From the Walter and Eliza Hall Institute for Medical Research, Post
Office, The Royal Melbourne Hospital, Parkville, Victoria, 3050, Australia and the Department of Physiology, University of
Melbourne, Grattan Street, Parkville, Victoria, 3052, Australia
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ABSTRACT |
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The p75 neurotrophin receptor (p75NTR) has been
shown to mediate neuronal death through an unknown pathway. We
microinjected p75NTR expression plasmids into sensory neurons in the
presence of growth factors and assessed the effect of the expressed
proteins on cell survival. We show that, unlike other members of the
TNFR family, p75NTR signals death through a unique
caspase-dependent death pathway that does not involve the
"death domain" and is differentially regulated by Bcl-2 family
members: the anti-apoptotic molecule Bcl-2 both promoted, and was
required for, p75NTR killing, whereas killing was inhibited by its
homologue Bcl-xL. These results demonstrate that
Bcl-2, through distinct molecular mechanisms, either promotes or
inhibits neuronal death depending on the nature of the death stimulus.
The neurotrophin receptor,
p75NTR,1 is required along
with the trkA receptor for high affinity NGF binding and
neuronal survival (1); however, more recently it was shown to be
involved in promoting cell death by an unknown pathway. Overexpression
of p75NTR increased neuronal death both in vitro and
in vivo (2-4), and lowering the expression of p75NTR was
shown to prevent neuronal death after growth factor withdrawal in
vitro, and after sciatic nerve axotomy (5, 6). Additionally,
p75NTR-dependent cell death occurred after activation by
NGF or brain-derived neurotrophic factor (BDNF) in cells, respectively,
lacking trkA or trkB expression (7-10).
p75NTR is a member of the tumor necrosis factor receptor (TNFR)
superfamily, showing homology in the extracellular ligand binding
domain and a cytoplasmic motif known as the "death domain," so
called because of the cytotoxic actions of proteins containing the
domain (11). The death signaling pathway of the TNFR family is well
characterized, typically involving other "death domain"-containing proteins (12), and is not inhibited by the anti-apoptotic proteins Bcl-2 or Bcl-xL. These Bcl-2 family members are well
characterized inhibitors of stress-induced cell death, blocking
neuronal cell death in a variety of models (13-15).
Herein we investigate the role of the death domain and Bcl-2 family
members in mediating p75NTR death signaling using microinjection technology to deliver expression plasmids into cultured neurons. We
show that the p75NTR death signaling pathway is unlike the TNFR death
signaling pathway as an intracellular juxta-membrane domain, and not
the death domain, of p75NTR is sufficient and required to mediated
death signaling. Secondly, the anti-apoptotic Bcl-2 family members that
have little influence on TNFR death signaling have profound effects on
the p75NTR death pathway.
Cell Culture--
Dorsal root ganglia were dissected from
postnatal day zero C57Bl/6 or Bcl-2-deficient mice and plated in 3-cm
tissue dishes precoated with poly-DL-ornithine (500 µg/ml, Sigma) and laminin (20 µg/ml, Life Technologies, Inc.) at a
density of 5000 neurons/dish (5). Cells were grown in Monomed medium
(CSL, Melbourne, Australia) containing 1% fetal bovine serum and
leukemia inhibitory factor (LIF, AMRAD, Australia) or nerve growth
factor (2.5 S NGF, 50 ng/ml, Alomone Laboratories). Survival of sensory
neurons was assessed by morphological criteria (5) and propidium iodide exclusion. Heterozygous mice containing a disrupted Bcl-2 gene, (D. Loh, Roche, Japan) were mated to produce litters contains homozygous
Bcl-2 knock-out mice. The genotypes of newborn mice were determined by
polymerase chain reaction, and results were confirmed by staining of
thymocytes with an anti-mouse Bcl-2 antibody (PharMingen) before
dissection and microinjection of neurons isolated from individual mice
of appropriate genotype.
