INSERM UMR623, Developmental Biology Institute of Marseille (IBDM), CNRS INSERM Université Méditerranée, Campus de Luminy Case 907, 13288 MARSEILLE Cedex 09, France
Author for correspondence (e-mail:
chris{at}ibdm.univ-mrs.fr)
Accepted 4 June 2004
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SUMMARY |
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Key words: Motoneuron, Survival pathways, Axon growth, In ovo electroporation
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Introduction |
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One apparent difference between vertebrates and invertebrates lies in the
degree to which cell death is predetermined on a cell-by-cell basis. Within
large populations of vertebrate neurons, the neurotrophic hypothesis suggests
that those neurons that survive do so as a result of successful competition
for access to target-derived neurotrophic factors, in an essentially
stochastic manner (Davies,
1996; Lewin and Barde,
1996
). However, in the nematode C. elegans, the
developmental pathway is more hard-wired, in that the same specific neurons
die in each animal (Sulston,
1983
). However, even in vertebrates, the action of extrinsic
regulators (e.g. neurotrophic factors) may be modulated by intrinsic factors
that predispose a given neuron to die or to survive. This would be of interest
for studies of both normal development and neurodegenerative pathologies.
Cell-death specification (CES) genes in C. elegans provide a
particularly interesting model (Ellis and
Horvitz, 1991). During development of the NSM neurons
(neurosecretory motoneurons) that innervate the pharynx, a precursor divides
to form two daughter cells, one of which normally undergoes programmed cell
death and the other of which becomes an NSM. In the absence of CES-2
activity, both sister cells survive and become NSM neurons. Thus, the
principal role of CES-2, which is a bZIP transcription factor related to the
PAR (proline acidic rich) family, is to predispose 50% of the NSM population
to cell death (Metzstein et al.,
1996
).
Vertebrate homologs of CES-2 have been identified. Although their sequence
similarity is distant, they share a consensus DNA-binding site
(Cowell et al., 1992;
Drolet et al., 1991
;
Falvey et al., 1995
;
Fonjallaz et al., 1996
;
Haas et al., 1995
;
Hunger et al., 1992
). One of
these, E4BP4 (also known as NFIL3), can act in different contexts as a
transcriptional repressor or activator
(Cowell et al., 1992
;
Lai and Ting, 1999
;
Zhang et al., 1995
). In pro-B
lymphocytes, which depend on the cytokine IL3 for their survival, E4BP4 is
induced by IL3, and is itself sufficient for survival in the absence of
cytokine (Ikushima et al.,
1997
; Kuribara et al.,
1999
). Thus, E4BP4 in pro-B cells, in contrast to CES-2 in the
nematode, is an anti-apoptotic factor.
We therefore asked whether E4BP4 might play a role in the survival of vertebrate motoneurons. We show that E4BP4 is expressed by motoneurons at the time at which their survival is being determined. Moreover, overexpression of E4BP4 in cultured motoneurons protects them against cell death. Importantly, overexpression in vivo reduces the number of dying motoneurons during development. Together with our observations that E4BP4 potently enhances motoneuron growth, these results define E4BP4 as a novel potential player in vertebrate motoneuron development and pathology.
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Materials and methods |
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For RT-PCR analysis, total RNA was isolated either from purified mouse motoneurons or from chicken spinal cords at various stages. cDNAs were synthesised with random oligonucleotide primers. The primers used for PCR amplification of E4BP4 sequences were: chicken, 5'-aaggatgctatgtattgggaga-3' and 5'-acagttgttgagctactgag3'; and mouse, 5'-taccagacatccaaggctgc-3' and 5'-ccaatcttgaatgttcgtcac-3'. All fragments were sequenced to verify the amplified sequences. The GAPDH control was amplified with the following PCR primers: 5'-gtcaacggatttggccgtat-3' and 5'-aatgccaaagttgtcatggatg-3'.
