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The Journal of Neuroscience, July 1, 2000, 20(13):5037-5044
Role of p38 Mitogen-Activated Protein Kinase in Axotomy-Induced
Apoptosis of Rat Retinal Ganglion Cells
Masashi
Kikuchi1, 2,
Lalitha
Tenneti2, and
Stuart A.
Lipton1, 2
1 Center for Neuroscience and Aging, The Burnham
Institute, La Jolla, California 92307, and 2 CNS
Research Institute, Brigham and Women's Hospital, Division of
Neuroscience, Children's Hospital, and Program in Neuroscience,
Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
p38 is a member of the mitogen-activated protein (MAP) kinase
superfamily and mediates intracellular signal transduction. Recent
studies suggest that p38 is involved in apoptotic signaling in several
cell types, including neurons. In the mammalian retina, ~50% of the
retinal ganglion cells (RGCs) die by apoptosis during development.
Additionally, transection of the optic nerve close to the eye bulb
causes apoptotic cell death of RGCs in adulthood. We investigated the
role of p38 in axotomy-induced apoptosis of RGCs. One day after
axotomy, activated (phosphorylated) p38 was visualized by
immunocytochemistry in the nuclei of RGCs, but not in control retinas.
Phosphorylated p38 was first detected on immunoblots 12 hr after
axotomy, reached a maximum at 1 d, and then decreased. To
investigate possible roles of p38 in RGC death, a p38 MAP kinase inhibitor, SB203580, was administered intravitreally at the time of
axotomy and repeated at 5 and 10 d. Assayed 14 d after
axotomy, SB203580 increased the number of surviving RGCs in a
dose-dependent manner (the minimum effective concentration was 1.6 µM). Furthermore, MK801, a selective inhibitor of NMDA
receptors, not only showed protective effects against RGC apoptosis but
also attenuated p38 MAP kinase activation in a dose-dependent manner.
Our findings imply that p38 is in the signaling pathway to RGC
apoptosis mediated by glutamate neurotoxicity through NMDA receptors
after damage to the optic nerve. p38 inhibitors could be potentially
useful for the treatment of optic nerve trauma and neurodegenerative diseases that affect RGCs, such as glaucoma.
Key words:
mitogen-activated protein kinase; p38; axotomy; retinal
ganglion cells; apoptosis; glutamate; NMDA; optic nerve
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INTRODUCTION |
Mitogen-activated protein (MAP)
kinases are serine/threonine kinases and play an instrumental role in
signal transduction from the cell surface to the nucleus. The mammalian
MAP kinases can be subdivided into extracellular signal-regulated
kinases (ERK), Jun N-terminal kinases (JNK), and p38 MAP kinases (p38). p38 is activated by dual phosphorylation on a Thr-Gly-Tyr motif in
response to environmental stress that can also activate JNK. Such
stress includes UV radiation, endotoxin, and hyperosmolarity, as well
as proinflammatory cytokines like tumor necrosis factor and
interleukin-1 (Freshney et al., 1994 ; Han et al., 1994; Lee et al.,
1994; Rouse et al., 1994; Raingeaud et al., 1995). ERK is activated by
mitogens and survival factors (Cano and Mahadevan, 1995; Cobb and
Goldsmith, 1995). Therefore, p38 and JNK are known as stress-activated
protein kinases. Recently, p38 activation has also been implicated in
mediating apoptosis in several cell types in various species (Kawasaki
et al., 1997; Kummer et al., 1997; Horstmann et al., 1998;
Castagné and Clarke, 1999). However, the role of p38 in apoptotic
pathways differs in a cell-type- and stimulation-dependent manner.
In the mammalian retina, bovine retinal pigment epithelial cells
manifest p38 immunoreactivity, which is activated by lipopolysaccharide
or interferon- (Faure et al., 1999), but the localization and role
of p38 in neurons of the mammalian retina remains unclear.