Microinjection--
Sensory neurons were injected into the
nucleus with a solution containing plasmid (100 µg/ml, with the
exception of Bcl-2, which was at 50 µg/ml), tetramethylrhodamine
dextran ("fluoro-ruby," 0.15%, Molecular Probes), and
phosphate-buffered saline. Where more than one plasmid was expressed in
a single condition, only one injection of solution containing all
plasmids was made, with the individual plasmid concentrations as
specified above. Approximately 70 neurons/well were injected, with two
or three wells comprising each condition. At least 2 h after
completion of the injections, the number of fluoro-ruby containing
cells that had survived the injection procedure was counted, and this
provided the time zero 100% value for each well.
Immunostaining--
Cells were fixed with 4% paraformaldehyde
for 15 min and then stained with an anti-rat-p75NTR antibody (MC192,
Roche Molecular Biochemicals) (3) or anti-human-Bcl-2 antibody
(Bcl-2/100, (PharMingen).
DNA Constructs--
The plasmid containing the full-length rat
p75NTR cDNA, p75NTR, is described (4), and all p75NTR plasmids are
modified versions of this original expression vector. A control
plasmid, p75NTRnc (no cytoplasmic domain) is missing the entire
cytoplasmic domain except the membrane anchoring Lys-274 and Arg-275.
p75NTRtr is truncated with an I308A substitution followed by a stop
codon, deleting the entire death domain. p75NTR Yeast Two-hybrid Methods--
The p75NTR death domain from
Leu-342 on was cloned into pGBT9 (CLONTECH) and
used to screen a C57Bl/6 P2 dorsal root ganglia neuron library cloned
into the HybriZap vector (Stratagene). No death domain proteins were
isolated. Interactions between the p75NTR death domain and other known
death signaling proteins (FADD death domain, full-length FADD, IAP1,
Trafs 1-6) cloned into pGAD10 (CLONTECH) were
tested by sequential yeast transformation for growth on media selective
for an interaction.
To investigate whether p75NTR signals death through interaction
with death domain containing proteins or via another pathway, we have
activated the p75NTR pathway in a similar fashion to experiments that
use ligand-free overexpression of TNFR to study apoptosis (18). We
overexpressed p75NTR in neurons by microinjecting a rat p75NTR
expression plasmid into the nucleus of mouse dorsal root ganglia
sensory neurons (Fig. 1) and cultured
them in the presence of the neural cytokine, LIF, to prevent
p75NTR-independent neuronal death. These neurons were chosen because we
had previously shown that their death was, at least in part, mediated
by p75NTR (5, 6). It was found after 16 h that approximately
20-25% of neurons injected with full-length p75NTR plasmid died (Fig. 2A), compared with neurons
injected with a control
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
retains Lys-274 and Arg-275 and the last 108 amino acids creating a cytoplasmic domain including the death domain but deleted for the 33-amino acid
juxta-membrane domain retained by p75NTRtr. All plasmids were
constructed using polymerase chain reaction to amplify desired coding
regions followed by subcloning the polymerase chain reaction products
into plasmid vectors. Details of primers used in construction of these
plasmids are available on request. Bcl-2 and Bcl-xL
plasmids are previously described (16, 17). The modified CrmA has the
caspase recognition sequence modified from wild-type Leu-Glu-Ala-Asp to
Asp-Gln-Met-Asp.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase plasmid or a truncated p75NTR
protein lacking the entire cytoplasmic domain (p75NTRnc) expressed to a
similar extent. The majority of the p75NTR-mediated death was observed
in the first 16 h (see Fig.
3C for example), comparable
with that reported for TNFR overexpression (18).
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Fig. 1.
Microinjection of sensory neuron with p75NTR
plasmid. A, photomicrograph of mouse postnatal day zero
sensory neuron being co-injected with rat p75NTR plasmid and
fluoro-ruby. B, phase-contrast photomicrograph of an
injected neuron that has been cultured for 24 h in NGF, showing
fluoro-ruby (panel C) and extensive rat p75NTR expression
(panel D) as revealed by immunofluorescence staining with
anti-rat-specific antibody. Expression of a Green Fluorescence Protein
plasmid (CLONTECH) was detectable as early as
6 h after injection, and surface rat p75NTR was strongly expressed
in over 80% of injected neurons by the end of the experiment.
