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Published Online
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March 25, 2002
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The Journal of Neuroscience, 2002, 22:RC216:1-7
RAPID COMMUNICATION
Neurodegenerative and Neuroprotective Effects of Tumor Necrosis
Factor (TNF) in Retinal Ischemia: Opposite Roles of TNF Receptor 1 and
TNF Receptor 2
Valerie
Fontaine1,
Saddek
Mohand-Said2,
Noelle
Hanoteau2,
Céline
Fuchs2,
Klaus
Pfizenmaier1, and
Ulrich
Eisel1
1 Institute of Cell Biology and Immunology, University
of Stuttgart, 70569 Stuttgart, Germany, and 2 Laboratoire
de Physiopathologie Cellulaire et Moléculaire de la Rétine,
Institut National de la Santé et de la Recherche Médicale
EMI 99-18, BP 426, 67091 Strasbourg, France
 |
ABSTRACT |
Tumor necrosis factor (TNF) is an important factor in various acute
and chronic neurodegenerative disorders. In retinal ischemia, we show
early, transient upregulation of TNF, TNF receptor 1 (TNF-R1), and
TNF-R2 6 hr after reperfusion preceding neuronal cell loss. To assess
the specific role of TNF and its receptors, we compared ischemia-reperfusion-induced retinal damage in mice deficient for
TNF-R1, TNF-R2, or TNF by quantifying neuronal cell loss 8 d after
the insult. Surprisingly, TNF deficiency did not affect overall cell
loss, yet absence of TNF-R1 led to a strong reduction of
neurodegeneration and lack of TNF-R2 led to an enhancement of
neurodegeneration, indicative of TNF-independent and TNF-dependent processes in the retina, with TNF-R1 augmenting neuronal death and
TNF-R2 promoting neuroprotection. Western blot analyses of retinas
revealed that reduction of neuronal cell loss in TNF-R1 / animals
correlated with the presence of activated Akt/protein kinase B
(PKB). Inhibition of the phosphatidylinositol 3-kinase signaling
pathway reverted neuroprotection in TNF-R1-deficient mice, indicating
an instrumental role of Akt/PKB in neuroprotection and TNF-R2
dependence of this pathway. Selective inhibition of TNF-R1 function may
represent a new approach to reduce ischemia-induced neuronal damage,
being potentially superior to strategies aimed at suppression of TNF
activity in general.
Key words:
Akt/PKB activation; neuronal TNF and TNF-R expression; retinal cell layers; ischemia; neuroprotection; knock-out mouse; immunohistology
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INTRODUCTION |
Tumor
necrosis factor (TNF) is upregulated in neurodegenerative disorders,
such as multiple sclerosis and Parkinson's and Alzheimer's disease,
as well as subsequent to brain trauma and ischemic injury (for review,
see Shoshami et al., 1999 ). Retinal ischemia is a frequent complication
in diabetic patients (Cunha-Vaz, 1998) and is believed to represent
many aspects of other brain ischemic insults. Development of a brain
ischemic lesion depends on the activation of many pathophysiological
processes, dominating basal or concomitantly induced neuroprotective
mechanisms (Maiese, 1998). Production and release of cytokines,
particularly of TNF, is one of the early cellular events subsequent to
an ischemic episode (Hangai et al., 1996; Shoshami et al., 1999).
