmed cell death, was originally
shown to be a cell suicide process with distinct morphological characteristics (Wyllie, 1987), enormous progress has been made in
unraveling the components of the death mechanism. Pioneering studies
performed on the nematode Caenorhabditis elegans identified a complement of genes related to cell death, including ced-3, ced-4, and ced-9. The first identified mammalian
homolog of CED-3 was the interleukin-1 -converting enzyme (ICE)
(Yuan et al., 1993). In turn, a search for ICE-related proteins
revealed an entire family of proteases in mammals. These comprise at
least 12 members termed caspases (for cysteine-containing,
aspartate-specific proteases) (Thornberry and Lazebnik, 1998). Among
them, activation of caspase-3 has been shown to participate in the
initiation of apoptosis (Thornberry and Lazebnik, 1998), especially in
neurons (Kuida et al., 1996; Armstrong et al., 1997; Yakovlev et al., 1997; Cheng et al., 1998; Namura et al., 1998).
In the present work, we examine the activation of caspase-3 in a line
of transgenic rats that carry the rhodopsin mutation S334ter.
Photoreceptors in these animals undergo rapid degeneration: >50% of
photoreceptors die in just 2 d, postnatal days (PD) 11 and 12. There is a significant increase in caspase-3 activation during the
rapid phase of photoreceptor degeneration. Furthermore, intraocular
administration of specific peptide active-site inhibitor of caspase-3
inhibits photoreceptor degeneration. These results indicate that a
caspase-3-dependent mechanism plays an important role in the demise of photoreceptors.
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MATERIALS AND METHODS |
Transgenic animals and intraocular injections.
Homozygous breeders of line 3 of transgenic rats that carry a murine
rhodopsin mutant S334ter (S334ter-3) were kindly provided by Dr.
M. M. LaVail (University of California, San Francisco, CA).
Heterozygous S334ter-3 rats were produced by mating homozygous breeders
with wild-type Sprague Dawley rats. All experiments were performed
using heterozygous S334ter-3 rats. Controls were age-matched Sprague
Dawley rats. Animals were kept in a 12 hr light/dark cycle at an
in-cage illuminance of <10 foot-candles (1 foot-candle = 10.76 lux). The in-cage temperature was kept at 20-22°C. Intraocular
injections were given directly into the vitreous by 32 gauge needles.
The caspase-3 inhibitor N-benzyloxycarbonal-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethyketone (z-DEVD-fmk) was purchased from Enzyme Systems Products (Dublin, CA).
Histology and Terminal dUTP Nick End Labeling. Animals were
killed by CO2 overdose, immediately followed by vascular
perfusion with mixed aldehydes (LaVail and Battelle, 1975). Eyes were
embedded in an Epon-Araldite mixture and sectioned at 1 µm thickness
to display the entire retina along the vertical meridian (LaVail and
Battelle, 1975). Retinal sections were examined by light microscopy. Twenty-seven transgenic animals from three litters were used for experiments described in Figure 1. Twelve wild-type Sprague Dawley rats
were used for experiments described in Figure 2. The terminal dUTP nick
end-labeling (TUNEL) method (Gavrieli et al., 1992) was used to detect
DNA fragmentation. Eyes were removed from 4% paraformaldehyde-perfused
animals (three transgenic and three control rats), cryoprotected with
20% sucrose, frozen in Tissue-Tek OCT compound (Miles, Elkhart, IN) in
powdered dry ice, and stored at  80°C. Cryosections of 10 µm were
cut through the entire retina along the vertical meridian and
thaw-mounted onto Super Frost Plus glass slides (Fisher Scientific,
Pittsburgh, PA). TUNEL was performed using an apoptosis detection
system (Promega, Madison, WI) according to the manufacturer's instructions.