In mammalian retinal ganglion cells (RGCs), as well as in other CNS
neurons, axotomy close to the cell body causes retrograde degeneration
and delayed RGC death (Aguayo et al., 1983; Villegas-Perez et al.,
1988; Thanos et al., 1993). This form of cell death appears to be
apoptotic in nature, similar to programmed cell death during retinal
development (Berkelaar et al., 1994; Garcia-Valenzuela et al., 1994;
Isenmann et al., 1997). It has been suggested that optic
nerve-axotomized RGCs die because of deprivation of neurotrophic factors from the superior colliculus. Exogenous application of neurotrophic factors such as BDNF and NGF are known to enhance RGC
survival after axotomy (Mey and Thanos, 1993; Peinado-Ramon et al.,
1996; Klocker et al., 1997; Yan et al., 1999). Recently, other studies
have reported that the intraocular level of glutamate is elevated after
optic nerve axotomy (Yoles and Schwartz, 1998), and abnormal activation
of glutamate receptors is involved in axotomy-induced RGC apoptosis
(Russelakis-Carneiro et al., 1996; Yoles et al., 1997). Here we
demonstrate that p38 is activated in RGCs after optic nerve axotomy and
that this activation is in the signaling pathway to RGC apoptosis
mediated by NMDA receptor-dependent neurotoxicity.
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MATERIALS AND METHODS |
Retrograde labeling of retinal ganglion cells. Adult
male Long-Evans rats weighing 200-250 gm were obtained from a local
breeder and, for all experimental manipulations, were anesthetized with 1-2% isoflurane and 70% N2O. RGCs were
retrogradely labeled with FluoroGold (hydroxystilbamidine; Molecular
Probes, Eugene, OR) to allow accurate counting of cell bodies, as
previously described (Vorwerk et al., 1999a,b). Rats were anesthetized
and placed in a small stereotactic instrument. The skull was exposed
and kept dry. The bregma was identified and marked, and a small window was drilled above the right hemisphere, leaving the dura intact. Using
a stereotactic measuring device and a Hamilton injector, hydroxystilbamidine solution was injected into four (4) regions of the
right superior colliculus using the following coordinates from the
bregma (anteroposterior, mediolateral, and depth, all listed in
millimeters): (1)  5.8, +1.0, 4.4; (2) 6.5, +0.7, 4.0; (3)
6.5, +1.0, 4.0; and (4) 7.3, +1.5, 3.7.
Axotomy. Optic nerve axotomy was performed on the left eye
4 d after retrograde labeling. After incision of the dorsolateral conjunctiva, the lateral extraocular muscle was transected, and the
optic nerve was exposed under a stereoscopic microscope. The optic
nerve was then transected at a distance of ~1 mm from the eye bulb.
During the operation, care was taken to avoid damage to the retinal
blood supply.
Drug application. Intravitreal injections were performed
using a 33 gauge needle attached to a 25 µl syringe after pupil
dilation with 1% atropine sulfate. The tip of the needle was inserted
through the dorsal limbus of the eye under stereomicroscopic
visualization. Injections were completed over a period of 1 min.
Intravitreal injections of SB203580 (Calbiochem, San Diego, CA), MK801
(Research Biochemicals International, Natick, MA) or control solutions
(as an equal volume of saline diluent) were performed on operated eyes
immediately after axotomy, and repeated 5 and 10 d after axotomy.
Quantification of axotomized RGC survival and histology. At
various time points, rats were given an overdose of pentobarbital, and
the eyes were removed. The retina was carefully dissected from the eye,
prepared as a flat whole-mount in a 4% paraformaldehyde solution, and
examined for stained ganglion cells by epifluorescence microscopy to
determine the density. The number of surviving RGCs in experimental and
control retinas was determined by counting hydroxystilbamidine-labeled
neurons in three standard areas of each retinal quadrant at one-sixth,
one-half, and five-sixths of the retinal radius, for a total area of
2.25 mm2, as previously described by
Kermer et al. (1998). RGC survival data from each group of animals is
presented as the mean density (RGCs/mm2)
and SD for n = 3-6 retinas. Statistical significance
of the data were determined by an ANOVA followed by a post
hoc Dunnett's test. For the histological studies, retinal
whole-mounts were prepared and stained by the method of Nissl using
cresyl violet (0.1%).
Immunohistochemistry. After enucleation, the eyes were
immersed in fixative composed of 4% paraformaldehyde in PBS, pH
7.4, at 4°C. Ten minutes later, the eyes were hemisected in the
fixative, and the anterior segment, lens, and vitreous body were
discarded. The remaining posterior eyecup was kept in fresh fixative
solution overnight at 4°C. The eyecup was then embedded in paraffin.
