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The Journal of Neuroscience, February 15, 1998, 18(4):1428-1439
Widespread Elimination of Naturally Occurring Neuronal Death in
Bax-Deficient Mice
Fletcher A.
White1,
Cynthia R.
Keller-Peck1,
C.
Michael
Knudson2,
Stanley J.
Korsmeyer2, 3, and
William D.
Snider1
1 Department of Neurology, Center for the Study of
Nervous System Injury, 2 Departments of Pathology and
Medicine, and 3 Howard Hughes Medical Institute, Washington
University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
The proapoptotic molecule BAX is required for death of sympathetic
and motor neurons in the setting of trophic factor deprivation. Furthermore, adult Bax / mice have more motor neurons
than do their wild-type counterparts. These findings raise the
possibility that BAX regulates naturally occurring cell death during
development in many neuronal populations. To test this idea, we
assessed apoptosis using TUNEL labeling in several well-studied neural
systems during embryonic and early postnatal development in
Bax/ mice. Remarkably, naturally occurring cell
death is virtually eliminated between embryonic day 11.5 (E11.5) and
postnatal day 1 (PN1) in most peripheral ganglia, in motor pools in the
spinal cord, and in the trigeminal brainstem nuclear complex.
Additionally, reduction, although not elimination, of cell death was
noted throughout the developing cerebellum, in some layers of the
retina, and in the hippocampus. Saving of cells was verified by axon
counts of dorsal and ventral roots, as well as facial and optic nerves
that revealed 24-35% increases in axon number. Interestingly, many of
the supernumerary axons had very small cross-sectional areas,
suggesting that the associated neurons are not normal. We conclude that
BAX is a critical mediator of naturally occurring death of peripheral
and CNS neurons during embryonic life. However, rescue from naturally
occurring cell death does not imply that the neurons will develop
normal functional capabilities.
Key words:
primary afferent neurons; motor neuron; apoptosis; programmed cell death; Bcl-2 gene family; BAX
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INTRODUCTION |
Naturally occurring cell death is a
widespread phenomenon serving a variety of fundamental functions in
multicellular animals. Within the developing nervous system, most
(although not all) classes of neurons seem to undergo this process to
some degree (Oppenheim, 1991 ). The traditional view has been that
neurons extend axons into target fields and then "compete" for
trophic factors, with the "losers" undergoing programmed cell
death. The recent introduction of sensitive techniques to detect DNA
fragmentation in situ has substantially refined this view.
For example, recent histological analyses in mice have revealed
naturally occurring cell death present in cells of the peripheral
nervous system before the period of target innervation (ElShamy and
Ernfors, 1996; Fariñas et al., 1996; White et al., 1996). Further
unexpected results have demonstrated that cell death is an ongoing
process during the proliferation of neuronal progenitors in the
ventricular zone of embryonic telencephalon and during the neuronal
migration of cells in the intermediate zone of the developing cortex
(Blaschke et al., 1996; see also Schindler et al., 1997). Taken
together, these results suggest that cell death occurs during several
phases of neural development and that the cell death may be more
extensive than appreciated previously. Interestingly, despite these new insights, the function served by naturally occurring cell death in the
nervous system remains obscure.
Major advances in understanding naturally occurring cell death have
been stimulated by the discovery of the BCL-2 (for review, see Merry
and Korsmeyer, 1997) and Caspase (for review, see Martinou and Sadoul,
1996) families of proteins that regulate apoptosis. Analysis of the
Bcl-2 gene family has revealed a broad range of both
promoters and suppressors of apoptosis. Many of these molecules are
present and active within the nervous system. For example, elimination
of BCL-2, a cell death inhibitor, leads to progressive degeneration of
both motoneurons and sensory neurons at early postnatal ages
(Michaelidis et al., 1996). Likewise, targeted disruption of
Bcl-xL, a Bcl-2 related gene,
reveals an early requirement for this molecule to prevent programmed
cell death (Motoyama et al., 1995). In addition to the death-inhibiting
members of the Bcl-2 gene family, death-promoting members,
BAX (Oltvai et al., 1993), BAK (Kiefer et al., 1995), and BAD (Yang et
al., 1995), have been characterized. Targeted disruption of the
Bax gene has shown that BAX is required for at least two
populations of neurons to undergo programmed death in the setting of
trophic factor or target deprivation (Deckwerth et al., 1996).
