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From the Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto, Japan.
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METHODS. Both RCS and Sprague—Dawley rats were used. Cryosections of the
retinas at various postnatal times were immunostained with antibodies
against caspase-1 (interleukin-1ß–converting enzyme, ICE) and
caspase-2 (Nedd2/Ich-1). Double staining with TdT-dUTP terminal
nick–end labeling (TUNEL), propidium iodide, and the antibodies was
also performed. To evaluate the time course of protein expression,
western blot analysis was carried out. The temporal profile of
caspase-like protease activity was studied using a fluorogenic
tetrapeptide substrate, acetyl-tyrosyl-valyl-alanyl-aspartic acid 
(4-methyl-coumaryl-7-amide) (Ac-YVAD-MCA). Intravitreal injection of a
caspase-1 inhibitor, acetyl-tyrosyl-valyl-alanyl-aspartic-aldehyde
(Ac-YVAD-CHO), at postnatal days 21 (P21) and P26 was performed to see
if this caused a decrease in apoptotic cell number at P28.
RESULTS. TUNEL-positive photoreceptors of RCS rats stained strongly with antibodies against caspase-1 and caspase-2. Double staining studies revealed that caspase-1 and caspase-2 were coexpressed in apoptotic cells. Western blot analysis showed that active forms of caspase-1–like and caspase-2–like proteases were upregulated at P28, concurrent with the peak in TUNEL-positive cells. The enzymatic activity of caspase-1–like protease was elevated in RCS rat retinas at P28, and the inhibitor of caspase-1 transiently reduced the number of the apoptotic photoreceptors.
CONCLUSIONS. Activation of caspase-like proteases plays an important role in photoreceptor apoptosis of RCS rats.
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Introduction |
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Apoptosis is a phenomenon underlying normal development and many pathologic conditions and is achieved through activation of a cascade called a "death program."3 4 In general, the apoptotic process is divided schematically into five steps: activation, propagation, commitment, execution, and, finally, cell death.3 Interleukin-1ß–converting enzyme (ICE)/caspase family proteins, identified by their homology with the nematode death gene ced-3, are executors of the apoptotic program in some vertebrate cells.4 Although the final step of apoptosis is invariably cell death, various signals contribute to the final common pathway, and intermediate steps differ depending on the etiology of the apoptosis and cell type.4 If the apoptotic process has not proceeded beyond the execution step, a cell can be rescued in some instances by blocking expression of apoptosis-related genes or by overexpression of bcl-2.4
Apoptosis plays a major role in the pathogenesis of many diseases in which photoreceptors degenerate, such as retinitis pigmentosa, retinal detachment, light injury, ischemic injury, and age-related macular degeneration.5 6 Therefore, it is important to know which genes are specifically expressed during apoptosis of photoreceptor cells. Herein, we will show that members of the caspase family may play a critical role in the apoptosis of photoreceptor cells in the RCS rat. In the present study, we concentrated on caspase-1 and caspase-2 because these enzymes are known to play an important role in neuronal apoptosis of ischemic brain injury and retinal development and apoptosis of rat pheochromocytoma-derived cell line cells (PC12).7 8 9
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsReferences |
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Antibodies
Antibodies used in this study were obtained from various sources:
Goat anti–caspase-1 (ICE) polyclonal antibody (M-19) and goat
anti–caspase-2 (Nedd2/Ich-1) polyclonal antibody (N-19) were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA); fluorescein
isothiocyanate (FITC)– and rhodamine-conjugated goat anti-rabbit IgG
from DAKO (Glostrup, Denmark); and FITC- and rhodamine-conjugated
rabbit anti-goat IgG from Jackson ImmunoResearch Laboratories (West
Grove, PA). These caspase antibodies recognize both proenzyme and
active forms of protease. Anti-phosphotyrosine antibody from Upstate
Biotechnology was used to distinguish photoreceptors from microglial
cells and peripheral macrophages.