Because of its diverse bioactivities, it is presently not clear under which conditions TNF promotes beneficial or deleterious effects on
neuronal tissues. An answer to this question might provide new
strategies to prevent pathologies resulting from ischemia. The
pleiotropic activities of TNF are mediated via two distinct receptors
(for review, see Wajant and Scheurich, 2001). In vivo, TNF
receptor 1 (TNF-R1) plays a prominent role in antibacterial responses
and in determining sensitivity to septic shock syndromes and is
involved in the cellular organization of secondary lymphoid tissues
(Pfeffer et al., 1993; Rothe et al., 1993; Kollias et al., 1999). The
role of TNF-R2 was unclear until the membrane form of TNF was
recognized as the physiological activator of this TNF receptor (Grell
et al., 1995). Several studies now point to an important contribution
of TNF-R2 to immune cell activation and endothelial functions, based on
a cooperative action with TNF-R1 (Küsters et al., 1997; Lucas et
al., 1997; Douni and Kollias, 1998; Kollias et al., 1999). However, the
role of TNF and its two cognate receptors in the CNS is far from being
understood. In vivo studies using different tools such as
TNF neutralizing antibodies, soluble TNF receptors, and TNF and TNF-R
knock-out and transgenic mouse models indicate an important function of TNF in inflammatory demyelinating diseases (Akassoglou et al., 1999)
but have not allowed us to draw firm conclusions regarding the role of
TNF produced in the CNS under trauma and ischemia (Cheng et al., 1994;
Bruce et al., 1996; Dawson et al., 1996; Nawashiro et al., 1997; Lavine
et al., 1998; Sherbel et al., 1999; Stahel et al., 2000). In the latter
condition, the available data suggest that TNF may be capable of
exerting opposite effects, which could depend on parameters such as the
site, degree, and duration of the ischemic period, the amount of TNF
production, the expression level of the two receptors, and the cellular
environment of affected neurons. Using a model of retinal
ischemia-reperfusion, we demonstrate involvement of the TNF/TNF-R
system and opposing actions of the two TNF-Rs, with TNF-R1 aggravating
neuronal damage and TNF-R2 promoting neuroprotection via an Akt/PKB
signal pathway.
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MATERIALS AND METHODS |
Animals and reagents. Mice were kept according to
federal regulations. TNF-R1/ (Rothe et al., 1993) and TNF-R2/
(Lucas et al., 1997) mice were from Horst Bluethmann (Hoffmann-La
Roche, Basel, Switzerland), and TNF/ (Moore et al., 1999) mice were from George Kollias (Pasteur Institute, Athens, Greece). Genotyping of
TNF-R1 knock-out mice was performed using PCR with primers TNF-R1-2883
5'-CTCTCTTGTGATCAGCACTG-3' and Neo-34 5'-TCCCGCTTC-AGTGACAACGTC-3', resulting in a 1 kb PCR product for the detection of the TNF-R1 null
allele, and TNF-R1-2883 and TNF-R1-4938
5'-AGAAATCTCAAGACAATTCTCTGC-3', resulting in a 500 bp fragment for the
wild-type allele. Genotyping of the TNF-R2 knock-out mice was performed
similarly as for TNF-R1 knock-out mice using TNF-R2-A primer
5'-CCTCTCATGCTGTCCCCGGATT-3' and Neo-IL4 5'-GCGCATCGCCTTCTATCGCC-3',
resulting in a PCR product of 700 bp for the detection of the TNF-R2
null allele, and TNF-R2-A and TNF-R2-B 5'-AGCTCCAGGCACAAGGGCGGG-3',
resulting in a 300 bp fragment for the wild-type allele. TNF knock-out
mice were kept in homozygosity. All mice were kept in a C57BL/6
background except for TNF-R1/, which were kept in a CD1xC57BL/6
background, with key experiments repeated in C57BL/6 background.
Sensitivity of wild-type CD1xC57BL/6 and heterozygous TNF-R2 knock-out
mice toward retinal ischemia was identical to that of C57BL/6, as
revealed from similar neuronal damage in all three retinal layers.
Tropicamide and oxybuprocaine chlorhydrate were from Cibavision
Ophthalmics (Toulouse, France). LY 294002 was from Calbiochem (Bad
Soden, Germany). Rabbit anti-TNF (H-156), mouse anti-TNF-R2 (D-2), and mouse anti TNF-R1 (H-5) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-Akt and mouse anti-phospho-Akt (Ser 473)
antibodies were from New England Biolabs (Beverly, MA). Mouse anti- -tubulin antibody was from Sigma (St. Louis, MO). Goat
anti-mouse IgG or anti-rabbit IgG conjugated to Alexa TM 488 were from
Molecular Probes Europe BV (Leiden, The Netherlands).
