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The Journal of Neuroscience, June 15, 1998, 18(12):4656-4662
Inhibition of CPP32-Like Proteases Rescues Axotomized Retinal
Ganglion Cells from Secondary Cell Death In Vivo
Pawel
Kermer,
Nikolaj
Klöcker,
Monika
Labes, and
Mathias
Bähr
Department of Neurology, Universität Tübingen, 72076 Tübingen, Germany
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ABSTRACT |
The majority of retinal ganglion cells (RGCs) degenerate and die
after transection of the optic nerve (ON) in the adult rat. This
secondary cell death can primarily be ascribed to apoptosis. Recent
work strongly suggests a decisive role for a family of cysteine
proteases, termed caspases, as mediators of neuronal apoptosis. In this
study, we investigated whether activation of caspases contributes to
delayed death of RGCs after axotomy. Intraocular application of various
caspase inhibitors rescued up to 34% of RGCs that would otherwise have
died 14 d after ON transection. Using a modified affinity-labeling
technique, we detected a 17 kDa protease subunit upregulated after
axotomy. Upregulation was prevented by caspase inhibitor treatment. The
17 kDa protein was identified as a CPP32-like protease by Western blot
analysis and affinity labeling with biotinylated
acetyl-Asp-Glu-Val-Asp-aldehyde, which specifically inhibits CPP32-like
caspases. In vivo application of the irreversible
caspase inhibitor
benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethylketone revealed
CPP32-like proteases to be major mediators of caspase-induced apoptosis
in axotomized RGCs, because this inhibitor showed an even higher
neuroprotective potential than the irreversible wide-range inhibitor
benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone. In
summary, the data presented here provide further insight into the
mechanisms of injury-induced neuronal apoptosis and could give rise to
more effective therapeutic intervention strategies in CNS trauma and
neurodegenerative diseases.
Key words:
caspases; protease-inhibitor; CPP32; apoptosis; retinal
ganglion cells; axotomy; neuroprotection
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INTRODUCTION |
Apoptosis or programmed cell death
(PCD) is a fundamental and essential process in the development and
tissue homeostasis of multicellular organisms. Dysregulation of
apoptosis, however, is involved in various diseases such as
neurodegenerative disorders and neuronal trauma (Thompson, 1995 ;
Vasilakos and Shivers, 1996). Investigation of the mechanisms
underlying apoptosis led to the discovery of various mammalian cysteine
proteases now called caspases (Yuan et al., 1993; Alnemri et al., 1996;
for review, see Cohen, 1997). Interleukin-1 -converting enzyme (ICE)
was first identified as an apoptosis-inducing protease attributable to
significant sequence homology with ced-3, the well known cell death
gene of the nematode Caenorhabditis elegans, which
mediates PCD during development (Yuan et al., 1993). In addition to
ICE, at least nine caspases have been found so far that can be divided
into three subgroups based on phylogenic relationship and sequence homology: ICE-like proteases, CPP32-like proteases, and Nedd2-like proteases (Alnemri et al., 1996; for review, see Cohen, 1997). Recent
data show that caspases are important mediators of neuronal apoptosis
(for review, see Martinou and Sadoul, 1996; Srinivasan et al., 1996;
Keane et al., 1997). Ischemic brain injury results in protease
upregulation (Bhat et al., 1996; Asahi et al., 1997; Kinoshita et al.,
1997), and inhibition of these proteases proved to be neuroprotective
in vitro and in vivo (Milligan et al., 1995; Schulz et al., 1996; Slee et al., 1996; Taylor et al., 1997). Cowpox
ICE-inhibitor CrmA, for instance, protected chicken dorsal root
ganglion cells from death after neurotrophic factor deprivation in vitro (Gagliardini et al., 1994). Moreover, naturally
occurring cell death of developing motoneurons in chicken was prevented by caspase inhibition in vivo (Milligan et al., 1995). In
addition, application of a protease inhibitor also had neuroprotective
effects in ischemic brain damage (Hara et al., 1997a).