TUNEL and Propidium Iodide Staining
DNA nick end-labeling was performed according to a slightly
modified method of Gavrieli et al.10
After rats
were anesthetized with an intraperitoneal injection of pentobarbital
(50 mg/kg), they were perfused transcardially with 100 ml of normal
saline supplemented with 1 U/ml heparin followed by 200 ml of 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The eyes were
enucleated and placed in 4% paraformaldehyde for 24 hours at
4oC. They were rinsed with phosphate-buffered
saline (PBS) and then transferred to 10%, 15%, and 20% sucrose in
0.1 M PBS for 3 hours at room temperature. Samples were then
snap-frozen in Tissue–Tek (Miles Laboratories, Elkhart, IN) on dry ice
in hexane and stored at -80oC. Cryostated
sections (10–15 µm) were thaw-mounted onto glass slides coated with
poly-L-lysine and air-dried for 2 hours at room temperature. After
cryosections were rinsed three times in 0.02 M PBS (pH 7.4), sections
were incubated with biotinylated terminal dUTP transferase and
biotinylated dUTP in TdT buffer (30 mM Tris, pH 7.2, 140 mM sodium
cacodylate, and 1 mM cobalt chloride) for 60 minutes at
37oC with terminal dUTP in the moisture chamber.
After rinsing, the sections were reacted with avidin–FITC and examined
with a Zeiss scanning laser confocal microscope (LSM 410 inverted Laser
Microscope; Zeiss, Oberkochen, Germany). Positive controls were
generated using DNase I in TdT buffer (1 µg/ml) before incubation
with terminal transferase and biotinylated nucleotides. Propidium
iodide (PI) staining was performed by incubating the sections with 50
µl PI (20 µg/ml) for 10 minutes at room temperature. TUNEL staining
of cells, compacted nuclear staining, nuclear fragmentation, and loss
of nuclear staining by PI were considered to be signs of apoptosis.
Immunohistochemistry
After rinsing with PBS, cryosections were incubated with 2%
normal goat or rabbit serum for 60 minutes at room temperature.
Incubation with the primary antibody (anti–caspase-1, 10 µg/ml, and
anti–caspase-2, 10 µg/ml) was carried out in a moisture chamber at
4oC overnight. FITC- or rhodamine-conjugated
secondary antibody was reacted at room temperature for 2 hours.
Quantitative Analysis of TUNEL and Immunohistochemistry
At each point of the time course study for TUNEL and
immunohistochemical staining, the number of TUNEL-positive- and
immunostained cells in the ONL was determined in 5 meridian sections
through the optic nerve. The numbers of cells were counted in 0.4-mm
lengths of the 1 mm from the optic nerve head on the superior
and inferior hemisphere using the Zeiss confocal microscope with the
"measure" function. Results are expressed as mean ± SEM.
Analysis of Western Blots
Samples (n = 5 at each time) were homogenized in
buffer containing Tris–HCl (pH 8.0), 250 mM NaCl, 0.5% NP-40, 0.1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, 20 µg/ml
leupeptin, and 10 µg/ml aprotinin. Sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out
as described by Laemmli.11
A 50-µg sample of
protein was loaded on each lane, electrophoresed with 10% acrylamide
gel, and then transferred to nitrocellulose membranes. After being
rinsed with Tris-buffered saline (pH 7.4) containing 0.25% Tween-20
(TBST), the membrane was incubated with the primary antibodies (0.5
µg/ml) in TBST for 1 hour at room temperature. After three washings,
the membrane was incubated for 1 hour at room temperature with
horseradish peroxidase–conjugated secondary antibody (Amersham,
Buckinghamshire, UK) diluted 1:500 in TBST. The membrane was washed
again and developed with the chemiluminescence ECL western blotting
system (Amersham).
Intravitreal Administration of Caspase-1 Inhibitor
A specific inhibitor of caspase-1,
acetyl-tyrosyl-valyl-alanyl-aspartate-aldehyde (Ac-YVAD-CHO), was
obtained from the Peptide Institute (Osaka, Japan). A 20-mM solution of
the Ac-YVAD in dimethyl sulfoxide was prepared, and 1 µl of the
solution was injected into the vitreous cavity of the RCS rats at P21
and P26 by means of a 30-gauge needle. As a control, 1 µl dimethyl
sulfoxide (vehicle) was injected in the same way. At P28, the eyes were
enucleated, and the TUNEL staining was performed as described above.