Measurement of caspase activities. Retinas were dissected,
snap-frozen in powdered dry ice, and stored at 80°C. Total protein was obtained by homogenizing retinas in a lysis buffer that contained 100 mM HEPES, pH 7.5, 10% sucrose, 1 mM EDTA,
20 mM EGTA, 0.2% 3[3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, 10 mM dithiothreitol, 10 µg of leupeptin, 2 µg/ml
pepstatin, 2 µg/ml aprotinin, 1 mM PMSF, and 0.03%
digitonin. The amount of total protein of each sample was determined by
the BCA protein assay (Pierce, Rockford, IL), and the samples were
stored at 80°C. Caspase-3 or caspase-1 activity was assessed by
measuring the cleavage of fluorogenic substrate Ac-DEVD-AMC or
Ac-YVAD-AMC, respectively, using a luminescence spectrometer
(LS-50B; Perkin-Elmer, Norwalk, CT). Cleavage of
N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (Ac-DEVD-AMC)
or N-acetyl-Tyr-Val-Ala-Asp-aminomethylcoumarin (Ac-YVAD-AMC) was measured for 900 sec for each sample of 100 µg of
total protein (accumulation of fluorescence was linear for at
least 2 hr). The rate of fluorescence accumulation was calculated as
the activity of a given enzyme. Experiments were repeated three times
with samples from three litters of transgenic or control animals.
Protein preparation and immunoblotting analysis. Retinas
were dissected, snap-frozen in powdered dry ice, and stored at
80°C. Pooled retinas were homogenized, and the concentration of
total protein in each sample was determined by the BCA protein assay (Pierce). Total protein of 100 µg from each sample was
electrophoresed on polyacrylamide gels and transferred to nitrocellular
membranes (Bio-Rad, Hercules, CA). Blots were stained briefly with
Ponceau S for visual inspection of transfer efficiency. Immunoblotting analysis was performed using polyclonal antibodies against the 12 kDa
subunit of the active form of caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA). Signals were visualized using an ECL kit (Amersham, Arlington Heights, IL) and recorded on Hyperfilm (Amersham). All experiments were repeated three times to verify the consistency of the results.
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RESULTS |
Photoreceptor degeneration in heterozygous
transgenic rats
The progressive photoreceptor degeneration in the
retinas of heterozygous S334ter-3 rats follows a pattern described by
M. M. LaVail (personal communication). Figure
1 shows representative light micrographs
of heterozygous S334ter-3 rat retinas from PD 6 to 20. The overall
appearance of the retina of a transgenic animal at PD 6 (Fig.
1A) is similar to an age-matched control (Fig.
2A), differing slightly
in stage of postnatal development. However, in the retina of a PD 8 S334ter-3 rat, 30-50 pyknotic nuclei in the outer nuclear layer (ONL)
are found in an entire retinal section (Fig. 1B), in
contrast to only one or two in the control ONL (data not shown). By PD
10, the retina of the transgenic rats differs greatly from that of
control: not only are many pyknotic nuclei found in the ONL in the
transgenic rats, but the inner segments of photoreceptors are now
disorganized (Fig. 1C). Substantial loss of photoreceptors
occurs at PD 11 (Fig. 1D). By PD 12, the ONL contains
only six or seven rows of nuclei, in contrast to 12-13 rows in normal
controls (Fig. 2C). The inner segments are even more
disorganized (Fig. 1E). By PD 14, the rows of nuclei in the ONL decreased to three or four in the S334ter-3 rats, and the
remaining inner segments are now short stumps (Fig.
1F). The nuclei in the ONL further decline to two or
three rows at PD 16. Only one row remains at PD 20 with residual inner
segments (Fig. 1G-I). No outer segments ever develop
in the heterozygous S334ter-3 rats.
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Figure 1.
Photoreceptor degeneration in the S334ter-3 rats.
Plastic-embedded sections of retinas from S334ter-3 rats of PD 6 (A), 8 (B), 10 (C), 11 (D), 12 (E), 14 (F), 16 (G), 18 (H), and 20 (I) were examined by light microscopy
(superior region). The ONL is indicated in each panel by a white
bar. Pyknotic nuclei in B and C
are indicated by white arrowheads. Loss of
photoreceptors becomes evident at PD 8. Peak of photoreceptor death
occurs at PD 11 and 12. Pyknotic nuclei are found mainly in the
proximal half of the ONL at PD 8 (B), 10 (C), and 11 (D). Some
photoreceptor nuclei are displaced to the subretinal space
(B-E). Sections were stained with toluidine
blue. Scale bar, 20 µm.