In the adult mammalian CNS, fiber tract lesions often induce secondary
degeneration and death of the parental neurons. For example,
transection of the optic nerve (ON) results in delayed death of ~80%
of retinal ganglion cells (RGCs), primarily attributable to apoptosis
(Garcia-Valenzuela et al., 1994; Rabacchi et al., 1994; Isenmann et
al., 1997). Therefore, it is of great interest and importance to
investigate the potential role of caspases in axotomy-induced
apoptosis. In the present report, we studied the in vivo
effects of protease inhibitors on the survival of axotomized RGCs and
analyzed the proteases mediating postlesional apoptosis in greater
detail by affinity labeling.
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MATERIALS AND METHODS |
Optic nerve transection. Surgery was performed as
described in detail elsewhere (Klöcker et al., 1997). Briefly,
adult female Sprague Dawley rats (200-250 gm) purchased from Charles
River Wiga (Sulzfeld, Germany) were anesthetized by intraperitoneal injection of chloral hydrate (0.42 gm/kg body weight). After skin incision close to the superior orbital rim, the orbita was opened, taking care to leave the supraorbital vein intact. After subtotal resection of the lacrimal gland, the superior extraocular muscles were
spread by means of a small retractor. The ON was exposed by
longitudinal incision of the eye retractor muscle and the perineurium. ON transection was performed ~2 mm from the posterior pole of the eye
without damaging retinal blood supply. Animals with persistent retinal
ischemia verified funduscopically were not included in the study.
Retrograde labeling of retinal ganglion cells. To allow
accurate counting of RGCs, cells were retrogradely labeled by the fluorescent tracers
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiI; Molecular Probes, Eugene, OR) and fast blue (FB; Dr. Illing
Chemie, Gross-Umstadt, Germany). The skull cartilage of rat pups
(postnatal day 7) was opened dorsal to the  fissure under
diethylether anesthesia, and both superior colliculi were instilled
with 5% DiI in dimethylformamide using a micropipette. Retrograde
labeling with FB was achieved by placing a small piece of gel foam
soaked in 2% aqueous FB at the ocular stump of the axotomized ON.
Drug administration. The irreversible wide-range protease
inhibitor
benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone
(ZVAD-fmk; Bachem) as well as the reversible and irreversible
inhibitors of CPP32-like caspases acetyl-Asp-Glu-Val-Asp-aldehyde and
benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethylketone (DEVD-CHO and
ZDEVD-cmk, respectively; Bachem) were dissolved in 2%
dimethylsulfoxide (DMSO; Sigma, Deisenhofen, Germany) in PBS. All these
inhibitors effectively block the intracellular activation of caspases,
as shown in several studies on apoptotic cell death in various types of
tissue (Roquet et al., 1996; Schulz et al., 1996; Slee et al., 1996;
Tomita et al., 1996; Armstrong et al., 1997). Whereas peptides with an
aldehyde moiety are reversible protease inhibitors, those with a
fluoromethylketone or chloromethylketone moiety bind to caspases
irreversibly (Armstrong et al., 1997; Cohen et al., 1997; Nicholson et
al., 1997). An additional benzyloxycarbonyl group (indicated as Z in
the above abbreviations) further improves their cell permeability (Zhu
et al., 1995; Armstrong et al., 1996; Slee et al., 1996; for review,
see Cohen, 1997).
For intraocular injection of inhibitors or vehicle (2% DMSO in PBS),
animals were anesthetized with diethylether. By means of a glass
microelectrode with a tip diameter of 30 µm, 2 µl of caspase
inhibitors or 2 µl of vehicle was injected into the vitreous space,
puncturing the eye at the cornea-sclera junction.