Assay of Caspase Activity
Caspase-1–like protease activity was assayed as described by
Nicholson et al.12
with slight
modification.13
Briefly, resected retinas were homogenized
in 10 mM Hepes–KOH, pH 7.2, 2 mM EDTA, 0.1% CHAPS, 5 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
pepstatin, 20 µg/ml leupeptin, and 10 µg/ml aprotinin. Homogenates
were centrifuged at 15,000g for 30 minutes, after which the
supernatants were transferred to new Eppendorf tubes. Aliquots of
extracts (300 µg protein in 100 µl extraction buffer) were
preincubated at 37°C for 30 minutes and then mixed with 5 µl of 10
mM tetrapeptide substrate, acetyl-tyrosyl-valyl-alanyl-aspartic acid
(4-methyl-coumaryl-7-amide) (Ac-YVAD-MCA; (Peptide Institute). Free
aminomethylcoumarin (AMC) accumulation, which resulted from cleavage of
the aspartate-AMC bond, was monitored in each sample at 37°C over 180
minutes using a spectrophotometer (Ultraspec III; Pharmacia, Cambridge,
England). The absorbance of the each sample at 370 nm was plotted
against time. Linear regression analysis of the velocity of each curve
yielded the activity for each sample. Data were expressed as a
percentage of the caspase-1–like activities in the samples compared to
those in the P28 SD rat retina.
Statistical Analysis
The data were analyzed statistically by one-way ANOVA followed by
Fisher’s post-hoc test. Probability values less than 0.05 were
determined to be statistically significant.
Results
TUNEL Staining
In both RCS and SD rats, TUNEL-positive cells were observed
in the ONL at P14. In SD rats, these cells were no longer observed at
P21. In contrast, TUNEL-positive cells increased in number in the ONL
of RCS rats at P21 to P45, with a peak at P28 (Figs. 1 A, 1B). At P28, the number of TUNEL-positive cells was approximately
250/0.4-mm section, and about one third of the total cell number in the
ONL at P28 was stained by TUNEL (Fig. 1B)
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TUNEL-Positive and Immunostained Cells
In RCS rats, quantitative analyses showed that the numbers of
cells stained with antibodies against caspase-1 and caspase-2 were very
similar during the follow-up period (Fig. 2C)
. Also, the time course of
TUNEL-positive cells showed a similar pattern of immunostaining. The
numbers of immunostained cells were almost equal to that of
TUNEL-positive cells at P14 and P21 (less than 6 cells/0.4-mm tissue
section), but there were approximately 10 times more TUNEL-positive
cells than immunostained cells at P28 (243.3 ± 8.0 TUNEL-positive
cells/0.4-mm tissue section, 18.2 ± 0.4 caspase-1–positive cells
and 13.7 ± 0.4 caspase-2–positive cells).
Double staining studies to identify TUNEL-positive cells and immunostained cells using anti–caspase-1 and anti–caspase-2 antibodies showed that these signals were colocalized in the same cell (Figs. 2A 2B) . Caspase-1 and caspase-2 were also double-stained in the same cell (graphic data not shown). However, all the anti–caspase-1–positive cells were not always stained with anti–caspase-2 antibodies. The results of double staining with the antibodies and TUNEL staining showed that expression of caspase-1 and caspase-2 was correlated with photoreceptor apoptosis in RCS rats. Anti-phosphotyrosine antibody–labeled cells did not show coexpression of caspase-1 or caspase-2 (graphic data not shown).
Immunoblot Analysis of Caspase-1– and Caspase-2–Like Proteases
As shown in Figure 3
A, anti–caspase-1 antibody depicted both 37.5-kDa and 20-kDa bands by
western blot analysis. The apparent molecular weight of the bands
agrees with that of caspase-1–like protease. Figure 3B
shows that
caspase-2–like protease is expressed in RCS rat retinas, based on the
finding that anti–caspase-2 antibody blotted at both 40.5- and
33-kDa bands. The active form of caspase-1 and caspase-2
consists of long and short fragments that are derived from proteolytic
processing of the proenzyme during apoptosis. Antibodies used in this
study reacted with the active form of the 20-kDa subunit of caspase-1
and the 33-kDa subunit of caspase-2. The level of expression of the
37.5 kDa caspase-1 subunit was constant, but that of the 20-kDa
subunit, the active form, was upregulated at P21, P28, and P35 RCS rat.
On the other hand, expression of the 40.5-kDa proenzyme was constant,
whereas that of the 33-kDa active form of caspase-2 was upregulated at
P14, P21, P28, and P35 in RCS rat retinas.