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Figure 2.
Photoreceptors in normal Sprague Dawley rats
during postnatal development. Plastic-embedded sections of retinas from
Sprague Dawley rats of PD 6 (A), 8 (B), 10 (C), 12 (D), 16 (E), and 20 (F) were examined by light microscopy (superior
region). The ONL is indicated in each panel by a white
bar. Development of rod outer segments is evident at PD 10 (C). Rod outer segments are approximately half of
the length of those in the mature retina by PD 12 (D). Photoreceptors in PD 16 (E) and 20 (F) animals
resemble closely those found in mature animals. Sections were stained
with toluidine blue. Scale bar, 20 µm.
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In the early stages of photoreceptor degeneration in the
S334ter-3 rats (PD 8-11), pyknotic nuclei are distributed mainly in
the proximal half of the ONL (Fig. 1B-D). As the
degeneration progresses, they are seen in the distal ONL as well (Fig.
1D,E).
During degeneration, some photoreceptor nuclei become dislocated to the
subretinal space next to the retinal pigment epithelial (RPE) cells
(Fig. 1B-E). No such dislocation is observed in
control animals (Fig. 2).
For comparison, Figure 2 shows representative light micrographs of
normal Sprague Dawley rat retinas from PD 6 to 20. At PD 6, the overall
morphological appearance of Sprague Dawley retina (Fig.
2A) is similar to that of the transgenic rats (Fig.
1A). At PD 8, the ONL (Fig. 2B)
thickens, and the inner segments are well organized (Fig.
2B). By PD 10, the outer segments of rod photoreceptors begin to develop (Fig. 2C). At PD 10, the rod
outer segments are approximately half of the length of those in mature retina (Fig. 2D). Photoreceptors in PD 16 (Fig.
2E) and 20 (Fig. 2F) animals are
similar to those found in mature normal animals. Only one or two
pyknotic nuclei are observed in the ONL from PD 10 through 20 (data not shown).
To determine whether photoreceptor death in the S334ter-3 rats was
apoptotic, we used the TUNEL method (Gavrieli et al., 1992) to detect
DNA fragmentation. As shown in Figure 3,
many cells in the ONL of a PD 11 transgenic rat are TUNEL-positive. The
distribution of TUNEL-labeled cells is denser in the proximal than in
the distal ONL (Fig. 3A). This is consistent with the
distribution of pyknotic nuclei (Fig. 1D). In
age-matched control animals (Fig. 3B), no TUNEL-labeled
cells are found in the ONL. In the inner nuclear layer (INL), a few
cells are TUNEL-positive in both control (Fig. 3B) and
transgenic (Fig. 3A) animals.
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Figure 3.
DNA fragmentation in degenerating photoreceptors
of the S334ter-3 rats. Cryosections (10 µm) from the retina of a PD
11 S334ter-3 (A) and a wild-type Sprague Dawley
rat (B) were TUNEL-labeled with fluorescein to
visualize the DNA fragmentation. In the S334ter-3 rat
(A), numerous cells in the ONL are labeled. The
distribution of labeled cells is denser in the proximal than in the
distal ONL. No labeled cells are found in the ONL of the control retina
(B). A few cells in the INL are also labeled in
the control (B) and transgenic
(A) rats. rpe, Retinal pigment
epithelium; onl, outer nuclear layer;
inl, inner nuclear layer; gc, ganglion
cell layer. Scale bar, 50 µm.
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Activation of caspase-3 in the S334ter-3 rat retina
during degeneration
The activity of caspase-3-like proteases was assessed by measuring
the cleavage of a fluorogenic substrate Ac-DEVD-AMC. Retinal samples
were collected from transgenic and age-matched Sprague Dawley control
animals at PD 8-14 and 16. A large increase in Ac-DEVD-AMC cleavage
was observed in transgenic animals at PD 11-14. It was close to
sixfold of control level at PD 11 and 12 and declined to approximately
twofold at PD 13 and 14 (Fig.