In situ hybridisation
Plasmids containing full-length chicken E4BP4 or islet 1
(ISL1) (chicken or rat) were used to synthesise digoxigenin-labelled
antisense riboprobes according to the supplier's protocol (Roche) and purified
on spin columns (Qiagen). In situ hybridisation on tissue sections was
performed as previously described
(Schaeren-Wiemers and Gerfin-Moser,
1993; Yamamoto and Henderson,
1999
). Briefly, chicken embryos of various stages were fixed
overnight (4% PFA in PBS), cryopreserved (20% sucrose in PBS) and embedded in
OCT (Miles). Cryosections (12-14 µm) were acetylated [0.1 M
triethanolamine/HCl (pH 8.0), 0.25% acetic anhydride, 10 minutes at room
temperature] and hybridised with riboprobe (150 ng/slide) overnight at
65°C. The sections were washed (1xSSC, 50% formamide, 0.1% Tween20,
2x45 minutes, 65°C) and blocked in the presence of 20% inactivated
goat serum prior to incubation overnight with AP-conjugated
anti-DIG-Fab-Fragments (Roche, 1:3000). After extensive washing, hybridised
riboprobes were revealed by performing a NBT/BCIP reaction. Negative controls
were performed using sense probes.
Whole-mount in situ hybridisation on E14 rat spinal cord was performed as
previously described (Garcès et
al., 2001). Riboprobes were hybridised at 68°C overnight.
Hybridisation was detected using AP-conjugated anti-DIG Fab-fragments and
NBT/BCIP. The spinal cords were flat-mounted as an `open book'
preparation.
Electroporation, culture and morphometrical analysis of primary rat motoneurons
Rat motoneurons were purified from E14 spinal cords using a
p75NTR antibody and magnetic beads as previously described
(Arce et al., 1999). Purified
motoneurons were resuspended in electroporation buffer [125 mM NaCl, 5 mM KCl,
1.5 mM MgCl2, 10 mM glucose, 20 mM HEPES (pH 7.4)] at a density of
50,000 cells in 50 µl of buffer and transferred to an electroporation
cuvette (4 mm). E4BP4-pCAGGS expression vector (3 µg) and/or GFP-pCAGGS (at
least 1 µg/µl in PBS) were added, gently mixed and incubated for 15
minutes at room temperature. Electroporation was performed with the following
protocol: 200 V, three pulses, 5 ms pulse length, 1 second interval (BTX, ECM
830). Immediately after electroporation, 500 µl of complete Neurobasal
culture medium (Life Technologies) with supplements (2% B27 supplement, 2%
horse serum, 0.5 mM L-glutamine, 25 µM ß-mercaptoethanol) were added.
The transduction rate judged by GFP expression after 2 days varied between 30
and 70% of surviving motoneurons. Previous experiments using GFP and tagged
proteins had shown that the degree of co-transfection obtained by this method
is high (>90%) (Raoul et al.,
2002
). The cells were distributed into four 16-mm wells and growth
factors or enzyme inhibitors were added either alone or in combination (GDNF 1
ng/ml, BDNF 10 ng/ml, CNTF 10 ng/ml, LY294002 10 µM). Given that
transfection is monitored by GFP expression, it was not possible to determine
directly the number of transduced motoneurons at the time of seeding. We
therefore quantified the survival rate by counting all GFP-positive
motoneurons in each culture dish after 24 hours or 48 hours, and expressing
them as a percentage of the number surviving in the presence of neurotrophic
factors at the same time. To trigger death, cells were treated with BDNF at
seeding, treated with soluble Fas ligand (0.1 µg/ml, Alexis) and Fas
enhancer (1 µg/ml, Alexis) after 24 hours, and counted 24 hours later. For
morphological analysis, pictures of motoneurons (80-200 of each condition)
were taken on an inverted fluorescence microscope after first counting the
cells for E4BP4 survival effects. Neurite outgrowth was analysed by
measuring the longest and the total neurite length of all GFP-positive
motoneurons in each experiment. Pictures were taken without prior fixation of
the cells. All processes were marked by hand and the length finally determined
by software (Visiolab, Biovision). Cell body growth was addressed by measuring
the pixel area of each GFP-positive motoneuron cell body. All pictures were
analysed and normalised using Lucia G software (Nikon).
Semi-quantitative RT-PCR for E4BP4
E4BP4 mRNA was quantified in cultures of dissociated cells from
E13 rat ventral spinal cord. For each data point, 300,000 cells were cultured
for 6, 16, 18 or 24 hours in the absence or presence of neurotrophic factors
(GDNF, 2 ng/ml; BDNF, 10 ng/ml; CNTF, 10 ng/ml). Total RNA was isolated by
using the Qiagen RNeasy protocol. The RNA was treated with DNAse (Invitrogen)
for 30 minutes, cleaned (RNeasy, Qiagen) and finally dried using a speed vac.