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Caspase-1 was first identified as the mammalian homologue of the ced-3 gene, initially identified in Caenorhabditis elegans. Caspase-1 is known to promote apoptosis during development of the nematode, and, currently, 10 Ced-3–related cysteine proteases, termed caspase, have been cloned.3 4 These family members participate in one of two distinct signaling pathways: activation of proinflammatory cytokines and activation of apoptotic cell death.4 All the known caspase family proteases are synthesized as inactive proenzymes, which require cleavage to liberate one large and one small subunit to form the active enzyme.4 Activation of one caspase can lead to cleavage and activation of another molecule of the same caspase, another caspase, or both, leading to an amplified apoptotic cascade.4 Indeed, in the Fas-mediated apoptosis of lymphoid cells, activation of caspase-3 via specific cleavage of the proenzyme by caspase-1 has been demonstrated.4 A number of experimental studies suggest that activation of the caspase family plays a critical role in the execution of apoptotic events.3 7 8 9 Caspase-1 was implicated in death caused by superoxide dismutase downregulation in PC12 cells but not in withdrawal of trophic factor support.9 Conversely, the antisense constructed to downregulate caspase-2 in PC12 cells inhibited cell death by withdrawal of trophic support but not oxidative stress.7 The activation of distinct caspases in the same cells thus can promote apoptosis initiated by the various stimuli.7
In the present study, immunohistochemical analysis showed that caspase-1 and caspase-2 were colocalized in the same cell. Furthermore, on immunoblot analysis and measurement of enzymatic activities (Figs. 3 4) , the active forms of caspase-1– and caspase-2–like proteases were shown to be upregulated in P28 RCS rat retinas. These results suggest that caspase-1 and caspase-2 are correlated with the photoreceptor apoptotic process in RCS rats. However, it remains to be determined how these two caspases are activated in photoreceptor degeneration. To estimate the potential role of caspase-1–like protease in photoreceptor apoptosis more directly, we examined in vivo effects of a specific inhibitor of caspase-1 on the number of apoptotic photoreceptors. When administrated into the vitreous, Ac-YVAD-CHO reduced the number of apoptotic cells (Fig. 5) . This suggests that caspase-1–like protease induced photoreceptor apoptosis in the RCS rat and further suggests that an inhibitor of such caspases could inhibit apoptosis in other photoreceptor degenerative diseases. In fact, inhibitors of caspase-1 and caspase-3 can inhibit neuronal apoptosis in brain injury induced by ischemia and by trauma.14
Compared with the number of TUNEL-positive cells, immunopositive cells were fewer in this study. It is difficult to give satisfactory explanation to this apparent discrepancy. However, there are four possible explanations or speculations for this. First, low level expression of apoptosis-related proteins may not be detectable by the immunohistochemical methods used in this study. Second, photoreceptor apoptosis proceeds very slowly in RCS rats. TUNEL-positive cells accounted for as many as one third of the total cell number in the ONL at P28; if these TUNEL-positive cells were digested rapidly by surrounding cells, all the photoreceptors would disappear within several days. However, because photoreceptors survive until P60,2 dying cells may accumulate and remain over a long period in the ONL. Thus, only a few cells that have recently entered the apoptotic process and that overexpress the caspase-like proteases may be present in any individual tissue sections. A third possibility is that numerous other mechanisms may be involved in the apoptotic process in RCS rats, and we may be aware of only a small portion of this apoptotic process. For example, caspases other than caspase-1 and caspase-2 may play a more important role in the apoptotic process of the RCS rat retina. Finally, it is likely that the persistence of apoptotic nuclei for long periods reflects a failure of phagocytosis.
To our knowledge, this is the first report of a correlation between photoreceptor apoptosis and the caspase family. Although this study suggests an important role for caspase-1 and caspase-2 in the process of photoreceptor apoptosis in the RCS rat retina, further studies are necessary to clarify the molecular mechanism underlying retinal degeneration in these animals.
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Footnotes |
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Supported by Grant-in-Aid 10470567 (NY) from the Ministry of Education, Culture, Sports and Science of the Japanese Government.
Submitted for publication June 24, 1998; revised October 28, 1998; accepted December 17, 1998.
Proprietary interest category: N.
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References |
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TopAbstractIntroductionMaterials and MethodsReferences |
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