Furthermore, motoneurons in the facial nucleus are increased in number
in these mice, suggesting an effect on naturally occurring neuron death
as well (Deckwerth et al., 1996). Finally, introduction of the
Bax null mutation into
Bcl-xL-deficient mice greatly reduces the
excessive apoptosis that occurs in the developing nervous system in
Bcl-xL-deficient mice (Schindler et al.,
1997).
The discovery that neuronal apoptosis is powerfully regulated by BAX
offers the opportunity to define periods of naturally occurring cell
death, determine which neuronal populations are regulated, and
determine the fate of supernumary neurons that ordinarily die during
nervous system development. We have addressed these issues in
Bax/ mice by studying several well-characterized neural
systems during embryonic and early postnatal life. We show here that
BAX primarily regulates the survival of postmitotic neurons. This
effect is seen in most populations of peripheral neurons and many
populations of CNS neurons. Perhaps surprisingly, many neurons in
BAX-susceptible populations are markedly atrophic, suggesting that
these cells may not function normally.
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MATERIALS AND METHODS |
Animals. Wild-type and Bax/ mice were
obtained from overnight matings [day of vaginal plug = embryonic
day 0.5 (E0.5)] of mice heterozygous for Bax (Knudson et
al., 1995). Pregnant females were killed by halothane overdose to
harvest embryos on E11.5, E12.5, E13.5, E14.5, E15.5, and E17.5.
Staging of embryos was verified by crown-rump length and degree of
limb development (Kaufman, 1992).
The genotyping of mice was performed by PCR using a set of three
primers: Bax exon 5 forward primer
(5'-TGATCAGAACCATCATG-3'), Bax intron 5 reverse primer
(5'-GTTGACCAGAGTGGCGTAGG3'), and Neo reverse primer
(5'-CCGCTTCCATTGCTCAGCGG-3'). Cycling parameters were 5 min at 94°C
for one cycle and 1 min at 94°C, 1 min at 55°C, and 1 min at 74°C
each for a total of 30-35 cycles. PCR products were resolved on a
1.5% agarose gel.
Staining and analysis of apoptotic figures. Whole staged
embryos were immediately frozen on dry ice and stored at 80°C.
Before use, embryos were embedded in Tissue-Tek OCT compound (Miles, Elkhart, IN) and sectioned at 18 µm on a cryostat. To detect cell death in the DRG, we stained sections with the terminal
deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)
method using the ApopTag fluorescent detection kit (Oncor,
Gaithersburg, MD) according to the instructions of the manufacturer.
Slides were then washed three times (5 min each) with PBS and
visualized with FITC.
The TUNEL method labels cells containing fragmented DNA, a hallmark of
apoptosis. Whether a cell exhibited positive labeling (FITC-labeled
3'-OH ends of fragmented DNA) was evaluated by examination of tissue
sections using epifluoresence at a magnification of 225×. Cells
undergoing apoptosis were recognized by an intensely fluorescent
nucleus. For lumbar DRG quantitative analysis, fluorescent cells were
counted in at least 20 lumbar DRG or spinal cord sections per animal,
and a mean number of TUNEL-positive (TUNEL+) cells per section was
determined. All observations reported are based on analysis of multiple
tissue sections from three to five Bax/ mice at each of
the ages indicated and from similar numbers of Bax+/+
littermates.
Histological analysis of myelinated-axon number and area.
After overdose with halothane, adult animals were perfused with 2%
paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate
buffer (PB), pH 7.4. The dorsal and ventral roots of lumbar levels 4 and 5 (L4/L5) and the optic and facial nerves were removed, soaked in
fresh fixative overnight, rinsed in PB, osmicated for 30 min in 1%
osmium tetraoxide, rinsed, dehydrated in ascending concentrations of
ethyl alcohol, and embedded in Epon resin. Semithin sections were cut
at 1 µm. A minimum of three Bax+/+ and three
Bax/ mice were studied.
Cross-sections of each nerve root were photographed, montages were
constructed, and myelinated axons were counted manually. For
determination of axonal cross-sectional areas, high-power images of
nerve roots were scanned into Macintosh Adobe Photoshop 4.0 using a
Polaroid Slide scanner. The images were calibrated and transferred into
the image analysis program, National Institutes of Health Image, for
axon area measurements. Measurements do not include the myelin sheath
surrounding the axons.