First strand cDNA was synthesised using random oligonucleotide primers
(Superscript II protocol, Invitrogen). The following primers were used for PCR
amplifications: rat E4BP4, 5'-gctctcggatgtgtctgagc-3' and
5'-tggggacctgctgctcgtct-3'; rat actin,
5'-ttgtaaccaactgggacgatatgg-3' and
5'-gatcttgatcttcatggtgctagg-3'. cDNA samples were diluted 10-fold
before amplification with the following protocol: 94°C for 30 seconds,
55°C for 30 seconds, 72°C for 30 seconds, 28 cycles (E4BP4)
or 22 cycles (actin). These cycle numbers were confirmed to correspond to the
proportional phase of amplification. After Southern blot transfer, radioactive
hybridisation was performed using 5'-labelled internal primers
(E4BP4, 5'-gagctacatgggtagctctttctccac-3'; and
actin, 5'-acctgacagactacctcatgaagatcc-3'). The intensity
of hybridised PCR bands was measured using Image J software. For each
individual experiment, the ratio of E4BP4 to actin intensities in
control conditions was calculated (termed Rc). The corresponding
ratio for the same cell preparation cultured with neurotrophic factors
(Rn) was then expressed as a fraction of the former. This intensity
ratio (Rn/Rc) was calculated for a total of 30
independent preparations and plotted as mean±s.e.m.
(Fig. 5C).
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Electroporation of chicken embryo spinal cord
Fertilised chicken eggs were incubated for 55-60 hours until they reached
stage 15 to 17 (Hamburger and Hamilton,
1951). After opening the shell, black ink (Waterman 1/10 in PBS,
0.22 µm filtered) was injected below the embryo and 100 µl PBS were
added above. Using a sharpened tungsten needle, a small hole was made in the
dorsal membrane of the spinal cord at somite level 22, providing access to the
neural tube. Freshly prepared DNA solution was injected into the neural tube
with a glass capillary. The DNA was prepared as follows: 15 µl
cE4BP4-pCAGGS (5-7 µg/µl water) + 3.5 µl GFP-pCAGGS (5 µg/µl
water) + 1.5 µl Fast Green (0.3% in water) + 2.2 µl 10xPBS. For
control electroporation, cE4BP4-pCAGGS was replaced by GFP-pCAGGS. The first
electrode was placed on the vitelline membrane (under PBS) and the second
under the membrane (after making a small hole into it) and beside the embryo,
so as to electroporate most efficiently the cells of the ventral horn.
Electroporation protocol: six pulses, 22-23 V, 30 ms pulse length, 1 second
interval (BTX, ECM 830). Finally, 200 µl PBS with antibiotics were layered
over the embryo and after closing the shell with tape, embryos were incubated
for 3-8 days.
TUNEL labelling of whole mount spinal cords and analysis
After removing viscera, the vertebral column of electroporated chicken
embryos (E6.5 or E7.5) was opened from the ventral side and electroporation
confirmed by visualizing co-electroporated GFP under a fluorescence binocular
(Zeiss). Only when GFP was visible in nerves that projected into the wings
were embryos retained for TUNEL analysis. Spinal cords were removed (if
necessary the dorsal side was opened) and fixed (4% PFA overnight).
Whole-mount TUNEL labelling was performed as described
(Yamamoto and Henderson,
1999). The NBT/BCIP reaction was stopped in PBSE (PBS, 5 mM EDTA)
and the spinal cords post-fixed in 4% PFA/PBS overnight. Finally, the spinal
cords were incubated in storage solution (80% glycerol, 0.5% PFA in PBS) and
flat-mounted under a dissecting microscope. The area of electroporation was
visualised by detection of the remaining GFP expression with a fluorescence
dissecting microscope. All TUNEL-positive nuclei in the GFP-positive area of
the electroporated and the contralateral side were counted (viewing from the
ventral side of the spinal cord) and compared with each other. The
TUNEL-positive nuclei of an area of the same length rostral and caudal of the
GFP positive area were analysed in the same way. All data were controlled by
recounting the positive nuclei after turning the spinal cords (viewing from
the dorsal side of the spinal cord).