Peroxidase-conjugated antibodies were from Jackson ImmunoResearch
Laboratories (West Grove, PA). The ECL detection kit was from Amersham
Biosciences (Little Chalfont Buckinghamshire, UK). Pentobarbital
and all other reagents used were purchased from Sigma (Deisenhofen, Germany).
Retinal ischemia-reperfusion. Retinal
ischemia-reperfusion was performed as described (Hughes, 1990).
Briefly, C57BL/6 [wild-type (wt)], TNF/, TNF-R1/, and
TNF-R2/ mice were anesthetized with an intraperitoneal injection of
pentobarbital (90 mg/kg), the pupil was dilated with a drop of
tropicamide, and a topical anesthesia was performed with oxybuprocaine
chlorydrate. Retinal ischemia was induced in the left eye by increasing
intraocular pressure to 150 mm Hg through air injection into the
anterior chamber for 45 min. For immunohistology and immunoblotting,
animals (n = 3) were killed after 6 or 24 hr of
reperfusion and immediately enucleated. For ischemic damage analysis,
left eyes (n = 10) were removed after 8 d of
reperfusion. For comparative purposes, non-ischemic retinas from each
mouse strain were also investigated. LY 294002 was dissolved in DMSO at
5 mg/ml diluted to 10 µM in 0.09% NaCl, and 1 µl was injected intravitreally using an UltraMicro-Pump (type UMP2)
equipped with a MicroSyringe Pump Controller (World Precision
Instruments, Sarasota, FL).
Immunohistological analyses. Eyes were fixed for 1 hr in 4%
paraformaldehyde in 5% sucrose, cryoprotected in graded sucroses (5, 10, and 20%), and sectioned transversally at 10 µm on a cryostat. Retinal sections were blocked for 1 hr in a solution containing 10%
normal goat serum, 1% bovine serum albumin, and 0.5% Triton X-100 in
saline phosphate buffer, pH 7.4. Anti-TNF (1:200), anti-TNF-R1 (1:500),
and anti-TNF-R2 (1:500) primary antibodies were applied overnight at
4°C. Staining was performed with Alexa TM 488-conjugated anti-mouse
IgG or anti-rabbit IgG (1:1000; 1 hr at room temperature). After
washing, 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (1 µg/ml in PBS) was applied for 10 min. Retinas were examined by
standard immunofluorescent microscopy. The specificity of each of the
TNF/TNR antibodies was checked and verified by complete lack of
staining in the respective knock-out strain.
Histological procedures and quantification of ischemic
damage. Eyes were fixed for 24 hr in 4% paraformaldehyde,
dehydrated, and embedded in paraffin. Sections (3 µm thick)
containing the retina and crossing the optic nerve were stained with
hematoxylin and eosin. Quantification of ischemic damage was assessed
by measuring cell densities in each retinal layer according to Hughes
(1990). They were expressed as a number of nuclei in a 30-µm-wide
band for the outer and inner nuclear layers and as a number of nuclei in a 150-µm-wide band for the ganglion cell layer. Retinal sections were examined from digitalized images. For each retina, two areas located 800 µm on both sides of the optic nerve were analyzed.
Protein extraction and Western blotting. After enucleation,
retinas were quickly removed and collected on ice in lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM
NaCl, 2 mM EDTA, 1% Nonidet P-40, 1 mM NaF, 1 mM
NaVO4, 10 µM bestatin, 10 µg/ml leupeptin, 4 µg/ml aprotinin, and 0.5 mM PMSF). Twenty micrograms of proteins were separated by SDS-PAGE on 10% gels and transferred on nitrocellulose membranes. Membrane blocking was performed 1 hr at room temperature in
5% skim milk in 0.1% Tween 20 (TBS-T) for anti-Akt, anti-tubulin antibodies, and in 5% BSA in TBS-T for anti-phospho-Akt antibody. For
each set of samples, membranes were tested for the three antibodies studied using a stripping step between each. Primary antibodies were
applied overnight at 4°C and were detected using
peroxidase-conjugated secondary antibodies. Immunoblots were visualized
using the ECL detection system.