Sections 5 µm in thickness were cut on a microtome and transferred
onto gelatin-coated glass slides. After rehydration, sections were treated for 30 min at room temperature with methanol to increase membrane permeability, followed by 4% hydrogen peroxide for 1 hr to
block intrinsic peroxidase activity. Then, sections were incubated with
20% normal goat serum (NGS) for 1 hr. After rinsing, the sections were
incubated overnight at 4°C in PBS with 0.3% Triton X-100, 1% NGS,
and one of the following specific antibodies: 1:1000 anti-p38
antibody (Santa Cruz Biotechnology, Santa Cruz, CA), 1:250
anti-phospho-specific p38 (New England BioLabs, Beverly, MA), or 1:50
anti-rat ED1 antibody (Serotec, Oxfordshire, UK). The sections were
then incubated with biotinylated anti-IgG (Sigma, St. Louis, MO) for 2 hr at room temperature. Color development was performed with a Vector
AEC substrate kit (Vector Laboratories, Burlingame, CA) or with a Sigma
Fast DAB kit. When immunoreactivity was exclusively localized to the
nucleus (see Fig. 5C), counterstaining was needed to define
the cell somata, and Meyer Hematoxylin was used.
Immunoblotting. Retinas were homogenized in SDS
sample buffer (2% SDS, 0.6% 2-mercaptoethanol, 10% glycerol, and 50 mM Tris, pH 7.2) and centrifuged. After estimation of
supernatant protein concentration with a Bio-Rad (Hercules, CA) Protein
Assay, aliquots containing 70 µg of protein were separated by
SDS-PAGE and transferred onto a nitrocellulose membrane (Hybond ECL,
Amersham Pharmacia Biotech). The membranes were then blocked with 25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.68 mM KCl, and 0.1% Tween 20 containing 5% nonfat milk for 1 hr at room temperature. Membranes were probed with 1:1500 anti-p38
antibody, 1:200 anti-p38 antibody (Santa Cruz Biotechnology), or
1:500 anti-phospho-specific p38 according to the instructions of the
manufacturer. The antibody-reactive bands were visualized by
chemiluminescent detection (ECL western detection kit; Amersham
Pharmacia Biotech).
Retinal cell cultures. Retinal cells were prepared from 6- to 10-d-old Long-Evans rats as described previously (Leifer et al.,
1984). Briefly, after dissociation in papain, retinal cells were plated
on poly-L-lysine-coated glass coverslips in Eagle's minimum essential medium. RGCs were identified by immunocytochemical staining using an anti-Thy-1 antibody (2G12), which is specific among
rat retinal cells for the ganglion cells (Leifer et al., 1984).
Assessment of NMDA-induced apoptosis in cultured RGCs. To
induce predominantly apoptosis (Bonfoco et al., 1995; Dreyer et al.,
1995), retinal cell cultures were exposed to 200 µM
NMDA/5 µM glycine for 18 hr in high calcium (3 mM) medium. For specific labeling of RGCs, the cultures
were incubated with anti-Thy-1 antibody for 1 hr. After three washes
with PBS, cells were incubated in goat anti-mouse IgG-FITC for 1 hr.
For assessment of apoptosis, retinal cells were fixed, permeabilized,
and stained with 20 µg/ml propidium iodide for 5 min, as previously
described (Ankarcrona et al., 1995). Briefly, coverslips containing the
cells were washed once with PBS and permeabilized with 85% methanol
for 10 min. After another wash with PBS, coverslips were fixed in
acetone for 5 min and subsequently stained with propidium iodide for 5 min in the dark. The coverslips were then mounted on glass slides in
glycerol:PBS (1:1), and visualized under epifluorescence microscopy. Apoptotic nuclei were scored in cells that were also stained by anti-Thy-1 and expressed as a fraction of total RGCs.
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RESULTS |
Effects of axotomy on RGCs
RGCs labeled retrogradely with hydroxystilbamidine showed a
characteristically fine-dotted pattern of fluorescence in the perikarya
(Fig. 1A). One day
after axotomy, no remarkable change in the fluorescence pattern of RGCs
was seen compared to that of controls (Fig. 1B). RGC
density, as assessed by the number of fluorescently labeled cells,
began to decline between 4 and 7 d after axotomy (Fig.