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Results |
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Cell-autonomous survival-promoting activity of E4BP4 for motoneurons
We took a gain-of-function approach to analyse the potential role of E4BP4
expressed in motoneurons. When motoneurons are purified from rat embryos and
cultured at low density without neurotrophic factors such as BDNF or GDNF,
50% undergo programmed cell death during the first day of culture
(Henderson et al., 1993
;
Henderson et al., 1994
); this
is generally considered to model cell death in vivo as neurons compete for
access to trophic support. We reasoned that this would provide a means of
assaying for factors that either exacerbated (pro-apoptotic) or inhibited
(anti-apoptotic) cell death. In order to overexpress the transcription factor
E4BP4 in motoneurons, we used a technique recently developed in our laboratory
for electroporation of neurons in suspension, which gives transduction rates
of at least 50% of surviving motoneurons, and a co-electroporation efficacy of
90% with reporter plasmids (Raoul et al.,
2002
). Only about 10% of the electroporated motoneurons survive
the electroporation procedure. However, they show normal healthy morphology
and retain normal survival and death responses, suggesting that they are
representative of the complete population of motoneurons.
Survival values were expressed as a percentage of the number of neurons surviving with neurotrophic support at the same time. When electroporated with GFP vector alone, more than 40% of motoneurons died after 1 day in culture in the absence of trophic support (Fig. 2A), as with non-electroporated neurons. Co-electroporation with the E4BP4 plasmid reduced this figure to less than 10%, even after 2 days (Fig. 2A). Thus, forced expression of E4BP4 has similar effects to those of exogenous trophic factors. In long-term cultures (5 days in vitro), the survival effect was barely significant (Fig. 2A), but it was not clear whether this was due to loss of E4BP4 expression or to limited duration of the E4BP4 effect. To determine whether E4BP4 and BDNF were acting on the same motoneuron population, we electroporated motoneurons with either GFP vector alone, or with both GFP and E4BP4 vectors and cultured them in optimal concentrations of BDNF. The ratio of the number of neurons expressing GFP alone to that expressing GFP plus E4BP4 was 0.95±0.05 (mean±s.e.m., n=6 independent experiments). Very similar results were obtained using other potent trophic factors for motoneurons such as GDNF or a combination of BDNF, GDNF and CNTF (see Materials and methods). These data strongly suggest that E4BP4 and neurotrophic factors act on the survival of the same population of motoneurons, although they do not formally exclude the possibility that E4BP4 keeps alive a different population while having a pro-apoptotic effect on BDNF-responsive cells.
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Many neurotrophic factors act through PI3 kinase to mediate their survival
effects, and in pro-B cells it has been shown that PI3K can act upstream of
E4BP4 to enhance survival (Kuribara et
al., 1999). Low concentrations (10 µM) of the PI3K inhibitor
LY294002 were added to electroporated motoneurons in different conditions.
LY294002 only slightly reduced survival of GFP-expressing motoneurons cultured
in basal medium (Fig. 2C) but
completely inhibited the trophic effect of BDNF (not shown). Inhibition of PI3
kinase completely blocked the anti-apoptotic effect of E4BP4
(Fig. 2C).
Effects of E4BP4 on cell size
In order to determine whether E4BP4 affected other aspects of motoneuron
development, we performed quantitative analysis of cell body area
(Fig. 3). As expected, addition
of BDNF tended to increase cell size. Surprisingly, the effect of E4BP4 on
cell size was even greater (Fig.
3A). Results from five independent experiments were combined by
calculating a median value (area attained by 50% of individual neurons) from
each data set (Fig. 3B). Whereas BDNF increased neuronal area by 16%, E4BP4 expression led to a 44%
increase. As BDNF and E4BP4 act on the same motoneurons (see above), this must
reflect the increase in size of individual neurons induced by E4BP4, and not
the survival of a subpopulation of large neurons. Addition of BDNF to cells
overexpressing E4BP4 did not further increase cell body size (7.3±0.3
pixels; n=3), suggesting that cells that responded to BDNF
represented a subpopulation of the E4BP4-sensitive neurons. As in the case of
the survival experiments, the effect of E4BP4 on cell size was blocked by 10
µM LY294002 (not shown).
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In vitro, therefore, E4BP4 has profound effects on neuronal growth. In our experiments, these were greater than those of BDNF or GDNF (not shown). However, this may simply reflect stronger stimulation of growth pathways by overexpression of E4BP4 than by application of exogenous growth factors. The non-parallelism of the E4BP4 and BDNF curves in Fig. 4C may reflect the fact that E4BP4 is active in all electroporated neurons, whereas only a fraction of them respond to BDNF.