Statistical analyses. Statistical analyses were performed as
described (Hughes, 1990) according to the parametric method of the
Student's t test for unpaired series for each variable.
After the variance analysis of each parameter, we performed Bartlett's test and compared the averages using Scheffe's t test.
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RESULTS |
Expression of TNF and its receptors was examined in non-ischemic
and ischemic retinas of C57BL/6 mice. Retinal ischemia was induced by
increasing intraocular pressure for 45 min, and retinas were analyzed 6 and 24 hr after reperfusion (Fig. 1). TNF
was found mainly in the inner retina, with a stronger expression in the
ganglion cell layer (GCL) compared with cells of the inner nuclear
layer (INL). Within the latter, the staining was stronger in neurons
such as amacrine cells close to the inner plexiform layer (IPL) (Fig.
1a, arrow). TNF expression was not found in the
outer nuclear layer (ONL) composed of photoreceptors (Fig. 1a,d). Significant increase in TNF expression was
observed after 6 hr of reperfusion, in particular in GCL and, to a
lesser extend, in INL cells adjacent to the inner plexiform layer (IPL)
(Fig. 1b,e). At this time point, TNF was also
detected within structures of the ONL resembling Müller glia
processes (Fig. 1b,c). After 24 hr, TNF
expression in ganglion cells was back to control levels (Fig.
1c,f). These results extend previous data
on induction of TNF after retinal ischemia (Hangai et al., 1996) and
identify the cell layers capable of TNF expression.
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Figure 1.
TNF and TNF-R upregulation after ischemia in the
murine retina. Cryosections of retinas from untreated or ischemic
C57BL/6 mice were analyzed by indirect immunofluorescence for TNF
expression (a-c) and costained with DAPI
to detect nuclear layers (d-f),
or TNF-R1 (g-i) and TNF-R2
(j-l) expression,
respectively. GCL, Ganglion cell layer;
IPL, inner plexiform layer; INL, inner
nuclear layer; OPL, outer plexiform layer;
ONL, outer nuclear layer; OS, outer
segment of photoreceptor cells; m, structures resembling
Müller glia processes.
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TNF-Rs were below the immunohistochemical detection level in
non-ischemic retina (Fig. 1g,j), which does not
rule out an expression level sufficient to respond to a TNF stimulus.
After 6 hr of reperfusion, cells localized in the inner INL and GCL
showed a strong signal for both TNF receptor types. TNF-Rs were also
found in the IPL and outer plexiform layer (OPL) as well as within
structures of the ONL resembling Müller glia processes (Fig.
1h,k). Twenty-four hours after
ischemia-reperfusion, expression of TNF-Rs in all retinal layers was
still discernable, but drastically reduced compared with 6 hr (Fig.
1j,l). At this time point, only a few strongly staining cells were found in the GCL. These results show that
TNF-Rs are present at a rather low expression level in the retina and
that they are strongly, but transiently, upregulated after an ischemic insult.
Histological analyses of the neuronal damage were performed in wt and
knock-out mice after 8 d of physiological reperfusion (Fig.
2). As a parameter of ischemia-induced
pathology, neuronal cell death was determined by counting the nuclear
densities in three distinct retinal layers: ONL, INL, and GCL. Control
retinas from the various mouse strains did not present significant
differences in cellular densities ranging between 91.4 ± 7.2 and
95.3 ± 12 for the ONL, between 29.5 ± 3.7 and 32.4 ± 3.7 for the INL, and between 18.5 ± 2.2 and 19.9 ± 4.4 for the GCL for the different strains of untreated animals (Fig.
3B). Ischemia-reperfusion in wt mice induced a reduction of the whole retinal thickness, with a
larger extent in the inner part of the retina, indicative of previous
neurological damage (Fig. 2). This was confirmed by cell nuclei
quantification, which showed significant (p < 0.001) neuronal death in all three retinal cell layers, with greater
damage in the GCL (36.7% reduction compared with controls) compared
with INL (22.2% reduction) and ONL (22% reduction) (Fig.
3A), which is in accordance with previous studies showing
that retinal ischemia affects all neuronal subtypes, with the GCL being
most susceptible (Hughes, 1990).