We used two different doses of ZVAD-fmk (400 or 4000 ng/injection,
which for all the inhibitors used resulted in final vitreous body
concentrations slightly above those used in vitro), each in
two different regimens for ZVAD-fmk-vehicle treatment: animals either
received (1) three intraocular injections of ZVAD-fmk-vehicle on days
4, 7, and 10 after axotomy or (2) four intraocular injections on days
0, 4, 7, and 10 after axotomy.
In another set of experiments we injected 4000 ng of DEVD-CHO or
ZDEVD-cmk, both of which are protease inhibitors specific for
CPP32-like caspases, on days 0, 4, 7, and 10 after axotomy (treatment
2).
Animals receiving 4000 ng Z-Phe-cmk served as additional controls.
Tissue processing. Fourteen days after ON transection,
animals received an overdose of chloral hydrate, and the eyes were removed. The retinae were dissected, flat-mounted on gelatin-coated glass slides, and fixed for 20 min in 4% paraformaldehyde in PBS. RGCs
were examined under the fluorescence microscope (Axiovert 35; Zeiss)
using a rhodamine filter (546/590 nm) for DiI fluorescence and a DAPI
filter (365/420 nm) for FB fluorescence, respectively. The number of
DiI- or FB-positive RGCs was determined by counting them in 12 distinct
areas of 62,500 µm2 each (three areas per retinal
quadrant at three different eccentricities of one-sixth, one-half, and
five-sixths of the retinal radius). Cell counts were performed
according to a double-blind protocol and by two different
investigators. Overestimation of RGC density by retinal shrinkage
attributable to intraocular injection was excluded (Klöcker et
al., 1998).
Affinity labeling. For this set of experiments retinae were
explanted on day 4 after lesion after the eyes had received an injection of 4000 ng of ZVAD-fmk on days 0 and 4 after axotomy. They
were homogenized immediately in ice-cold lysis buffer containing 150 mM NaCl, 50 mM Tris, pH 8.0, 2 mM
EDTA, and 1% Triton X-100 containing 0.1 mM PMSF and 2 µg/ml pepstatin, leupeptin, and aprotinin. The cell suspension was
lysed on ice for 20 min, and cell debris was pelleted for 15 min at
14,000 × g. The protein concentration of the
supernatant was determined using the BCA reagent (Pierce, Rockford,
IL), and 200 ng of protein was subjected to affinity labeling with
biotinylated protease inhibitors.
Biotin-Ac-Tyr-Val-Ala-Asp-chloromethylketone (bio-YVAD-cmk; Bachem) and
biotin-Ac-Asp-Glu-Val-aspartic acid aldehyde (bio-DEVD-CHO; Bachem)
were added to a final concentration of 20 nM, and the
suspension was incubated at room temperature (RT) for 30 min with
constant agitation. Labeled proteins were then affinity-purified on
streptavidin (SA) M-280 dynabeads (diameter, 2.8 µm; density, 1.3 gm/cm; concentration, 10 µg/µl; Dynal, Oslo, Norway). Twenty
microliter dynabeads were washed once with binding and washing buffer
(BWB; 5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, and 1 M NaCl) and added to the lysate, which had been adjusted to 1× BWB by adding 1 volume of 2× BWB. The mixture was again incubated at RT for 30 min with constant shaking. Bound proteins were washed four
times with BWB using a magnetic separator, resuspended in 1×
denaturing protein loading buffer, and subjected to SDS-PAGE.
Western blotting. After separation by SDS-PAGE (Ausubel et
al., 1987) of either lysate (50 µl) or purified, affinity-labeled samples, proteins were transferred to a nitrocellulose or
polyvinylidene difluoride (PVDF) membrane and blocked with 5% skim
milk in PBS-Tween 20 (PBS-T; 0.1% Tween 20). Labeled proteins were
detected by incubating with SA-HRP (1:5000 in PBS-T) for 1 hr, washing
three times in PBS-T, and applying the ECL-Plus reagent (Amersham,
Arlinghton Heights, IL) according to the supplier's instructions.