Regulation of E4BP4 in cultured motoneurons by neurotrophic factors
Levels of E4BP4 in pro-B cells are upregulated by survival factors. To
determine if this was the case in motoneurons, we looked for regulation of
E4BP4 mRNA and protein in cultured ventral spinal neurons from E13 rat as a
function of neurotrophic support. The choice of embryonic age reflected our
findings (Fig. 1) that levels
of E4BP4 are lower at the beginning of the cell death period. Cells were
cultured (or not) with a cocktail of growth factors composed of BDNF, CNTF and
GDNF (see Materials and methods). We analysed expression levels of
E4BP4 in these cultures 6 hours after plating by semi-quantitative
RT-PCR (Fig. 5A). To avoid high
PCR cycle numbers, we then performed specific radioactive hybridisation to
detect RT-PCR products. Under these conditions, the expression level of
E4BP4 in motoneurons was not changed by treatment with trophic
factors (Fig. 5C). At later
time points (16, 18 or 24 hours after plating; data not shown) levels remained
constant. Similarly, no change in E4BP4 levels was observed when we
tested BDNF (10 ng/ml), CNTF (10 ng/ml), GDNF (1 ng/ml), HGF (10 ng/ml), NT3
(3.8 ng/ml), BMP7 (10 ng/ml) or CT1 (10 ng/ml) alone (data not shown). To look
for regulation of E4BP4 protein, we performed western blot analysis on
dissociated rat E13 spinal neurons. Again, E4BP4 levels were not altered in
the presence of a cocktail of BDNF, CNTF and GDNF
(Fig. 5B,C). We therefore
conclude that E4BP4 expression levels in motoneurons are not regulated by
neurotrophic factors in vitro.
E4BP4 is a neuronal survival factor in vivo
The potency of these unexpected functional effects of E4BP4 led us to ask
whether E4BP4 might affect the survival of motoneurons during the cell death
period in vivo. For this, we used electroporation in ovo to transduce
motoneuron precursors at early stages, with the aim of analysing effects on
survival at later stages (Fig.
6A; see Materials and methods). When expression plasmids encoding
GFP were introduced into one side of the brachial neural tube at E2.5,
distinct fluorescence in motoneurons could still be detected up to 6 days
later. Although this may in part reflect the intrinsic stability of the GFP
protein, it also suggested that unintegrated plasmids are sufficiently stable
in these conditions to have functional effects during the motoneuron cell
death period. Therefore this approach, which has been much used for studies of
early development, will be of general interest for studying later phenomena
such as cell death.
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Overexpression of GFP alone did not affect motoneuron cell death (Fig. 6D). However, E4BP4 clearly modified the pattern of dying motoneurons. In the single embryo illustrated (Fig. 6C), three representative zones are apparent. In zone 1, expression of E4BP4 in a position that was dorsal to motoneurons undergoing cell death did not affect the TUNEL signal (asterisk). By contrast, in zone 2, E4BP4 expressed in motoneurons that were actively undergoing cell death significantly reduced the number of TUNEL-positive profiles (compare black and open arrowheads). Last, in zone 3, where no PCD was normally detected by TUNEL, overexpression of E4BP4 in motor columns did not induce apoptosis (black arrow).
To quantify effects of E4BP4 on motoneuron death, we defined for each embryo a zone of apparently continuous GFP expression in the ventral horns (`electroporated area' in Fig. 6A,D) and counted the number of TUNEL-positive nuclei on each side. The result was expressed as a ratio of treated to untreated sides. To eliminate the possibility of asymmetric TUNEL labelling resulting from other causes, two GFP-negative regions immediately rostral and caudal to this zone in each spinal cord were also counted (Fig. 6A; `control area' in Fig. 6D). Overexpression of E4BP4 led to a 45% reduction in the number of TUNEL-positive profiles (Fig. 6D), demonstrating its potent anti-apoptotic activity in vivo. Given that probably not all motoneurons in the `electroporated zone' expressed GFP, this may correspond to saving of an even higher percentage of those motoneurons effectively transduced.
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Discussion |
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In common with E4BP4 in pro-B lymphocytes, but in contrast to CES-2 in
C. elegans, E4BP4 in motoneurons is anti-apoptotic in vivo and in
vitro. The complete resistance to Fas-triggered cell death conferred by E4BP4
provides a particularly striking demonstration of this. The different
functions of CES-2 in NSM neurons and E4BP4 in pro-B cells or motoneurons most
probably result from different genetic interactions with downstream effectors:
CES-2 inhibits the anti-apoptotic CES-1 transcription factor
and thereby triggers cell death, whereas the fusion protein E2A-HLF, which is
thought to act through a similar mechanism to E4BP4, activates the
anti-apoptotic CES-1 homolog Slug and therefore promotes survival
(Ikushima et al., 1997;
Inukai et al., 1999
;
Metzstein et al., 1996
;
Metzstein and Horvitz, 1999
).