Bar = 100 µm in panels A-C,
and 200 µm in panel D.
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Fig. 2.
Effect of p75NTR overexpression on mouse
sensory neuron survival. A, overexpression of p75NTR
resulted in a significant (* p < 0.5) increase in
neuronal cell death when compared with p75NTRnc or control
-galactosidase plasmid. Shown is a representative experiment, and
from eight separate experiments there was an average increase in cell
death of 21%. Neurons are cultured in the presence of
survival-promoting growth factor LIF. B, schematic of
different p75NTR proteins expressed from plasmids injected into neurons
(i), and their effect on neuronal survival compared with
p75NTR (100%) and p75NTRnc (0%) after 16 h (ii). Only
the proteins containing the juxta-membrane portion killed; whereas the
construct that contains only the death domain was ineffective.
Error bars indicate S.E., n = 3.
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Fig. 3.
Effect of Bcl-xL and
Bcl-2 expression plasmids on p75NTR-mediated killing.
A, Bcl-xL and p75NTR plasmids were co-injected
into neurons in the presence of LIF, and survival 16 h after
injection was assessed. Bcl-xL was able to totally rescue
neurons from p75NTR-mediated cell death. Note also that neurons
injected with Bcl-xL had increased survival compared with
Fig. 2A. B, although the survival of neurons in
the presence of LIF was not affected by expression of Bcl-2 plasmid
alone (compare with Fig. 2A), in combination with p75NTR
there was a significant increase in cell death. The ability of Bcl-2 to
promote cell death because of full-length p75NTR is not seen in the
presence of p75NTRnc. C, Bcl-2 and Bcl-xL have
opposite effects on the p75NTR-mediated killing within the first
16 h after injection. D, microinjection of either
p75NTR alone or p75NTR and Bcl-2 constructs failed to increase neuronal
death in the presence of NGF. E, death induced by withdrawal
of NGF is inhibited by expression of the microinjected Bcl-2 plasmid.
, uninjected and,
, Bcl-2-injected neurons in NGF;
,
uninjected and,
, Bcl-2-injected neurons after factor withdrawal. *,
p < 0.05; ** p < 0.01; ***
p < 0.001; error bars indicate S.E.,
n = 3.
Because the cytoplasmic tail of p75NTR is required for the cytotoxic
effect, we next examined whether deletion of the death domain also
abolished the ability of p75NTR to kill. Surprisingly, the neuronal
death observed after expression of p75NTR with a truncated cytoplasmic
tail (p75NTRtr), lacking the death domain, was not significantly
different from that observed with full-length p75NTR (Fig.
2B). Furthermore, a deletion p75NTR protein (p75NTR), containing the death domain but deleted for the juxta-membrane portion,
did not induce significant death (Fig. 2B). Thus, the death
domain was not required for p75NTR killing, and the juxta-membrane cytoplasmic tail is necessary and sufficient for killing. The p75NTR
death domain has recently been shown to have a different tertiary
structure to the death domain of the TNFR family and does not
self-associate in vitro (19), supporting the finding that
the p75NTR death domain does not function to induce death. Using the
yeast two-hybrid method to find p75NTR death domain binding partners,
we found no evidence of specific interaction with other death
domain-containing proteins (see "Materials and Methods"). Together,
these results suggest that an alternative pathway to one involving
death domain adapter proteins, such as TRADD and FADD, may be
responsible for p75NTR-mediated killing.
To further explore whether p75NTR killing was different from TNFR killing pathways, we examined whether overexpression of the anti-apoptotic Bcl-2 family proteins could inhibit killing by p75NTR. Bcl-2 and Bcl-xL are well characterized inhibitors of growth factor withdrawal and stress-induced apoptosis (15). However, both proteins are poor inhibitors of CD95/Apo1/Fas and TNFR-mediated apoptosis (20, 21).