In a second set of experiments retinae were homogenized as described
above. Lysates were extracted, and 50 µg of protein was separated on
a 12% SDS gel without previous inhibitor incubation. Proteins were
transferred on PVDF membranes as described above and incubated with
rabbit-anti-CPP32 (1:2000; 3 hr at RT; Upstate Biotechnology, Lake
Placid, NY) followed by goat anti-rabbit HRP (1:2000; 1 hr at RT).
Statistics. Data are given as mean ± SEM. Statistical
significance was assessed using one-way ANOVA followed by the Duncan test. For expressing RGC survival-promoting effects of protease inhibitors, we defined RGC rescue rate (RRR; Klöcker et al., 1997): RRR = (Nther  Ncon)/(Ntot Ncon) × 100, where
Ntot is the number of RGCs in unlesioned
retinae, Ncon is the number of RGCs surviving
without therapy, and Nther is the number of RGCs surviving after a given therapy.
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RESULTS |
Axotomy-induced retrograde death of RGCs
In unlesioned control retinae, retrograde labeling of RGCs from
the superior colliculi by the fluorescent tracer DiI resulted in a
typical fine-dotted pattern of fluorescence of the RGC perikarya (Klöcker et al., 1997). The mean RGC density was 2080 ± 69 cells/mm2. This number is in agreement with cell
densities observed by other investigators (Eschweiler and Bähr,
1993; Mansour-Robaey et al., 1994). Within 14 d after ON
transection, it decreased to 17% of control, as determined by
retrograde FB labeling from the axon stump, which displayed a uniform
labeling throughout the retina as observed with DiI but a more diffuse
staining of perikarya (Table 1, Fig.
1). As has been observed earlier,
lesion-induced RGC death was more pronounced at the inner retinal
radius with 14% surviving RGCs compared with 24% of control at the
outer retinal radius (Klöcker et al., 1997). RGCs appeared
shrunken and hardly exposed any dendritic processes (Fig.
1a). Comparison of DiI and FB data are possible because it
has been shown that both dyes have a similar label efficacy (Eschweiler
and Bähr, 1993). Animals with persistent retinal ischemia or
lacking uniform labeling of RGCs throughout the retina were excluded
from the study.
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Table 1.
Neuroprotective effects of intraocular treatment with
various caspase inhibitors on RGCs 14 days after ON transection in the
adult rat
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Figure 1.
Representative micrographs of flat-mounted retinae
at corresponding areas of one-half of the retinal radius 14 d
after ON transection. RGCs are labeled retrogradely with the
fluorescent tracer fast blue. a, Axotomy without
treatment. b, Injection of 4000 ng of ZVAD-fmk on days
0, 4, 7, and 10 after lesion. c, Injection of 4000 ng of
ZDEVD-cmk on days 0, 4, 7, and 10 after lesion. Note the higher density
of RGCs in c when compared with a and
b. Scale bar, 100 µm.
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Effects of ZVAD-fmk on survival of axotomized RGCs
Whereas three intraocular injections of vehicle did not
significantly influence the survival of axotomized RGCs, three
intraocular injections of 400 ng of ZVAD-fmk on days 4, 7, and 10 after
lesion led to a significantly higher mean RGC density with an RRR value of 12% (Table 1, Fig. 1). This rescue effect could not be improved by
raising the dose of ZVAD-fmk to 4000 ng (RRR 12%). However, starting
ZVAD-fmk treatment immediately after surgery (4 intraocular injections
on days 0, 4, 7, and 10 after axotomy) significantly increased the RRR
to 17% at 400 ng/injection and to 22% at 4000 ng/injection, again
without statistical difference between the two doses. An additional
vehicle injection had no effect (data not shown). The dependence of RGC
rescue by ZVAD-fmk on the different treatment regimens is illustrated
in Figure 2. In comparison with axotomy
without therapy, many labeled RGCs appeared healthy and displayed a
normal shape and dendritic arborization (Fig. 1b).