No expression of Slug, Snail or their direct homologs has been described in
motoneurons, and so the pathway downstream of E4BP4 may be novel.
The actions of E4BP4 are most probably cell-autonomous, as expected for a
transcription factor. It can act in low-density cultures to enhance the
survival of those cells that express it, and in vivo when expressed more
dorsally in the spinal cord it does not affect motoneuron survival at the same
rostrocaudal level. Thus, E4BP4 is probably an intrinsic determinant of cell
survival. Indeed, the role of transcriptional events in controlling the
response of neurons to death and survival factors is becoming progressively
more apparent (Brunet et al.,
2001; Wiese et al.,
1999
). Two examples from our laboratory include the requirement
for transcription of REG-2 in the CNTF survival pathway
(Nishimune et al., 2000
) and
the upregulation of nNOS in a motoneuron-specific death pathway
triggered by the Fas receptor (Raoul et
al., 2002
). Of particular interest in the present context is the
report that NGF-mediated neuronal survival requires CREB
(Lonze et al., 2002
;
Riccio et al., 1999
), which
participates in similar transcription factor complexes to E4BP4 and is also
required in the IL3 survival pathway (Chen
et al., 2001
). Neurons, which are essentially irreplaceable cells,
may have developed slower, but more tightly controlled, mechanisms to regulate
their numbers.
The effects of E4BP4 on growth of cell bodies and axons were at least as
striking as those on survival. What determines the final size of a neuron, or
indeed any particular cell type, has been little studied
(Conlon and Raff, 1999).
However, recent reports point to a crucial involvement of the PI3 kinase
pathway (Backman et al., 2001
;
Groszer et al., 2001
;
Heumann et al., 2000
;
Kwon et al., 2001
;
Markus et al., 2002
;
Namikawa et al., 2000
). This
fits well with our observations that all effects of E4BP4 in motoneurons were
completely inhibited by the PI3K inhibitor LY294002, suggesting that either
E4BP4 activates the PI3K pathway, or PI3K needs to be activated by other means
in order to cooperate with E4BP4 in promoting survival and growth. Indeed,
E4BP4 can itself be phosphorylated in certain situations
(Chen et al., 1995
;
Zhang et al., 1995
).
Unfortunately, the biochemical studies required to distinguish between these
possibilities are not accessible with the low quantities of material available
using electroporated motoneurons.
The literature suggested another potential level of involvement of PI3K: in
pro-B cells, PI3K acts upstream of E4BP4: survival factors activate PI3K and
thereby upregulate E4BP4 (Kuribara et al.,
1999). However, unexpectedly, E4BP4 levels in motoneurons did not
change following treatment with classical growth and survival factors. Our
results suggest that E4BP4 is not simply a read-out or signalling intermediate
for exogenous survival signals, but rather may have significant
cell-autonomous functions, as in the nematode.
Our gain-of-function studies in vitro and in vivo gave results consistent
with our deduction from the expression pattern in vivo. Nevertheless, it will
obviously be of interest to determine by loss-of-function studies to what
extent this pathway is active during normal development. We ruled out the use
of a dominant-negative approach because of the non-specificity of
heterodimerisation of transcription factors, and because interpretation would
be complicated by the ability of E4BP4 to function either as a trans-repressor
or a trans-activator (Cowell et al.,
1992; Lai and Ting,
1999
; Zhang et al.,
1995
). Null-mutant mice for E4BP4 have not been reported.
In conclusion, the parallels between the expression of CES-2 in
neurosecretory motoneurons of the C. elegans pharynx and of E4BP4 in
rat and chicken spinal motoneurons provide another example of the evolutionary
conservation of elements of cell death and survival pathways. However, as in
many other cases, the function of E4BP4 seems to have been modified during
development. In particular, E4BP4 functions in vertebrate neurons as an
intrinsic survival factor, in accordance with its expression pattern in
developing spinal cord and its regulation. The pathways activated by E4BP4
therefore represent potentially interesting targets for therapeutic strategies
aimed at nervous system regeneration and repair. Given its function in other
systems as a clock gene (Doi et al.,
2001; Mitsui et al.,
2001
), it is also interesting to speculate that E4BP4 may also be
involved in the timing of cell death during development.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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