We found that overexpression of Bcl-xL protected neurons against p75NTR-induced death (Fig. 3A), supporting the hypothesis that p75NTR signals through an alternative pathway to TNFR-induced apoptosis. Whereas Bcl-2 overexpression alone had no effect on cell survival in the presence of LIF (Fig. 3B), Bcl-2 in combination with p75NTR overexpression, surprisingly, enhanced the neuronal death seen with p75NTR overexpression alone, yet in combination with p75NTRnc it had no effect (Fig. 3B). The results are surprising because Bcl-2 is functionally indistinguishable from Bcl-xL in most cell-death systems (16, 22), yet in this assay we find that they have opposite effects (Fig. 3C). The cell death observed with p75NTR and Bcl-2 overexpression was totally ablated if the cells were cultured in NGF, confirming that signaling though trk may inhibit p75NTR activity (Fig. 3D). Bcl-2 was able to protect against neuronal death induced by NGF withdrawal (Fig. 3E). Thus, at the same expression levels in the same neuronal population, Bcl-2 was able to inhibit or enhance neuronal cell death depending on the nature of the death signal.
To determine whether the paradoxical effect of Bcl-2 on p75NTR-induced
killing was related to its known anti-apoptotic activity, inactive
Bcl-2 mutants were utilized (17). Like wild-type Bcl-2, expression of
the Bcl-2 mutants did not affect neuronal survival. In combination with
p75NTR expression, the enhanced killing effect seen with Bcl-2
co-expression was abrogated by expression of the mutant G145E Bcl-2,
suggesting the protein was nonfunctional (Fig. 4A). Thus, an intact BH1
homology region is required for both the survival and death promoting
activities of Bcl-2. Using a W188A mutated Bcl-2, we found that
co-expression with p75NTR not only abolished the increased p75NTR
killing but, more importantly, protected neurons from any
p75NTR-induced death (Fig. 4B), reminiscent of that seen
with Bcl-xL, despite being unable to protect against growth
factor withdrawal-induced death (17). Thus, the mechanism by which
Bcl-2 participates in the death pathway is separable from its function
in the survival pathway.
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Bcl-2 has previously been observed to increase cell death when highly
expressed both in vitro and in vivo (23-25).
Thus, it is possible that the high level of Bcl-2 is able to
"prime" the death pathway such that an apoptotic stimulus via
p75NTR results in rapid cell death. We examined whether Bcl-2 may not
only enhance p75NTR killing but may be essential for p75NTR killing to
proceed by testing whether lowering endogenous Bcl-2 would inhibit
p75NTR-mediated killing. We used a Bcl-2 antisense plasmid, which has
previously been shown to effectively lower Bcl-2 expression after
microinjection into neurons (26, 27). When the Bcl-2 antisense plasmid
was injected at the same time as the p75NTR plasmid, no diminution of
the death signal was seen. If, however, the neurons were microinjected in the presence of NGF, to give time for the antisense to deplete endogenous Bcl-2, and then switched into LIF the next day to permit p75NTR killing, there was a significant decrease in the ability of
p75NTR to kill neurons with lowered Bcl-2 (Fig.
5A); in fact, the level of
killing was not significantly different from that of neurons expressing
p75NTRnc (Fig. 5A). The specificity of the Bcl-2 antisense
was demonstrated by its inability to affect neuronal survival when
expressed alone or in conjunction with p75NTRnc (data not shown). In
addition, a control vector expressing green fluorescent protein had no
affect on p75NTR killing (data not shown).
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The requirement for endogenous Bcl-2 for p75NTR killing was also demonstrated in neurons from newborn mice lacking Bcl-2, which showed a significant reduction in cell death induced by p75NTR compared with wild-type litter mates (Fig. 5B), supportive of the Bcl-2 antisense experiments.
To investigate whether the p75NTR-Bcl-2 death-signaling cascade was
dependent on caspase activation, a modified CrmA, designed to inhibit
downstream group II caspases, such as caspase 3 (28, 29), was
overexpressed together with p75NTR. The modified CrmA was able to block
the killing induced either by p75NTR alone or by co-expression of
p75NTR and Bcl-2 in wild-type neurons (Fig. 5C), suggesting
that p75NTR-Bcl-2-induced apoptosis utilizes a caspase-dependent pathway.