1C). By 14 d, a substantial number of fluorescently
labeled RGCs appeared to have been lost (Fig. 1D). Additionally, at 7-14 d after axotomy, we observed a number of other
cells labeled with hydroxystilbamidine in the ganglion cell layer (GCL)
and other retinal layers, although the pattern of fluorescence was
quite different from that of RGCs (Fig. 1C,D, white
arrows). These cells had small somata and fine tortuous processes,
and were morphologically typical of microglia (for specific
immunostaining, see below). In the adult rat retina, microglial cells
phagocytose the debris of degenerating RGCs (Perry et al., 1983) and
thus can become fluorescently labeled (Thanos et al., 1992, 1993).
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Figure 1.
Representative photographs of flat-mounted
retinas. Hydroxystilbamidine-labeled RGCs in corresponding regions of
an unlesioned control retina (A), and after
axotomy at 1 d (B), 7 d
(C), and 14 d (D). In
addition to RGCs, intensely labeled microglial cells with
characteristic shapes began to appear at approximately day 7 (C) and increased in number by day 14 (D). Representative presumed microglial cells are
marked by white arrows in C and
D. No microglial cells were labeled with
hydroxystilbamidine in the control retina (A) or
at 1 d after axotomy (B). Scale bar, 50 µm.
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Morphologically, in whole-mounted retinas stained with cresyl violet,
GCL of unlesioned control retinas manifest no apoptotic profiles or
pyknotic nuclei (Fig.
2A). In contrast, by
10 d after axotomy, multiple degenerating cell bodies with
pyknotic nuclei were apparent in the GCL, and the cell density in this
layer appeared to be greatly diminished (Fig.
2B).
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Figure 2.
Photomicrographs of representative regions of the
GCL in whole-mounted retinas stained with cresyl violet. Normal
uninjured control retina (A) versus degenerated
retina 10 d after axotomy (B). At 10 d
after axotomy, cell density was decreased, and pyknotic nuclei
(arrows) appeared in the GCL. In contrast, no pyknotic
profiles were visible in the control GCL. Scale bar, 30 µm.
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When hydroxystilbamidine fluorescence in the GCL was quantified (Fig.
3), in unlesioned control retinas the
mean RGC density was 2533 ± 104 cells/mm2 (mean ± SD). Within
14 d of axotomy, the mean RGC density decreased to 348 ± 107 (14% of control, Fig. 3).
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Figure 3.
Effects of axotomy on the density of RGCs. Density
(in cells per square millimeter; mean ± SD) of
hydroxystilbamidine-labeled RGCs at different time points after
axotomy: 0, 1, 4, 7, and 14 d (n = 3-6
retinas in each group).
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Activation of p38 in RGCs after optic nerve axotomy
We initially investigated which isoform of p38 is predominant in
the adult rat retina. In control retinas, p38 displayed strong
immunoreactivity by immunoblotting (Fig.
4A, left
lane), whereas p38 was virtually undetectable (Fig.
4A, lanes 2, 3). We next used a second
anti-p38 antibody for immunoblotting (the gift of Dr. J. Han, The
Scripps Research Institute), but it, too, did not detect p38
immunoreactivity in control retinas or after axotomy (data not shown).
Immunoblotting also revealed that p38 was not phosphorylated/activated
in control retinas (Fig. 4B, top
panel). Phosphorylated p38 was first detected 12 hr after axotomy, reached a maximum at 1 d, and then slowly decreased. By
14 d, phosphorylated p38 had virtually disappeared (Fig.
4B, top panel).
Nonetheless, total p38 immunoreactivity (including both phosphorylated
and unphosphorylated forms) did not change substantially after axotomy
(Fig. 4B, bottom panel). This result was
expected because many cell types (see below) contain unactivated (unphosphorylated) p38, and RGCs comprise only 1% of the retinal population. Importantly, 1 d after axotomy, when p38
phosphorylation had reached its maximum, neither RGC density nor the
pattern of staining with hydroxystilbamidine had yet changed (Fig.