Analysis of cranial nerve VII soma size. Adult animals of
known genotype were deeply anesthetized with a
ketamine/xylazine/acepromazine (3:3:1) cocktail and perfused
transcardially with phosphate buffer followed by 4% paraformaldehyde.
Heads were removed and post-fixed 2 hr before the brainstem and caudal
telencephalon were removed. After being rinsed overnight in
phosphate-buffered sucrose (5% sucrose), brains were dehydrated via a
graded series of alcohols and embedded in Paraplast X-tra (Fisher
Scientific, Houston, TX). Twelve micrometer serial sections were cut
through the entire facial nucleus and mounted on Superfrost Plus slides
(Fisher Scientific). Sections were stained with 0.5% cresyl violet. To
determine soma size, we analyzed 400 neurons from each
Bax/ and Bax+/+ animal. Every fourth section
was photographed and scanned into the Adobe Photoshop program. After
being encoded to blind genotype, every cell with a clear nucleoli was
traced and imported into the National Institutes of Health Image
analysis program to determine soma area. No attempt was made to account
for the tissue shrinkage caused by histological processing.
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RESULTS |
BAX regulates developmental death of neurons in spinal ganglia and
spinal cord
DRGs and spinal cord
In control mice, abundant TUNEL+ cells were detected in lumbar
DRGs between E12.5 and E14.5 (Fig. 1).
Relatively few apoptotic figures were present before E12.5 or after
E14.5 (data not shown). In sharp contrast with control mice, virtually
no cell death was detected between E12.5 and E14.5 in the
Bax/ mice (Fig. 1). Quantification confirmed the visual
impression of virtual elimination of naturally occurring cell death in
the Bax/ animals (Fig. 2).
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Figure 1.
Photomicrographs of FITC-labeled cells using the
TUNEL method in ventral spinal cord and DRG of wild-type and
Bax null mutant mice (E12.5-E14.5). Note that a
considerable number of FITC-labeled cells are seen throughout wild-type
DRG and ventral spinal cord, whereas FITC-labeled cells are absent in
Bax null mutant littermates. DRG, Dorsal
root ganglion; VH, ventral horn of spinal cord. Scale bar, 50 µm.
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Figure 2.
Histograms comparing the number of TUNEL-labeled
cells in DRG and motor pools of wild-type and Bax null
mutant mice. The total number of TUNEL-labeled cells present in DRG and
ventral spinal cords of staged wild-type (gray
bars) and Bax null mutant (black bars) mice is shown. Cell number is expressed as a mean number of TUNEL-labeled cells present in each section of DRG and ventral spinal cord. Note that TUNEL-labeled cells were virtually absent on all
days examined in Bax null mutants.
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To examine whether BAX was necessary for developmental motoneuron
death, we quantitated the number of apoptotic figures present on
different embryonic days in lumbar spinal cords of Bax/
and Bax+/+ mice. At E11.5, lumbar spinal cords of both
genotypes lacked apoptotic figures. Beginning at E12.5, lumbar spinal
cords of Bax+/+ mice exhibited large numbers of apoptotic
figures (Fig. 1, top left). In contrast,
Bax/ mice of the same age exhibited few, if any, TUNEL+
cells (Fig. 1, top right). Similar findings were
observed in E14.5 Bax/ and Bax+/+ spinal
cords (Fig. 1, bottom). The period of naturally occurring
cell death seemed to be primarily over by E15.5. Surprisingly, analysis
of lumbar spinal cords in Bax+/+ mice at E13.5 demonstrated
few apoptotic figures (Fig. 1, middle). This observation
appears to be consistent with the early and late periods of motoneuron
cell death present in chick cervical spinal cord (Yaginuma et al.,
1996).
Few TUNEL+ cells were observed outside the motor pools between E11.5
and postnatal day 1 (PN1) in either Bax+/+ or
Bax/ mice. However, we did find occasional TUNEL+ cells
in the ventricular zone of both genotypes. These data suggest that the
deletion of BAX does not affect survival of proliferating cells. By
PN4, increased cell death outside motor pools was observed in both
Bax+/+ and Bax/ mice. Whether these TUNEL+
cells were interneurons or glia was not determined.