4A). Because
Ac-DEVD-AMC is also cleaved by caspase-1, we ruled out this possibility
by using a caspase-1-specific fluorogenic substrate, Ac-YVAD-AMC.
Substrates with a sequence of YVAD, based on the recognition sequence
for caspase-1 YVHD, have high selectivity for caspase-1 over caspase-3.
In fact, the tetrapeptide aldehyde Ac-YVAD-CHO is highly selective,
possessing an affinity for caspase-1 six orders of magnitude higher
than for caspase-3 (Fernandes-Alnemri et al., 1995; Margolin et al.,
1997). As shown in Figure 4B, no significant
alteration in Ac-YVAD-AMC cleavage was observed from PD 8 to 16 in the
S334ter-3 rats, compared with the controls, indicating that caspase-1
is not significantly activated during photoreceptor degeneration.
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Figure 4.
Activities of caspase-3-like and caspase-1-like
proteases in the retina. Activities of caspase-3-like or caspase-1-like
proteases were determined by measuring the cleavage of Ac-DEVD-AMC or
Ac-YVAD-AMC, respectively. Retinas were collected at PD 8-14 and 16 from S334ter-3 or normal Sprague Dawley rats. Intensity of fluorescence
was measured on a Perkin-Elmer LS-50B luminescence spectrometer at 30 sec intervals for 900 sec. The rate of accumulation of fluorescence was
calculated as the activity of a given enzyme. Data are presented as
mean ± SD (n = 3). ***p < 0.001; *p < 0.01 (Student's t
test).
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Caspases-3 is synthesized as a 32 kDa inactive proenzyme and is cleaved
at Asp-28-Ser-29 and Asp-175-Ser-176 to generate a subunit of 17 kDa
and a smaller one of 12 kDa after activation (Nicholson et al., 1995).
To assess the amount of active caspase-3 during photoreceptor
degeneration, we measured the amount of the 12 kDa subunit in retinas
of the S334ter-3 rats by immunoblotting analysis. Figure
5A displays the relative
amounts of the 12 kDa subunit during photoreceptor degeneration.
Significant increases were found at PD 10-12 in the transgenic rats,
coincident with the rapid phase of photoreceptor loss in the retina.
The amounts of the 12 kDa subunit in the transgenic rats during the
rapid degeneration phase are much higher than those in the age-matched Sprague Dawley control rats (Fig. 5B).
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Figure 5.
p12 subunit of activated caspase-3 during
photoreceptor degeneration in the S334ter-3 rats. Immunoblotting
analyses were performed to detect the p12 subunit of the active form of
caspase-3, using polyclonal antibodies against the p12 subunit of
caspase-3. A, In the retinas of S334ter-3 rats,
significant increases in the p12 subunit are found at PD 10-12, which
coincides with the rapid degeneration phase. B, The
amounts of the 12 kDa subunit in the retina of PD 10 (P10) and 12 (P12) S334ter-3 animals
(Tg) are significantly higher than age-matched Sprague
Dawley rats (SD).
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Protection of photoreceptors by peptide caspase
inhibitor z-DEVD-fmk
To test whether caspase-3 is important for photoreceptor death in
the S334ter-3 rats, we used an irreversible inhibitor for caspase-3,
z-DEVD-fmk. Six S334ter-3 rats (littermates) were injected at PD 9 with
50 µg of z-DEVD-fmk (in 1 µl of DMSO) into the left eyes and 1 µl
of DMSO into the right eyes. The eyes were collected at PD 20, and
retinas were examined by light microscopy. As shown in Figure
6, the retina of a PD 20 normal animal is
fully developed and intact. The ONL contains 12-13 rows of nuclei, and
the outer and inner segments are well organized (Fig.
6A). The retina of a S334ter-3 rat treated with DMSO
(Fig. 6B) corresponds to those without any treatment
(Fig. 1I): only a single row of nuclei remain in the
ONL (Fig. 6B). In contrast, the ONL of the
z-DEVD-fmk-treated retina from the other eye of the same animal
contains four or five rows of nuclei (Fig. 6A). In
all six animals, the ONL in the control eyes has one or two rows of
nuclei, whereas the treated eyes have three to five rows.