1B). Moreover, when phosphorylated p38 reached its
maximum, no p38 immunoreactivity was observed (Fig.
4A, right lane), in contrast to the
presence of p38.
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Figure 4.
A, Immunoreactivity of p38 isoforms
in adult rat retinas by immunoblotting. Strong immunoreactivity for
p38 was present in control retinas (left lane),
whereas p38 was virtually undetectable in control retinas
(middle lane) and 24 hr after axotomy (right
lane). B, Temporal profiles of p38 activity by immunoblotting.
Total p38 immunoreactivity (including both phosphorylated and
nonphosphorylated forms) did not change after axotomy (bottom
panel). p38 was not phosphorylated in control retinas,
but phosphorylation was detected 12 hr after axotomy and reached a
maximum at 24 hr before slowly decreasing (top panel).
By 14 d after axotomy, phosphorylated p38 immunoreactivity had
essentially disappeared.
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In the next series of experiments, we investigated the localization of
p38 and phosphorylated/activated p38 in the adult rat retina. In
control retinas, p38 immunoreactivity was seen in virtually all
cells in the GCL, inner plexiform layer (IPL), outer plexiform layer
(OPL), and in some cells in the proximal portion of the inner nuclear
layer (INL; Fig. 5A,
control). However, no cells in control retinas stained
positively for phosphorylated/activated p38 (Fig. 5B,
control). In contrast, 1 d after axotomy,
phosphorylated/activated p38 localized to cell nuclei in the GCL layer
(Fig. 5B,C, arrows). Nonetheless, the
distribution of total p38 immunoreactivity did not change (Fig.
5A, 1 day). By 14 d after axotomy, when both phosphorylated/activated p38 immunoreactivity and most RGCs themselves were gone (Fig. 5B, 14 days), p38 total immunoreactivity
was still observed in the GCL, IPL, and OPL, and in some cells in the
proximal portion of the INL (Fig. 5A,B, 14 days).
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Figure 5.
Localization of total and phosphorylated/activated
p38 immunoreactivity in the adult rat retina at various time points
after axotomy. Immunohistochemistry with anti-total p38 antibody
(A) and with anti-phosphorylated p38 antibody
(B) in control and axotomized retinas at 1 and
14 d after insult. A, In control retina, p38
immunoreactivity was seen in virtually all cells in the GCL, IPL, and
OPL, and in some cells in the proximal portion of the INL.
B, C, One day after axotomy,
phosphorylated/activated p38 was visualized in cell nuclei in the GCL
(black arrows), but not in control (nonaxotomized)
retinas or in retinas 14 d after axotomy. Panel C
shows the GCL at higher magnification 1 d after axotomy (400×).
D, Microglial cells 1 d after axotomy were
visualized with anti-rat ED1 antibody (black arrows).
The localization and morphology of ED1-positive microglial cells were
quite different from that of the phosphorylated p38-positive cells (in
B, black arrows point to RGCs; in
D, black arrows point to microglia). Anti-phospho-p38
antibody labeled RGCs but virtually no microglia. To better visualize
this localization, counterstaining was performed in C
with Meyer Hematoxylin. Scale bars: A,
B, D, 75 µm; C, 50 µm.
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Previously, Walton et al. (1998) had demonstrated that
microglial cells, rather than neurons, manifest p38 activation after global forebrain ischemia. Therefore, we visualized activated microglia
with anti-ED1 antibody (Fig. 5D). One day after axotomy, ED1-positive cells had a different distribution compared to the p38-activated cells (Fig. 5D, black arrows).
Virtually none of the microglial cells, by both histological and
morphological criteria, coincided with cells that stained positively
with anti-phosphorylated p38 (Fig. 5B, 1 day).
Effect of p38 inhibition on survival of axotomized RGCs
After demonstrating that p38 was activated/phosphorylated in
the nuclei of axotomized RGCs during an early phase of apoptosis, we
next examined whether treatment with SB203580, an inhibitor of p38 (Lee
et al., 1994; Cuenda et al., 1995), could prevent axotomy-induced RGC
apoptosis. SB203580 was administered intravitreally at the time of
axotomy and repeated at 5 and 10 d. Assayed 14 d after
axotomy, SB203580 increased the number of surviving RGCs in a
dose-dependent manner (Fig. 6). A
significant degree of neuroprotection was observed after injection of
as little as 0.2 nmol of SB203580, corresponding to an intravitreal
concentration of 1.6 µM, given the volume of the rat
vitreous has been reported to be ~120 µl (Hughes, 1979).