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Figure 2.
Histology of retinal sections. a,
Wild-type (wt) control retina. b-e,
Retinal sections 8 d after ischemia-reperfusion. Retinas from wt
(b) and TNF/ (c) looked
very similar. TNF-R2/ retinas (d) displayed a
more degenerative morphology; TNF-R1/ retinas
(e) were very well preserved. OS,
Outer segments; OPL, outer plexiform layer;
IPL, inner plexiform layer; GCL ganglion
cell layer. Scale bar, 13 µm.
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Figure 3.
Differential sensitivity of TNF/ and TNFR/
mice to retinal ischemia-reperfusion-damage. A,
Neuronal degeneration in different retinal cell layers.
ONL, outer nuclear layer;
INL, inner nuclear layer;
GCL, ganglion cell layer. Data are
expressed as percentage reduction in neuronal cell number after 8 d of reperfusion compared with non-ischemic animals of the same mouse
strain. B, Quantitative and statistical analyses of
neuronal cell densities. A neuronal cell count was performed on
histological sections of ischemic retinas after 8 d of reperfusion
as well as on control retinas. Treatment with LY294002 in solution
alone without ischemia had no effect on the retinal cell numbers
(GCL, 19.7 ± 1.5; INL, 28.3 ± 2.1; ONL, 90.3 ± 4.7). For the ONL and INL, 2.5%
of the total retinas were counted; for the GCL, 12.5% were counted.
Values presented are expressed as the mean ± SD of the number of
nuclei per field (n = 20). Statistical analyses:
(1) control (c) compared
with ischemia (i) in each group tested;
(2) i compared with
i in wt mice. NS, Not
significant. *p < 0.05; **p < 0.01; ***p < 0.001.
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After ischemia, each of the three mutant mouse strains displayed
a distinct pattern of cell degeneration (Fig.
3A,B). In comparison with wt mice,
TNF-R1/ mice displayed no neuronal loss in the INL (0 vs 22.2%)
and a significantly reduced damage in ONL (12.3 vs 22%) and GCL (15 vs
36.7%). Unexpectedly, retinas from TNF-R2/ mice were more strongly
affected by ischemia than those from wt mice, showing an increased
neuronal loss in all three cellular layers (ONL, 37.8 vs 22%; INL, 37 vs 22.2%; GCL, 51.4 vs 36.7%), suggesting a neuroprotective role of
TNF-R2 for retinal neurons. Interestingly, in mice with a deletion of
the TNF gene itself, the overall neuronal damage after retinal ischemia
was not significantly different from wt mice (Fig.
3A,B).
To identify potential mechanisms involved in TNF-R2-mediated
neuroprotection, we investigated the presence and activation of the
protein kinase Akt/PKB, which has previously been shown to participate
in TNF-induced anti-apoptotic pathways (Ozes et al., 1999). Akt/PKB was
uniformly detectable in lysates of ischemic retinas of all mouse
strains investigated (Fig. 4, top
panel). The protein levels did not change after ischemia.
However, phosphorylated and thus activated Akt/PKB was detected
exclusively in ischemic retinal extracts from TNF-R1/ animals after
6 hr of reperfusion, but not in extracts from wild-type or TNF/ or
TNF-R2/ animals (Fig. 4, middle panel),
indicative of a TNF/TNF-R2 dependence of Akt/PKB activation in this
tissue. After 24 hr, Akt/PKB phosphorylation was no longer detectable
in the TNF-R1/ mouse or in any other of the mouse strains tested
(data not shown). The importance of Akt/PKB activation for protection
from ischemic damage was shown by intravitreal application of the
phosphatidylinositol 3-phosphate kinase inhibitor LY294002 in
TNF-R1/ mice, resulting in marked enhancement of retinal damage
(Fig. 3B).
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Figure 4.