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Figure 6.
Protection of photoreceptors by z-DEVD-fmk.
Plastic-embedded sections of retinas from a normal rat
(A) and a S334ter-3 rat treated with DMSO
(B) or z-DEVD-fmk (C) at PD
20 were examined by light microscopy. The normal retina has well
developed outer and inner segments. The ONL has 12 or 13 rows of nuclei
(A). In the DMSO-treated retina, the ONL has only
one row of nuclei, and inner segments become short stumps
(B). In the z-DEVD-fmk-treated retina from the
same animal (C), the ONL has four or five rows of
nuclei. The inner segments are better preserved, although still
shortened and disorganized. Some dislocated cells are found in the
subretinal space next to the RPE. Sections were stained with toluidine
blue. OS, Outer segment; IS, inner
segment; OPL, outer plexiform layer. Scale bar, 20 µm.
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To verify that z-DEVD-fmk did inhibit caspase-3 activation in the
retinas of S334ter-3 rats, we measured the 12 kDa caspase-3 subunit
after inhibitor injection. Three transgenic rats were injected with 50 µg of z-DEVD-fmk (in 1 µl of DMSO) into the left eyes and 1 µl of
DMSO into the right eyes at PD 9, and retinas were collected at PD 11. As shown in Figure 7, the amount of the 12 kDa subunit in retinas treated with z-DEVD-fmk is significantly lower than in those treated only with DMSO. Nevertheless, it is still
higher than in the normal Sprague Dawley rats.
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Figure 7.
The p12 subunit of activated caspase-3 after
intraocular injection of caspase-3 inhibitor z-DEVD-fmk. PD 9 S334ter-3
rats were injected with 50 µg of z-DEVD-fmk (in 1 µl of DMSO) to
the left eyes and 1 µl of vehicle (DMSO) to the right eyes. Retinas
were collected at PD 11. Retinas of age-matched Sprague Dawley rats
served as control. Immunoblotting analyses were performed to detect the
p12 subunit of the active form of caspase-3, using polyclonal
antibodies against the p12 subunit of caspase-3. In the S334ter-3 rats,
the amount of the p12 subunit is much less in inhibitor treated retinas
(Tg, Ihb +) than in vehicle-treated retinas (Tg,
Ihb ), although it is still higher than normal Sprague Dawley
controls (SD, Ihb ).
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DISCUSSION |
Since the discovery of mutations in the rhodopsin gene in human
with retinitis pigmentosa (Dryja et al., 1990a,b; Farrar et al., 1990),
various mutations of the visual pigment have been introduced into the
genomes of animals as models of these disorders (Olsson et al., 1992;
Steinberg et al., 1996, 1997; Petters et al., 1997). These transgenic
animals are valuable tools for retinal degeneration research. In the
present work, we used a line of transgenic rats that carry a murine
rhodopsin mutation, S334ter (Steinberg et al., 1996, 1997). These
animals undergo a degenerative course leading to the death of >90% of
all photoreceptors in <2 weeks. At the peak rate of photoreceptor
death, the loss of photoreceptors reaches three or four rows of nuclei
in the ONL in 1 day, or close to 30% of the photoreceptors in the
entire retina (Fig. 1). It is estimated that in the rat retina one row
of nuclei in the ONL represents about 1 million
photoreceptors. Thus, at the peak of degeneration, the death rate is
3-4 million rods/d. The massive photoreceptor death in these animals
provides an opportunity to investigate the biochemistry of
photoreceptor degeneration.
Histological analysis shows that photoreceptor degeneration begins at
the proximal portion of the ONL, because pyknotic nuclei are found
mainly in the proximal half of the ONL from PD 8 through 11 (Fig. 1).
In addition, more TUNEL-labeled cells are observed in the proximal than
in the distal ONL at PD 11 (Fig. 2). The reason why cells in the
proximal ONL are more susceptible to degeneration is not clear. It is
possible that these cells are more advanced in development than those
in the distal portion of the ONL and therefore die sooner.
The displacement of photoreceptor nuclei to the subretinal space (Fig.