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Figure 6.
Quantitative assessment of the neuroprotective
effect of the p38 inhibitor, SB203580. SB203580 was injected into the
vitreous at the time of axotomy, injections were repeated 5 and 10 d later, and RGC survival was assessed 14 d after axotomy. At
doses of 0.2 nmol (corresponding to a concentration of 1.6 µM in the vitreous) or higher, SB2030580 produced
significant prevention of RGC death in the axotomy model
(*p < 0.01 by ANOVA with Dunnett's post
hoc comparison). Data are expressed as mean cell density ± SD (cells per square millimeter) of hydroxystilbamidine-labeled RGCs
(n = 3-6 retinas in each group).
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Involvement of the p38 signaling pathway in NMDA-induced
neuronal apoptosis
We next investigated if glutamate neurotoxicity via activation of
the NMDA receptor could effect apoptosis via the p38 signaling pathway
in RGCs. To address this question, we first examined the effects of
SB203580 on NMDA-induced RGC apoptosis in vitro. Treatment with 1 µM SB203580 significantly increased the
survival of RGCs in the face of an NMDA exposure capable of causing
apoptosis (Fig. 7). This finding
suggested that p38 activation is involved in RGC apoptosis induced by
NMDA. Then, we tested the effect on axotomy of the noncompetitive NMDA
receptor antagonist MK801 in vivo. MK801 was injected into
the vitreous in the same manner as SB203580. MK801 not only increased
the number of surviving RGCs 14 d after axotomy (Fig.
8A), but also inhibited
p38 phosphorylation/activation in a dose-dependent manner (Fig.
8B). The addition of SB203580 to MK801 did not offer
further protection above that of MK801 alone in this paradigm (Fig.
8A).
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Figure 7.
Inhibition of p38 activity protects cultured RGCs
from NMDA-induced apoptosis. Retinal cells were either treated or not
treated with 1 µM SB203580 during exposure to 200 µM NMDA, and treatment with SB203580 resulted in
significant protection of the RGCs from apoptosis
(*p < 0.01 by ANOVA with Dunnett's post
hoc comparison). Data are mean ± SEM of three independent
experiments.
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Figure 8.
Effects of MK801 on p38 phosphorylation and RGC
survival after axotomy. A, Protective effects of MK801
against axotomy-induced RGC apoptosis in vivo. MK801 was
injected into the vitreous at the time of axotomy, and repeated
injections were made 5 and 10 d later (n = 3-5 retinas in each group). A dose of 2 nmol of MK801 was
administered, corresponding to an intravitreal concentration of 16 µM. RGC survival was assessed 14 d after axotomy.
Data are expressed as mean cell density ± SD (cells per square
millimeter) of hydroxystilbamidine-labeled RGCs. Treatment with MK801
significantly attenuated RGC death compared to animals not receiving
MK801, but the degree of protection was not enhanced by the addition of
2 nmol of SB203580 in each injection (*p < 0.01 by
ANOVA with Dunnett's post hoc comparison).
B, Representative immunoblots from one of three
experiments showing that the level of phosphorylated p38 observed
1 d after axotomy decreased with increasing concentrations of
MK801.
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DISCUSSION |
In the present study, we demonstrate that the p38 MAP kinase
signaling pathway is involved in RGC apoptosis after axotomy. Previous
reports have described the time course of survival of RGCs after
axotomy (Mey and Thanos, 1993; Villegas-Perez et al., 1993;
Peinado-Ramon et al., 1996; Klocker et al., 1997). In general, the loss
of RGCs is more severe when the optic nerve is transected close to the
eye globe (Villegas-Perez et al., 1993). In our studies we found that
after axotomy near the eye, the density of RGCs remained approximately
constant for ~4 d, and then ~86% of RGCs died over the ensuing
10 d. This time course is similar to that of other reports
(Peinado-Ramon et al., 1996; Klocker et al., 1997; Koeberle and Ball,
1998). We next studied the time course of p38 activity after axotomy
but preceding cell death.
p38 has four isoforms, designated p38 (Han et al., 1994; Lee et al.,
1994), p38 (Jiang et al., 1996), p38 (Lechner et al., 1996; Li et
al., 1996), and p38 (Jiang et al., 1997; Wang et al., 1997).