BAX regulates developmental death of cranial ganglia and brain
Trigeminal and cochleovestibular ganglia
Cell death was readily detected in the trigeminal ganglion from
E11.5-PN4 in wild-type mice. Thus, naturally occurring death is
detected over a longer developmental time frame in this ganglion than
in DRGs. Remarkably, almost no TUNEL+ cells were detected in the
trigeminal ganglia of Bax/ mice at any of the ages
studied. Representative tissue sections are shown in Figure
3.
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Figure 3.
Photomicrographs of trigeminal ganglion
(E11.5-E17.5) stained with cresyl violet (left) and
corresponding sections labeled using the TUNEL method
(center and right) in wild-type and
Bax null mutant littermates, respectively. Note that a
considerable number of FITC-labeled cells are seen throughout wild-type
trigeminal ganglion at every age shown (center), whereas
FITC-labeled cells are absent in Bax null mutant
littermates (right). Scale bar, 50 µm.
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We also studied the cochleovestibular ganglia that were assessed at
E13.5-E17.5. During that time frame, abundant TUNEL+ cells could be
seen in wild-type mice. Similar to the situation in the DRG and
trigeminal ganglion, TUNEL+ cells were virtually absent in
Bax/ mice (Table 1).
Trigeminal brainstem nuclear complex and cerebellum
Brainstems were examined at ~48 hr intervals between E13.5 and
PN4. As expected from the results described above for ventral motor
pools, the numbers of TUNEL+ cells in the facial nucleus were markedly
reduced in Bax/ mice (data not shown). We next focused
on structures in the brainstem associated with sensory processing.
Analysis was concentrated on the trigeminal brainstem nuclear complex
(TBNC) that is the central target of trigeminal primary afferents.
Results at PN1 are shown in Figure 4
(top). In control mice, large numbers of TUNEL+ cells were
observed in the postnatal period. The developmental cell death in this
brainstem structure was virtually eliminated in Bax/
mice.
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Figure 4.
Cresyl violet staining and TUNEL labeling in
postnatal TBNC and cerebellum of wild type and Bax null
mutants. Low- and high-power photomicrographs of cresyl violet staining
in TBNC (top, left) and corresponding
high-power TUNEL-labeled sections (top,
right) at PN1 are shown. Notice the absence of
TUNEL-labeling in Bax/ TBNC (top,
rightmost). Photomicrographs of the cerebellar MZ
stained with cresyl violet and the TUNEL method at PN1
(middle) and PN4 (bottom) in wild-type
and Bax null mutant mice are shown. Note the extensive
labeling at PN1 and PN4 throughout the MZ of wild-type cerebellum.
TBNC, Trigeminal brainstem nuclear complex;
MZ, marginal zone; EGL, external granule
cell layer; D, dorsal; L, lateral. Scale
bars, 50 µm.
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Apoptotic figures were also readily detected in the cerebellum of
wild-type mice at PN1 (Fig. 4, middle). TUNEL+ cells were observed throughout the marginal zone (MZ) of the cerebellum. This is a
zone in which immature Purkinje cells are found during migration to
their final position. However, no TUNEL+ cells were seen in this region
in Bax/ mice. Three days later at PN4, apoptotic figures
could be detected in both the MZ and external granule cell layers in
wild-type mice (Fig. 4, bottom). In contrast, in Bax/ mice, only a few TUNEL+ cells were noted in these
regions. We repeatedly observed such differences in multiple sections
within the developing cerebellum in all pairs of animals examined.
Eye
The developing eye was analyzed in detail (Fig.
5). Here, the pattern of naturally
occurring cell death is more complex than in the periphery. Abundant
cell death was observed as early as E11.5 in the developing layers of
both the wild-type and Bax/ eye (see Fig. 5,
top) and continued through PN1. It is clear that many cells
(precursors, postmitotic neurons, and non-neuronal cells) in both the
developing eye and the associated optic nerve (Fig. 5,
middle) are TUNEL+. Thus, cell death during this period seems to be BAX-independent.
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Figure 5.