1) is likely to be a phenomenon related to the degenerative process,
because it is observed only in the retinas of transgenic animals
undergoing photoreceptor degeneration.
The first indication that caspases are involved in photoreceptor death
derives from Drosophila, in which mutations in the rhodopsin
gene also cause photoreceptor degeneration (Steele and O'Tousa, 1990;
Colley et al., 1995; Kurada and O'Tousa, 1995). Experiments in two
strains of Drosophila with rhodopsin mutations, ninaERH27 and
rdgC306, showed that eye-specific
expression of baculovirus protein p35 not only protects photoreceptors
but also preserves their normal function (Davidson and Steller, 1998).
p35 blocks cell death after viral infection and is regarded as
antiapoptotic (Friesen and Miller, 1987; Clem et al., 1991). Purified
recombinant p35 inhibits a broad range of caspases, including
caspase-1, -2, -3, and -4 (Bump et al., 1995; Xue and Horvitz, 1995).
The demonstration by Davidson and Steller (1998) that p35 protects
photoreceptors indicates that in Drosophila photoreceptor
cell death likely is mediated by a caspase-dependent mechanism.
Caspase-3 was first cloned using the sequence of an expressed sequence
tag in the sequence database that shared similarity with ICE and was
named CPP32 (Fernandes-Alnemri et al., 1994). Subsequently, two other
groups independently identified it and named it Yama (Tewari et al.,
1995) and apopain (Nicholson et al., 1995). Among known caspases, it
has the highest homology to C. elegans CED-3 in both amino
acid sequence and substrate specificity. It is recognized as one of the
key executioners of apoptosis. Moreover, activation of caspase-3 is
important for the initiation of apoptosis in neurons. For instance, in
caspase-3 knock-out mice, there is a selective defect in cell death in
the CNS that leads to a doubling of brain size (Kuida et al., 1996). This finding indicates that caspase-3 plays a critical role during morphogenetic cell death in the brain. In cultured mouse cerebellar granule neurons, K+-serum withdrawal also activates
caspase-3, inducing apoptosis (Armstrong et al., 1997). In addition,
caspase-3 is found to contribute to apoptosis in brain neurons after
traumatic brain injury or experimental cerebral ischemia (Yakovlev et
al., 1997; Cheng et al., 1998; Namura et al., 1998). Last, in the
retina caspase-3 is involved in ganglion cell death after optic nerve
transection (Kermer et al., 1998).
The present work establishes a role of caspase-3 in the photoreceptor
degeneration in S334ter-3 rats. A clear relationship among the increase
in Ac-DEVD-AMC cleavage, the increase in the 12 kDa subunit of
caspase-3, and the course of photoreceptor degeneration indicates a
correlation between caspase-3 activation and photoreceptor degeneration. That inhibition of caspase-3 by the irreversible caspase-3 inhibitor z-DEVD-fmk protects photoreceptors provides further
evidence that caspase-3 activation promotes the death of photoreceptors.
Our present work leaves unanswered the question of whether
photoreceptor death by causes other than rhodopsin mutations is also
mediated by the same mechanism. However, preliminary experiments (R. Wen, unpublished observations) do indicate that caspase-3 is involved
in photoreceptor death in other animal models as well, suggesting a
common mechanism for photoreceptor degeneration in these disorders.
Further analysis of the involvement of caspases not only will deepen
our understanding of photoreceptor cell death but also might provide
the basis for a new approach to therapies for retinal degenerative
disorders. The present work represents a step toward that goal.
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FOOTNOTES |
Received Feb. 3, 1999; revised March 30, 1999; accepted April 5, 1999.
This work was supported by the Foundation Fighting Blindness and a
grant from the Paul and Evanina Mackall Foundation Trust. R.W. is a
recipient of a Research to Prevent Blindness career development award.
We thank Dr. Matthew M. LaVail and Nancy Lawson for homozygous
S334ter-3 rats.
Correspondence should be addressed to Dr. Rong Wen, Department of
Ophthalmology, D-603 Richards Building, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104.
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TOP
ABSTRACT
INTRODUCTION
Compound via MeSH