Relatively little is known about the potential role of p38 and
p38 in apoptosis. However, p38 and p38 have been shown to be
involved in the apoptotic pathway in several cell types, including
neurons, at least in vitro (Kawasaki et al., 1997; Kummer et
al., 1997). Here we demonstrate strong immunoreactivity for p38, but
not p38 in the normal retina. These control retinas, however, did
not manifest phosphorylated/activated p38 activity. After axotomy,
anti-phospho-p38 immunoreactivity, signifying activated p38, was
visualized only in the GCL. Because the phosphorylated/activated p38
disappeared from the GCL as the RGCs died, it is likely that this cell
type contained the activated form of p38.
The question arises, however, why total p38 activity did not appear to
decrease in the retina after the demise of the RGCs after axotomy. In
adult rats, the population of RGCs represents <1% of total retinal
cells. Additionally, 40-50% of the cells in the rodent GCL are
displaced amacrine cells rather than RGCs (Perry, 1981). Fourteen days
after axotomy, total p38 immunoreactivity was still seen in the GCL,
IPL, OPL, and in some cells in the proximal portion of the inner
nuclear layer (INL; Fig. 5A). It is likely, however, that
the p38 immunoreactivity that remained 2 weeks after axotomy
represented staining of amacrine cells, a population that far
outnumbers the RGCs. In fact, 14 d after axotomy
phosphorylated/activated p38 immunoreactivity had disappeared as had
the RGCs. Thus, 2 weeks after axotomy total p38 immunoreactivity reflected mainly the inactive (unphosphorylated) p38 that was present
in the amacrine cells. In contrast, phosphorylated/activated p38 was
observed only in cells injured by axotomy, i.e., the RGCs, and only at
earlier time points.
Recently, Walton et al. (1998) reported that phosphorylated p38
increases in activated microglia after forebrain ischemia. We therefore
studied p38 activity in microglia in the retina. After optic nerve
axotomy, microglia are known to be activated and, in our study, some
became fluorescent by phagocytosis of degenerating
hydroxystilbamidine-labeled RGCs (Thanos et al., 1992, 1993). However,
we found that few if any microglia were labeled by anti-phospho-p38
compared to RGCs.
Next, we investigated if a p38 antagonist could protect RGCs from
apoptosis after axotomy.
4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) imidazole (SB203580) is an inhibitor of p38 MAP kinase (Cuenda et al.,
1995). Among the various p38 isoforms, p38 and p38 are sensitive
to this pyridinyl imidazole derivative, whereas p38 and p38 are
not (Cuenda et al., 1997). In general, pyridinylimidazoles, such as
SB203580, inhibit p38 activity by blocking the ATP-binding site (Wilson
et al., 1997). Thus, SB203580 does not inhibit the phosphorylation of
p38. It is important to note that at ~10-fold higher concentrations
of SB203580 than used here to protect RGCs (we used 1.6 µM in vivo in Fig. 6 and 1.0 µM in vitro in Fig. 7), this drug
can also inhibit JNK (Harada and Sugimoto, 1999). Similar to p38, JNK
can also participate in apoptotic signaling in some cell types.
However, at ~1 µM SB203580 appears to be
relatively specific for p38 (Harada and Sugimoto, 1999), although we
acknowledge that another mode of action of the drug cannot be
completely eliminated. Additionally, we demonstrated that p38
activation/phosphorylation preceded RGC apoptosis and that
pharmacological prevention of death with another drug (an NMDA receptor
antagonist, see below) also prevented p38 activation/phosphorylation.
Taken together, therefore, our results suggest that p38 activation
in RGCs occurs early after axotomy and plays an important role in apoptosis.