TUNEL-labeled cells in the developing eye of
wild-type and Bax null mutant mice at E11.5
(top), the optic nerve in a Bax null mutant at E17.5 stained with cresyl violet and the corresponding TUNEL-labeled section (middle), and TUNEL-labeled cells
in the neural layers of the retina in wild-type and Bax
null mutant mice at PN4 (bottom). Numerous TUNEL-labeled
cells were observed in both the developing neuroepithelium of the eye
(top) and in the glial cells of the optic nerve
(middle, arrows) of wild-type and Bax/ animals. In contrast, TUNEL-positive cells were
absent in the RGC layer of the Bax null mutant at PN4, a
time at which peak cell death is occurring (bottom).
Asterisk, Optic chiasm; RGC, retinal ganglion cell
layer; vh, vitreous humor. Scale bars, 50 µm.
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Between PN1 and PN4, abundant TUNEL+ cells were also seen throughout
the developing retina in both wild-type and Bax null tissue
(Fig. 5, bottom). However, in the retinal ganglion cell (RGC) layer, TUNEL+ figures were absent in Bax/ mice.
This time period has been established previously as the period of
highest developmental death in the wild-type RGC layer (Young, 1984). Thus, BAX seems to influence the death of RGCs in the postnatal period.
Elimination of death in Bax/ RGCs results in a
substantial increase in the size of the optic nerve (see below).
Hippocampus
At PN3, the time of greatest programmed cell death in developing
mouse hippocampus (Reznikov, 1982), abundant TUNEL+ figures are
apparent in Bax+/+ mice. Apoptosis was particularly
prominent in CA2 and CA3. Apoptotic figures were seen in both the
granule and molecular cell layers. As we observed in the other regions analyzed, virtually no TUNEL+ figures are seen in Bax/
mice (n = 3; data not shown).
BAX may not influence survival of non-neuronal cells
Sciatic and optic nerve
The majority of glial cells associated with the optic
(Barres et al., 1992, 1993; Raff et al., 1993) and the sciatic (Jessen et al., 1994; Dong et al., 1995; Gavrilovic et al., 1995; Trachtenberg and Thompson, 1996) nerves are dependent on an axon-derived signal for
prevention of cell death during embryonic and postnatal periods of
development. To determine whether programmed cell death in glial cells
is also affected by the deletion of Bax, we examined optic
and sciatic nerves at PN4 for the presence of apoptotic figures. In
contrast to the elimination of programmed cell death in the DRG and the
RGC layer, the mean number of TUNEL+ cells per section (~5) in at
least 10 sections of each Bax null mutant optic and sciatic
nerve (n = 4) did not differ from that of wild-type littermates (n = 3; data not shown). These results
suggest that the bulk of the naturally occurring cell death in
non-neuronal cells is not prevented by deletion of BAX at this age.
Many neurons in adult Bax/ mice are atrophic
The virtual elimination of apoptosis in many regions of the
nervous system during the period of naturally occurring cell death raises the question of mature neuron fate. To begin to address this
issue, we examined nerve morphology and myelinated-axon number in the
optic nerve and in L4/L5 dorsal and ventral roots. Additionally, we
measured both the number and area of myelinated axons in the facial
nerve as well as the cross-sectional area of facial motoneurons.
Low-power photomicrographs of typical optic nerves from adult wild-type
and Bax null mutant mice are shown in Figure
6. Optic nerves in Bax null
mutant mice were dramatically enlarged; cross-sectional areas were
increased by 30%. The increase in nerve diameter seems to be caused by
greater numbers of both small myelinated (Table 2) and unmyelinated axons. Whether
supernumerary retinal ganglion cell axons innervate normal targets in
the lateral geniculate and superior colliculus is unknown.
Interestingly, there was not only an increase in the number of very
small myelinated axons but also an across-the-board reduction in axon
size (see below).
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Figure 6.
Semithin sections of myelinated axons in the optic
nerve of adult wild-type and Bax null mutant mice. The
dramatic increase in size of the optic nerve from a Bax
null mutant mouse when compared with that from a wild-type littermate
is shown (top, at low magnification). That the increase
in optic nerve diameter is attributable to the increased number of
axons present in the Bax null mutant when compared with
a Bax+/+ mice is shown (bottom, at high
magnification). Note also the across-the-board reduction in the size of
myelinated axons. Scale bars, 50 µm.