Phosphorylated Akt/PKB is present in the retina of
TNF-R1/ mice subsequent to an ischemic insult. Retinas from C57BL/6
wt (lane 1), TNF/ (lane 2),
TNF-R1/ (lane 3), and TNF-R2/ (lane
4), without or with ischemia, were analyzed by Western
blotting for Akt/PKB (total protein, top panel)
and reprobed for phosphorylated Akt (middle
panel). The results were normalized by reprobing with
antibodies specific for -tubulin (bottom
panel).
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DISCUSSION |
We addressed the multifactorial nature of ischemic lesions and
provide new mechanistic insights with respect to the contribution of
TNF, which is induced after ischemia in the retina (Fig. 1) and
promotes at the same time neurodegeneration and neuroprotection. We
present, for the first time, experimental evidence for an antagonistic function of the two TNF-Rs during ischemia-reperfusion damage of
retinal neurons. In a TNF-R2-deficient situation, TNF was found to
aggravate cell death in three different retinal layers. This indicates
not only that TNF-R1 signaling is sufficient for TNF-mediated retinal
damage, but further suggests that the observed enhancement of pathology
is probably caused by an unbalanced activation of TNF-R1. The
importance of TNF-R1 in mediating neuronal cell death in
vivo is underlined by the absence (INL) or the significant reduction (ONL, GCL) of cell death in the different retinal layers of
TNF-R1/ mice. Furthermore, the reduced lesion size in TNF-R1/ as compared with TNF/ mice, the latter presenting with damage similar to that of wild-type animals, reveals (1) a TNF -independent component in ischemia-induced lesions and (2) a requirement for TNF-R2
signals antagonizing this TNF-independent neuronal damage, resulting in
full (INL) or at least substantial protection of the affected neuronal
populations. Because the neuroprotective activity of TNF via TNF-R2
became apparent only in mice lacking TNF-R1, we suggest that
TNF-mediated protection is blurred by concomitantly ongoing
degenerative signals emanating from TNF-R1.
Our finding of a TNF-independent pathway of retinal degeneration is not
unexpected, because several other members of this large cytokine family
have been implicated in neurodegenerative processes. First, Lymphotoxin
binds to TNF-R1 (Wajant et al., 1998) and thus could potentially
substitute for TNF in retinal TNF-R1 signal pathways. However, at
present the role of Lymphotoxin in neurodegenerative diseases is
controversial (Suen et al., 1997; Sean Riminton et al., 1998).
Second, two recent studies showed, in addition to TNF, involvement of
Fas ligand (Martin-Villalba et al., 1999, 2001) and TRAIL in brain
ischemia induced by arterial occlusion (Martin-Villalba et al., 1999).
Although for the latter two members of the TNF family an involvement in
retinal degeneration remains to be verified experimentally, the
TNF-independent damage observed in the present study is in accordance
with a contribution of one or both of these pro-apoptotic cytokines.
An important question relates to the immediate cellular targets of the
antagonistic TNF actions, i.e., neuronal versus non-neuronal cells.
This is of particular relevance because effects on the vasculature are
now considered essential to initiate ischemic tissue damage (for
review, see Petty and Wettstein, 2001). In fact, our data do not rule
out TNF actions on the vasculature, in particular those contributing to
tissue damage. However, the wide staining for both TNF-Rs in several
retinal neuronal layers indicates that neuronal cells are involved and
potentially direct targets of TNF action. In support of this reasoning,
at least for the neuroprotective function of TNF, a direct and
TNF-R2-dependent action on human neuronal cells has been shown recently
in vitro using an antisense oligonucleotide approach to
downregulate TNF-R2 expression (Shen et al., 1997). Our in
vitro studies with primary cortical neurons from TNF-R1 and TNF-R2
knock-out mice now corroborate these findings and clearly show a direct
TNF action on these primary murine neurons, with TNF-R1-deficient, but
not TNF-R2-deficient, neurons being protected from glutamate-induced
excitotoxicity (L. Marchetti and U. Eisel, unpublished data). These
data provide further evidence for the differential role of the two
TNF-Rs expressed on neuronal cells.