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Figure 2.
Rescue effects of ZVAD-fmk and ZDEVD-cmk treatment
on axotomized RGCs 14 d after ON transection. Three intraocular
injections on days 4, 7, and 10 after axotomy (white
bars) of either 400 or 4000 ng of ZVAD-fmk showed identical
survival-promoting effects on lesioned RGCs when compared with
untreated axotomy. An additional injection on day 0 after axotomy
(gray bars) significantly improved the
postlesional survival of RGCs. Treatment with 4000 ng of ZDEVD-cmk on
postlesional days 0, 4, 7, and 10 resulted in a RGC rescue rate of
34%, which was statistically significant when compared with four
injections of ZVAD-fmk (gray bars). RRR = (Nther Ncon)/(Ntot Ncon) × 100, where
Ntot is the number of RGCs in unlesioned
retinae, Ncon is the number of RGCs
surviving without therapy, and Nther is the
number of RGCs surviving after a given therapy. Data are given as
mean ± SEM. Asterisks indicate significant
differences between intraocular injection on days 4, 7, and 10 and
additional injection on day 0 after ON transection.
p < 0.05.
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Activation of CPP32 and CPP32-like caspase after
ON transection
To identify potential active proteases involved in the death of
axotomized RGCs, we modified the affinity-labeling technique developed
by Thornberry et al. (1994). Retinae were lysed on day 4 after ON
transection and incubated with the biotinylated protease inhibitors
YVAD-cmk or DEVD-CHO. With magnetic streptavidin beads it was possible
to extract biotin-labeled active caspase subunits from total protein
lysates before separation on SDS-PAGE.
Using bio-YVAD-cmk (Fig. 3a)
we detected a labeled protein migrating at the molecular weight of 17 kDa. After axotomy, the amount of that protein was increased in
comparison with unlesioned control retinae. By intraocular injection of
4000 ng of ZVAD-fmk on days 0 and 4 after lesion this upregulation
could be prevented (Fig. 3a). One known caspase that is
cleaved to 17 and 12 kDa subunits, respectively, is CPP32 (Enari et
al., 1996). Although YVAD-cmk is a potent inhibitor of ICE-like
caspases (KiICE, 0.76 nM;
Rotonda et al., 1996), no cleavage product of ICE was detectable in any
of our experiments.
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Figure 3.
Affinity labeling of activated retinal proteases
14 d after ON transection. a, Affinity labeling
with biotinylated YVAD-cmk. Note the upregulation of an active protease
with a molecular weight of 17 kDa after axotomy. Under treatment with
ZVAD-fmk this upregulation is clearly inhibited. b,
Affinity labeling with biotinylated DEVD-CHO, a specific inhibitor for
CPP32-like caspases. Again, one can detect the identical regulation of
an active protease at 17 kDa as described in a.
c, Western blot with an antibody against CPP32. Note the
identical regulation of active CPP32 as described in a
and b. ctrl, Untreated control;
axo, axotomy without treatment; ZVAD,
injection of 4000 ng of ZVAD-fmk on days 0 and 4 after axotomy.
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In another set of experiments we used a biotinylated derivate of
DEVD-CHO (Fig. 3b), a protease inhibitor specific for
CPP32-like caspases (KiCPP32, 0.35 nM; Rotonda et al., 1996). Again we observed a change in
the amount of a 17 kDa protease subunit as seen with bio-YVAD-cmk (Fig.
3a), supporting the hypothesis of an involvement of CPP32
and CPP32-like caspases in cell death after axotomy.