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DISCUSSION |
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Bcl-2 has been shown to be cleaved by caspases, with the cleavage product being capable of promoting apoptosis in vitro (30). Cleavage of Bcl-2 may occur in our system but the mutated Bcl-2 proteins would be similarly cleaved, arguing that cleavage of Bcl-2 is unlikely to be the dominant mechanism by which Bcl-2 promotes killing in our system. In addition, if caspase cleavage was the dominant mechanism, Bcl-xL might also be expected to promote death in our system as Bcl-xL has been shown to be cleaved in a similar way (31).
Bcl-2 and Bcl-xL are likely to have a very similar tertiary
structure based on sequence similarity. The NMR structure of
Bcl-xL has been determined, showing a hydrophobic cleft
formed by seven helixes where other Bcl-2 family proteins interact
(32). Bcl-xL, and Bcl-2, may function differently by
specific interaction with proteins that mediate p75NTR-induced death.
For example, pro-apoptotic Bcl-2 family members, Bak and Bad, interact
strongly with Bcl-xL but weakly with Bcl-2 (33, 34). Also,
Bcl-xL, but not Bcl-2 as yet, has been shown to interact
with Apaf-1, the mammalian CED-4 homologue, to regulate apoptosis via
caspase-9 activation (35). Mutation of Bcl-2 at Gly-145 or Trp-188 may
affect its interaction with pro- and anti-apoptotic proteins by
conformational changes disrupting access to the cleft. This suggests
that the conformation of the Bcl-2 protein is integral to the
paradoxical functions observed herein and that Bcl-2 participates in
the p75NTR-killing pathway and stress-induced survival by different
molecular mechanisms.
The different neuronal phenotype observed in Bcl-2- and Bcl-xL-deficient animals is consistent with our results that Bcl-2 and Bcl-xL can have quite different functions in regulating neuronal death. Mice deficient for Bcl-xL have a major loss of neurons shortly after their differentiation (36), whereas Bcl-2-deficient mice show only minor loss of neurons during embryogenesis and early neonatal life (37).
The latter phenotype would support the idea that during development Bcl-2 plays two contrasting roles: under conditions where there are appropriate neuritic connections leading to trk signaling, Bcl-2 may act to enhance neuronal survival, as has been shown for trigeminal sensory neurons in vitro (26, 27); alternatively, as observed, Bcl-2 may also be capable of promoting neuronal death at other stages, for example when neurons may become more dependent on neural cytokine (e.g. LIF, CNTF, OsM) support in early postnatal development (38, 39). The LIF family signal through Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathways, which can be inhibited by the neurotrophin-activated mitogen-activated protein (MAP) kinase pathway (40). Thus, it is possible that the in vivo switch of neuron dependence between neurotrophins and neural cytokines may prime the neurons to cell death. Activation of p75NTR then may occur through NGF or BDNF signaling as has been recently shown (10, 41).
The apparent paradoxical actions of Bcl-2 may be a program by which
rapid selection of cell survival or death occurs during neuronal
development and after nerve injury. There are high levels of Bcl-2 in
neurons of both the central and peripheral nervous systems during
periods of developmental cell death (42), and activation of p75NTR,
also expressed widely in the nervous system during development, in the
presence of high Bcl-2, would lead to rapid apoptosis.
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ACKNOWLEDGEMENTS |
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We thank Dr. D. C. Huang, Walter and Eliza Hall Institute (WEHI), for helpful discussions, critical reading of the manuscript, anti-human Bcl-2 antibody, and the Bcl-2 and Bcl-xL plasmids; Dr. Cris Print, WEHI, for assistance with Bcl-2 genotyping; and Dr. P. Ekert, WEHI, for modified CrmA plasmid.
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FOOTNOTES |
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* This work was supported in part by the National Health and Medical Research Council of Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: +61 3 9345-2537; Fax: +61 3 9347-0852; E-mail: bartlett{at}wehi.edu.au.
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ABBREVIATIONS |
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The abbreviations used are: p75NTR, p75 neurotrophin receptor; NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; TNFR, tumor necrosis factor receptor; LIF, leukemia inhibitory factor.
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