Concerning the potential mechanisms involved in TNF-R2-mediated
neuroprotection, our data point to an important function of protein
kinase Akt/PKB. Akt/PKB has been reported to play an important role in
cell survival pathways by interfering at several levels with
pro-apoptotic signals (Yuan and Yankner, 2000) and to be activated in
response to TNF (Ozes et al., 1999). Akt/PKB is supposed to be critical
in protection from reperfusion injury (Yellon and Baxter, 1999) and is
known to mediate, at least in part, the effect of brain-derived
neurotrophic factor, one of the most effective survival factors in the
retina (Klöcker et al., 2000). The failure of TNF/ mice to
stimulate Akt/PKB kinase activity and the kinetics of TNF/TNF-R
induction after ischemia, which coincides with retinal Akt/PKB
phosphorylation, supports TNF dependence on Akt/PKB activation in retinal neurons in vivo. These findings are in accordance
with the proposed role of Akt/PKB in neuroprotection (Yellon and
Baxter, 1999; Klöcker et al., 2000; Yuan and Yankner, 2000). Very
recent data show that TNF protects, via Akt/PKB activation, axotomized retinal ganglion cells from retrograde cell death, and therefore they
fully support our findings (Diem et al., 2001). Our data also show that
in retinal neurons, activation of Akt/PKB is dependent on a TNF-R2
signal pathway. We further suggest that lack of detectable Akt/PKB
activation in the retina of wt mice could reflect a repression of this
TNF-R2-dependent pathway by TNF-R1-mediated signals.
A neuroprotective role of TNF in a model of focal cerebral ischemia was
already suggested previously from data with mice double-deficient for
both TNF receptors (Bruce et al., 1996). At variance with our data,
however, is a subsequent study from the same laboratory analyzing
hippocampal ischemia-reperfusion damage, indicating a TNF-R1
dependence of the neuroprotective effect (Gary et al., 1998). The two
models, retinal and focal cerebral ischemia, differ not only with
respect to methodological aspects and time course of development of the
lesions, but probably as important, they differ with respect to
cellular composition in the regions affected by the ischemic insult. In
the retinal ischemia model, we have obtained evidence that the distinct
neuronal cell populations present in the various retinal layers differ
with respect to sensitivity to the ischemic insult and expression of
(Fig. 1) and protection by TNF/TNF-R2 signals (Fig. 3). Therefore, the
opposite role of TNF-R1 determined in the two models, disease promoting
in the case of retinal ischemia (this paper) and disease amelioration in the case of hippocampal damage (Gary et al., 1998), may reflect this
different cellular composition in the targeted areas and a differential
cellular response to TNF exposition. However, very recent data obtained
in an experimental autoimmune encephalomyelitis model also
suggest an antagonistic action of the two TNF receptors (Kassiotis and
Kollias, 2001), which is in full accordance with our data obtained in
retinal ischemia and strengthen our hypothesis of a differential role
of the two TNF receptors in vivo. On the basis of our data,
we propose that blocking TNF-R1 function and selectively activating
TNF-R2 could represent a promising new approach in preventing
irreversible neuronal loss by ischemic insults, at least in the retina.
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FOOTNOTES |
Received July 27, 2001; revised Jan. 11, 2002; accepted Jan. 23, 2002.
This work was supported by Hertie Foundation and Volkswagenstiftung to
K.P. and U.E. and by Fédération Française des
Aveugles. We are indebted to Horst Bluethmann (Hoffmann-La Roche,
Basel, Switzerland) for providing TNF-R1/ and TNF-R2/ mice and
to George Kollias (Pasteur Institute, Athens, Greece) for providing TNF/ mice. We thank Nouredine Sadeg for assistance with the statistical analysis.
Correspondence should be addressed to Ulrich Eisel, Institute of Cell
Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. E-mail:
ulrich.eisel{at}po.uni-stuttgart.de.
This article is published in
The Journal of Neuroscience, Rapid Communications Section,
which publishes brief, peer-reviewed papers online, not in print. Rapid
Communications are posted online approximately one month earlier than
they would appear if printed. They are listed in the Table of Contents
of the next open issue of JNeurosci. Cite this article as:
JNeurosci, 2002, 22:RC216 (1-7). The
publication date is the date of posting online at
www.jneurosci.org.
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