Indeed, active CPP32 could be detected by a specific antibody that
detected the same purified 17 kDa band after membrane stripping (data
not shown), as well as in Western blotting experiments with fresh
lysates. As shown in Figure 3c, axotomy resulted in an
increase of the 17 kDa fragment on blots treated with anti-CPP32. In
agreement with the affinity-labeling experiments, this upregulation was blocked to a great extent by caspase inhibition with ZVAD-fmk. Again,
there was a baseline activity of CPP32 in unlesioned controls. These
results further underline the importance of CPP32 and CPP32-like caspases for neuronal apoptosis after axotomy in vivo.
Specific inhibition of CPP32-like caspases promotes survival of
axotomized RGCs
After having demonstrated an axotomy-induced activation of CPP32
and CPP32-like caspases, we were interested in the neuroprotective effect of CPP32-inhibitors in vivo. In fact, intraocular
injection of DEVD-CHO significantly promoted the survival of axotomized RGCs. Four injections (on days 0, 4, 7, and 10 after ON transection) of
4000 ng of DEVD-CHO rescued 20% of the RGCs that would otherwise have
died after axotomy (Table 1). This rescue rate did not differ significantly from the RRR achieved by ZVAD-fmk after the identical treatment protocol.
Under identical experimental paradigms, four injections of ZDEVD-cmk,
an irreversible inhibitor of CPP32-like caspases, resulted in a further
improvement of rescue effects. Compared with ZVAD-fmk or DEVD-CHO
treatment the number of surviving RGCs increased significantly with a
rescue rate of 34% (Table 1; Figs. 1c, 2).
To exclude potential rescue effects caused by halogenic methylketones,
we applied Z-Phe-cmk in an additional set of control experiments. In
agreement with Armstrong et al. (1997) no significant neuroprotective
effect could be observed under these conditions when compared with
axotomy without treatment.
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DISCUSSION |
In a number of studies, caspases have been shown to play a
decisive role in mediating neuronal apoptosis (for review, see Martinou
and Sadoul, 1996). We demonstrated for the first time that inhibition
of caspases can rescue RGCs from secondary death after axonal lesion.
Moreover, we identified CPP32-like proteases as major mediators of
caspase-regulated apoptosis in axotomized RGCs.
Because of good surgical accessibility, the retino-tectal projection
in the rat serves as a convenient model to study degenerative and
regenerative processes in the mammalian CNS (Villegas-Perez et al.,
1988; Mey and Thanos, 1993; Bähr and Bonhoeffer, 1994; Bähr
and Wizenmann, 1996). Axotomy of all RGCs by ON transection results in
the delayed death of ~80% of RGCs (Eschweiler and Bähr, 1993; Mansour-Robaey et al., 1994; Cui and Harvey, 1995). Experimental evidence indicates that this secondary death can be ascribed primarily to apoptosis (Garcia-Valenzuela et al., 1994; Rabacchi et al., 1994;
Isenmann et al., 1997), which can be prevented in adult mice
overexpressing the antiapoptotic proto-oncogene bcl-2 (Cenni et al.,
1996).
Recent in vivo data demonstrate that inhibition of caspases,
which are activated in ischemic brain injury (Bhat et al., 1996; Asahi
et al., 1997; Kinoshita et al., 1997), can protect the affected neurons, thus possibly representing a new therapeutic option in neuronal trauma (Hara et al., 1997a). To test whether similar mechanisms underlie neuronal apoptosis induced by axonal injury, after
ON transection in rats we applied the irreversible wide-range protease
inhibitor ZVAD-fmk, which has a good cell permeability attributable to
its benzyl-oxycarbonyl group (Zhu et al., 1995; Cohen, 1997) and is
proven to support RGC survival in vitro (Chen et al., 1997).
In the first approach, we designed a treatment regimen consisting of
three single intraocular injections on days 4, 7, and 10 after lesion
because (1) apoptotic death of axotomized RGCs can first be detected at
day 4 and reaches a maximum at day 7 after axotomy (Isenmann et al.,
1997); and (2) other neuroprotectants antagonizing apoptosis, such as
brain-derived neurotrophic factor, show the same rescue effects when
injected on day 0, 3, or 5 after lesion (Mansour-Robaey et al., 1994).
ZVAD-fmk treatment, starting on day 4 after surgery, led to a
significant increase in the number of surviving RGCs when compared with
axotomized but untreated or sham-treated retinae. In another set of
experiments, ZVAD-fmk was additionally injected immediately after
axotomy, resulting in a further significant increase in RRR to 22%.
This suggests an early activation of proteases during the first 4 d after ON transection, which is supported by in vitro data
showing an activation of caspases within hours after the apoptotic
stimulus (Faleiro et al., 1997; Harvey et al., 1997; Keane et al.,
1997).
To characterize the proteases activated after ON transection, we used
bio-YVAD-cmk, which preferentially binds subunits of ICE-like proteases
in a modified version of the affinity-labeling technique first
developed by Thornberry et al. (1994). As has been observed in other
models of neuronal apoptosis (Srinivasan et al., 1996; Keane et al.,
1997), we detected an active protease with a molecular weight of ~17
kDa that was clearly upregulated after axotomy. ZVAD-fmk treatment
could inhibit this upregulation, suggesting a specific mode of action
for this protease inhibitor. To date, the only caspase known to be
cleaved into 17 and 12 kDa subunits is CPP32 (Enari et al., 1996; Keane
et al., 1997). Therefore, we repeated the affinity-labeling experiments
using bio-DEVD-CHO, which specifically binds activated CPP32-like
proteases (Enari et al., 1996). Under the same experimental conditions,
bio-DEVD-CHO also detected the upregulation of a 17 kDa protein after
axotomy, as did bio-YVAD-cmk. Moreover, using an antibody against
CPP32, we were able to demonstrate activation of CPP32 after lesion. From the data of the affinity labeling and Western blotting, it is not
possible to identify the retinal cell type in which CPP32 and/or
CPP32-like caspases are activated. Still, the rescue of axotomized RGCs
in vivo by caspase inhibition strongly suggests caspase
activation in RGCs.
Our findings are in agreement with results derived from ischemic brain
lesions in which an important role for CPP32-like proteases has been
shown (Asahi et al., 1997; Hara et al., 1997a). The dramatic developmental malformations in the CNS of CPP32 knock-out mice, such as
excess cell masses found in several regions of the brain, including the
retina, suggest a central role of CPP32 in neuronal apoptosis (Kuida et
al., 1996). ICE and ICE-like caspases, however, seem to play only a
minor (Asahi et al., 1997) or indirect role (Bhat et al., 1996; Hara et
al., 1997b), a view that is supported by the apparently normal
phenotype of mice lacking the ICE gene (Kuida et al., 1995; Li et al.,
1995). The hypothesis that ICE-like proteases are not essential in
neuronal apoptosis would be in agreement with our data, because we
failed to detect any known cleavage products of ICE-like proteases in
our affinity-labeling experiments. This is all the more surprising,
because we first used bio-YVAD-cmk, which has an affinity >10,000-fold
for ICE-like proteases compared with CPP32-like caspases (Rotonda et
al., 1996; see Results). However, we cannot exclude the possibility
that our method was not sensitive enough to detect proteases other than
those from the CPP32-like family. It is also possible that there are
proteases apart from CPP32 that are cleaved to a 17 kDa product,
detectable with bio-YVAD-cmk and bio-DEVD-CHO. Using an antibody
against CPP32 in Western blot experiments, however, it was possible to
identify CPP32 as a regulated caspase. To further clarify the role of
CPP32-like proteases in our model, we compared the neuroprotective
potential of the wide-range protease inhibitor ZVAD-fmk with the
specific CPP32-like caspase inhibitors DEVD-CHO and ZDEVD-cmk in
vivo. DEVD-CHO rescued approximately the same number of axotomized
RGCs from cell death as ZVAD-fmk, whereas intraocular injection of the
irreversible inhibitor ZDEVD-cmk further enhanced the rescue rate.
These results support a central role of CPP32 and CPP32-like proteases
in our lesion model.
It is worth noting that we detected a baseline expression of activated
caspases in unlesioned control retinae. Because the complete range of
substrates processed by caspases is not yet known, we hypothesize that
caspases could also be active in cell metabolism under physiological
conditions. An alternative explanation would be the immediate
activation of caspases during eye removal and retina preparation,
independent of the respective experimental group.
Overall, complete rescue of axotomized RGCs could not be achieved by
either ZVAD-fmk or ZDEVD-cmk treatment. Suboptimal dosage of the
protease inhibitors is unlikely, because 400 and 4000 ng injections
rescued the same number of RGCs, indicating inhibitor saturation. It is
more likely that multiple pathophysiological pathways underlie
secondary death of RGCs after axotomy. Of course, caspases are not the
only known mediators of neuronal apoptosis. Pathways leading to the
activation of protein kinases and transcription factors resulting in
subsequent gene induction that can also give rise to apoptosis are
reviewed by Lavin et al. (1996). In this context it is worthwhile to
note that the apoptosis-related gene bax is upregulated in RGCs after
transection of the rat ON within hours (Isenmann et al., 1997).
Glutamate-dependent neurotoxicity (excitotoxicity) (Choi, 1988; Lipton
and Rosenberg, 1994), which might be at least in part independent of
caspase activation, also contributes to delayed death of axotomized
neurons as has been shown for cat RGCs (Russelakis-Carneiro et al.,
1996). Considering the complex and multiple pathways leading to
apoptosis, it is not surprising that factors showing a neuroprotective
effect in our model failed to rescue the total RGC population when
applied alone. Treatment strategies that combine neuroprotectants
antagonizing different presumptive pathways, which result in secondary
death, may prove to be much more effective (Klöcker et al.,
1998).
One point that remains elusive in this context is whether there is a
long-term effect of caspase inhibition on the survival of axotomized
RGCs. For BDNF, it has been shown that short-term therapy is not
sufficient to guarantee long-lasting RGC survival after ON transection
(Mey and Thanos, 1993; Mansour-Robaey et al., 1994). Thus, BDNF might
only delay secondary cell death. Whether this holds true for
irreversible caspase inhibitors as well or whether they are capable of
actually preventing apoptosis and supporting neuronal long-term
survival is not yet known.
In summary, we demonstrate here for the first time that CPP32-like
caspases are major mediators of apoptosis in RGCs after ON transection
and that inhibition of these proteases in our distinct model of
neuronal trauma increases the number of surviving neurons after axonal
lesion. Together with recent findings in other traumatic CNS injury
paradigms (Yakovlev et al., 1997), these results provide evidence for
the activation of CPP32-like caspases as a general mechanism of
neuronal apoptosis. Consequently, inhibition of CPP32-like caspases
might be a very promising strategy in the treatment of various forms of
brain injury, including cerebral trauma and neurodegenerative diseases.
| |
FOOTNOTES |
Received Oct. 2, 1997; revised Feb. 18, 1998; accepted March 26, 1998.
This work was supported by grants from the Bundesministerium für
Bildung, Wissenschaft, Forschung und Technologie (Neurotraumatologie to
M.B. and 01KS 9602 to N.K.). We thank S. Thomsen for technical assistance and M. Rott and J. B. Schulz for critical readings of
this manuscript.
Correspondence should be addressed to Mathias Bähr, Department of
Neurology, Universität Tübingen, Hoppe-Seyler-Strasse 3, 72076 Tübingen, Germany.
| |
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CALEB Binds via Its Acidic Stretch to the Fibrinogen-like Domain of Tenascin-C or Tenascin-R and Its Expression Is Dynamically Regulated after Optic Nerve Lesion
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Top
Abstract
Introduction