Next we investigated possible upstream pathways for p38 activation
leading to RGC apoptosis. RGC apoptosis after optic nerve axotomy is
thought to occur, at least in part, because of a lack of neurotrophic
factor support from the superior colliculus (Mey and Thanos; 1993,
Peinado-Ramon et al., 1996; Klocker et al., 1997; Yan et al., 1999) and
also because of excessive glutamate levels possibly caused by the
injury (Yoles and Schwartz, 1998). In the present study, we focused on
glutamate neurotoxicity because p38 had been reported to participate in
neuronal cell death pathways triggered by glutamate in in
vitro cerebellar granule cell, striatal, and hippocampal models
(Kawasaki et al., 1997; Vincent et al., 1998; Mukherjee et al., 1999).
Additionally, RGCs were the first neurons to be shown to be vulnerable
to glutamate-induced cell death (Lucas and Newhouse, 1957). Initially,
we prepared cultured RGCs to test whether p38 MAP kinase activation is
involved in NMDA-induced RGC apoptosis. Treatment with SB203580
protected RGCs from NMDA-induced apoptosis, suggesting that p38
activation plays an important role in this form of RGC death.
Furthermore, intravitreal administration of the noncompetitive NMDA
receptor antagonist MK801 prevented RGC apoptosis caused by axotomy in our study as well as in other reports (Russelakis-Carneiro et al.,
1996; Yoles et al., 1997). Importantly, in our axotomy model, MK801
also inhibited phosphorylation of p38 in a dose-dependent manner,
suggesting that p38 activation after axotomy is mediated by stimulation
of NMDA receptors.
Recently, p38 inhibitors have been shown to be effective in animal
models of arthritis, bone resorption, and endotoxic shock (Badger et
al., 1996), and several human clinical trials are currently in progress
for these entities. Optic nerve axotomy permits rapid analysis of RGC
death and is therefore a valuable model for diseases producing RGC
apoptosis, including glaucoma. We and our colleagues recently reported
that glutamate neurotoxicity may mediate, at least in part,
glaucomatous damage to RGCs (Dreyer et al., 1996; Dkhissi et al., 1999;
Dreyer and Lipton, 1999; Vorwerk et al., 1999b). Here we show that
inhibition of p38 MAP kinase activity can ameliorate glutamate-related
RGC apoptosis in vitro and axotomy-induced RGC death
in vivo. In fact, this represents the first in
vivo model with a p38 inhibitor to show efficacy in the adult
mammalian central nervous system. Therefore, we speculate that p38
inhibitors may be potentially useful for the treatment of retinal
diseases such as glaucoma.
| |
FOOTNOTES |
Received Feb. 15, 2000; revised April 4, 2000; accepted April 20, 2000.
This work was supported in part by National Institutes of Health Grants
R01 EY05477, R01 EY09024, and P01 HD29587 (S.A.L.), and by the Japan
Eye Bank Association (M.K.). We thank Dr. Jiahuai Han of The Scripps
Research Institute (La Jolla, CA) for the generous gift of anti-p38
antibody and recombinant p38 protein.
Correspondence should be addressed to Dr. Stuart A. Lipton, Center for
Neuroscience and Aging, The Burnham Institute, 10901 North Torrey Pines
Road, La Jolla, CA 92037. E-mail: slipton{at}burnham-inst.org.
| |
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M. P. Lafuente, M. P. Villegas-Perez, P. Sobrado-Calvo, A. Garcia-Aviles, J. Miralles de Imperial, and M. Vidal-Sanz
Neuroprotective Effects of {alpha}2-Selective Adrenergic Agonists against Ischemia-Induced Retinal Ganglion Cell Death
Invest. Ophthalmol. Vis. Sci.,
August 1, 2001;
42(9):
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[Abstract]
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S. Schumacher, M. Jung, U. Norenberg, A. Dorner, R. Chiquet-Ehrismann, C. A. O. Stuermer, and F. G. Rathjen
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
J. Biol. Chem.,
March 2, 2001;
276(10):
7337 - 7345.
[Abstract]
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S. Yamagishi, M. Yamada, Y. Ishikawa, T. Matsumoto, T. Ikeuchi, and H. Hatanaka
p38 Mitogen-activated Protein Kinase Regulates Low Potassium-induced c-Jun Phosphorylation and Apoptosis in Cultured Cerebellar Granule Neurons
J. Biol. Chem.,
February 9, 2001;
276(7):
5129 - 5133.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION