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In contrast to the large increase in cross-sectional area of optic
nerves, little increase was detected in the area of L4/L5 dorsal or
ventral roots. However, counts of myelinated axons in L4/L5 dorsal
roots revealed an increase of 24% in Bax null mutant nerves
compared with control nerves. Similarly, 34% more axons were detected
in the ventral roots of Bax/ animals (Table
3). These findings are thus consistent
with our observations of the virtual elimination of apoptosis in
retina, DRG, and spinal cord.
Photomicrographs of semithin sections of facial nerves stained for
myelin from Bax/ and wild-type mice are shown in Figure 7. Similar to observations in
Bax/ L4/L5 roots, there was no increase in the
cross-sectional area of the facial nerve of null mice. However, there
were 35% more myelinated axons in the facial nerve of
Bax/ animals versus littermate controls (Table 3). This
finding is consistent with the increase in facial neuron numbers
demonstrated previously (Deckwerth et al., 1996). Strikingly, however,
supernumerary motor axons do not appear to be normal. Most facial motor
axons have large cross-sectional areas consistent with a preponderance
of  motor neurons projecting axons to the facial muscles. However,
in Bax/ mice, abundant small, thinly myelinated axons
were observed (Figs. 7, 8,
top). We hypothesize that the smallest axons are from
neurons saved from apoptosis. An additional finding, however, was that
axons in all size ranges were reduced in size; mean axon caliber in the
facial nerve of Bax/ mice is 14% smaller than that of
wild-type littermates.
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Figure 7.
Semithin sections of myelinated axons in the
facial nerve of adult wild-type and Bax null mutant mice
at low magnification. The insets show, at higher
magnification, that many axons in Bax null mutant nerve
are markedly atrophic. Scale bar, 50 µm.
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Figure 8.
Cross-sectional area of axons and soma of
Bax/ and wild-type facial nucleus. Size-frequency
histograms of axon area in adult wild-type and Bax null
mutant facial nerves (top) are shown. Size-frequency histograms of motoneuron cross-sectional area in the facial nucleus of
Bax null mutant and wild-type littermates
(bottom) are shown.
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The significant decrease in myelinated axon area in adult
Bax/ facial nerves suggested that motoneurons within the
facial nucleus would also be atrophic. To test this hypothesis, we
measured the cross-sectional area of motoneurons in the facial nucleus of Bax/ and wild-type littermates. The results are shown
in Figure 8 (bottom). Again, two abnormalities were
observed, (1) an across-the-board reduction in all soma sizes with a
mean somal reduction of 17% and (2) an unexpectedly large increase in
the numbers of neurons in the smallest size ranges.
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DISCUSSION |
The most striking consequence of the Bax
deficiency is the virtual elimination of developmental cell death in
many populations of neurons. The uniform reduction in cell death in the
neural systems examined suggests that these results will generalize to other regions of the nervous system. A potential objection to our
interpretation is that the absence of TUNEL+ staining may not imply
that apoptosis has been eliminated. In theory, neurons could die during
development without exhibiting the endonucleosomal DNA fragmentation
that allows readily detectable nick-end labeling using terminal
transferase (see Clarke, 1990). Although this possibility cannot be
formally excluded, the fact that supernumerary neurons are present in
Bax/ mice make this possibility unlikely.
BAX regulates survival of PNS neurons with differing
trophic requirements
In Bax/ animals, cell death in the DRG and in
trigeminal, sympathetic, and cochleovestibular ganglia during
midembryonic and early postnatal development was virtually eliminated
(see Table 1). Interestingly, these ganglia contain specific
subpopulations of neurons that require different neurotrophin family
members for survival. As we detected virtually no TUNEL+ cells in any of these ganglia in Bax/ mice, we conclude that BAX is
not linked in a direct way to any specific member of the neurotrophin
family. This result is surprising given the large number of BCL-2
family members and in vitro evidence in chick that both
BCL-2 (Allsop et al., 1993) and BAX (Middleton et al., 1996)
differentially regulate neurotrophin-responsive, as opposed to
cytokine-responsive, peripheral neuron populations. The extent to which
our results will generalize to cytokine-dependent peripheral ganglia
such as the ciliary, or glial-derived neurotrophic factor-dependent (GDNF-dependent) ganglia such as the enteric, remains to be
determined.
The powerful effects of BAX on peripheral neuron survival are
underscored by the dramatic ability of peripheral neurons from Bax/ mice to survive in vitro in the absence
of exogenous neurotrophic molecules. For example, we (Deckwerth et al.,
1996) have shown previously that sympathetic ganglion cells from
Bax/ mice can be cultured in the absence of any
exogenous growth factor, including NGF, for more than 21 d. More
recently, we have found that DRG neurons from Bax/ mice
can be maintained in vitro for at least 14 d in the
absence of any neurotrophin (Lentz et al., 1997). Furthermore, both
NGF- and neurotrophin-3-dependent populations survive in
Bax/ cultures, as demonstrated by the fact that when either factor is added, a subpopulation of surviving neurons responds. It is important to point out, however, that surviving neurons cultured
in the absence of neurotrophins are not normal. In fact, Bax/ mice may offer a convenient means of separating
survival versus growth-promoting effects of neurotrophic factors for
many populations of PNS neurons (see below).
BAX regulates survival of several populations of CNS neurons
BAX protein is widely distributed within the CNS, consistent with
a role in regulation of CNS neuron survival (Krajewski et al., 1994).
Indeed, Bax deficiency decreased neuronal death in several
populations of CNS neurons examined. In particular, death of spinal
motoneurons between E11.5 and PN1 was virtually eliminated in
Bax/ mice. Similarly, there seemed to be a powerful
effect on retinal ganglion cells during the postnatal period of
naturally occurring cell death. Effects on brainstem, cerebellar, and
hippocampal cell death were also observed. These effects are
particularly interesting because most populations of CNS neurons are
thought to require multiple factors for survival during
development.
Reduction of cell death in the CNS results in a 50% increase in
motoneuron number (Deckwerth et al., 1996) and a 35% increase in motor
axon number (Table 3). Although it is important to point out that very
atrophic motoneurons could have been missed in material analyzed at the
light microscopic level, the number of supernumerary motoneurons in
Bax/ animals approximately corresponds to the number of
dying postmitotic motoneurons determined previously in studies of mouse
spinal cord and brainstem (Lance-Jones, 1982; Oppenheim, 1991).
However, deletion of Bax did not appear to effect the
substantial number of proliferative cells that die during neurogenesis
(Blaschke et al., 1996; Galli-Resta and Ensini, 1996), because we did
not detect differences between Bax/ and wild-type mice
in the number of TUNEL+ cells present in the ventricular zone. Thus,
the increased numbers of cells observed in Bax null mice can
be explained simply as the result of the elimination of postmitotic
cell death.
In this regard, it is interesting to consider differences between
Bax/ mice and mice with a targeted deletion of CPP32
(Caspase-3). Deletion of CPP32 effectively eliminates death of
proliferative cells (Kuida et al., 1996). Moreover, CPP32 may also
regulate survival of non-neuronal elements within the CNS (Keane et
al., 1997). The end result is a brain that is grossly hypertrophied. In
contrast, the brains of Bax/ mice are not significantly
larger than control. The fact that BAX is regulating a period of death that occurs after cell proliferation, combined with the fact that survival of non-neuronal cells may not be increased in
Bax/ mice, may in part explain this outcome.
Although the number of TUNEL+ cells is dramatically reduced in the CNS
of Bax/ mice, in vitro data suggest that the
consequences of the Bax deficiency may be somewhat different
for the PNS and CNS. When disassociated PN0 Bax/
superior cervical ganglia (SCG) neurons are cultured in the absence of
trophic factors, these cells demonstrate an ability to survive for at
least 23 d (Deckwerth et al., 1996). In striking contrast,
disassociated cultures of PN4 Bax/ cerebellar granule
cells are able to survive only 7 d after potassium deprivation, an
apoptosis-inducing event (Miller et al., 1997). These findings suggest
that BAX may be a more powerful regulator of apoptosis of peripheral
neurons than of central neurons. In fact, differences between PNS and
CNS neurons in signal transduction pathways related to survival have
recently been demonstrated (Meyer-Frank et al., 1995). It is possible
that the variation in the survival-promoting effects of BAX deficiency
is related to the differences in signal transduction pathways used by
CNS and PNS neurons in vitro.
Neurons saved from naturally occurring cell death may not
function normally
It is interesting to consider the fate of neurons saved from
apoptosis in the Bax null mutant mice. There are two
apparent possibilities. First, supernumerary neurons may be poorly
connected with their target structures and may therefore be unable to
obtain trophic support. In this scenario, one would expect to find
substantial numbers of markedly atrophic neurons and axons, as well as
a population of cells of relatively normal size and morphology.
Alternatively, supernumerary neurons might form appropriate connections
with their peripheral target structures and participate fully in normal functions. This hypothesis would predict increased competition for
trophic support, resulting in uniformly smaller soma and axon cross-sectional areas.
In support of the first possibility, a striking finding in
Bax/ mice is the presence of markedly atrophic neurons
and axons. Furthermore, although the size of all motoneurons is reduced
in Bax/ mice (see below), there was also a unexpected
increase in the number of very small caliber axons. In fact, motor
axons with cross-sectional areas of less than 3 µm2 were more than twice as frequent in
Bax/ mice than in control mice. Indeed, axons in this
small size range account for the majority of supernumerary axons
encountered in these mice. This latter observation suggests that the
supernumerary neurons are unable to acquire target-derived trophic
factors that regulate axon caliber (Munson et al., 1997).
During innervation of peripheral structures, target-derived factors
influence both the survival and growth of developing neurons. The
presence of supernumerary, but markedly atrophic, neurons and axons
suggests that these two effects are disassociated in Bax/ mice. Other data are consistent with this view. For
example, Bax/ facial motoneurons survive axotomy in the
neonatal period, although the resultant cells, deprived of connection
with muscle, are atrophic compared with contralateral motoneurons
(Deckwerth et al., 1996). Similarly, Bax/ DRG neurons,
which can survive NGF deprivation in vitro, are markedly
atrophic and exhibit vastly reduced neurite outgrowth when compared
with Bax/ DRG neurons grown in the presence of NGF
(Lentz et al., 1997). Bax/ sympathetic ganglia neurons
also survive NGF withdrawal in vitro, although the cells
undergo reductions of 90% in mRNA and protein synthesis (T. Deckwerth
and E. M. Johnson, Jr., personal communication). Thus, deletion of
BAX is able to compensate for the survival-promoting but not the
growth-promoting effects of target-derived factors.
Complicating the interpretation that atrophic neurons are not fully
connected is the fact that motoneurons and associated axons in all size
ranges, including the very largest, are smaller in Bax null
mutants than in wild-type littermates. Because there is an increase of
35% in facial motor axons of Bax null mutant animals, this
finding is consistent with an across-the-board reduction in the amount
of trophic support available per neuron. The possibility must also be
considered, however, that BAX itself may be involved in regulation of
cell and axon size. Although BAX and other BCL-2 family members have
traditionally been considered regulators of apoptosis, a recent study
suggests that BCL-2 may influence neuronal morphology (Chen et al.,
1997).
The phenotype of mice overexpressing the antiapoptotic protein BCL-2 is
very similar to the phenotype of mice with a Bax deletion. Both eliminate apoptotic cell death in facial motoneurons, retinal ganglion cells, and other neuronal populations. However, Martinou and
colleagues (Martinou et al., 1994) reported that neuron specific enolase-Bcl-2 (NSE-Bcl-2) mice exhibited
hypertrophied brains that they attributed to an increase in the overall
number of neurons. Unlike brains of NSE-Bcl-2 mice, however,
gross observations of brains from Bax null mice do not
reveal any discernible differences in size compared with the brains of
wild-type littermates. This evidence raises the possibility that
neuronal size may be enhanced by overexpression of BCL-2 and reduced by
deletion of BAX. Overall, we favor the interpretation that supernumary
neurons in Bax/ mice are not fully connected to their
targets and that, in addition, the absence of BAX interferes with the
growth of normally connected neurons.
Conclusions
In summary, we have shown that deletion of BAX eliminates much of
the postmitotic neuronal death that occurs in both PNS and CNS during
development, but the resultant supernumerary neurons and axons are
atrophic. BAX mutants therefore constitute a valuable model for
understanding the mechanisms as well as the role of cell death in the
developing nervous system. Importantly, these mice may represent a
valuable tool to test the effects of inhibition of apoptosis in
neurodegenerative disorders.
| |
FOOTNOTES |
Received Sept. 11, 1997; revised Nov. 18, 1997; accepted Dec. 4, 1997.
This work was supported by National Institutes of Health Grants NS31768
and P5O-AG05681 to W.D.S. and HD27500 to S.J.K. and by the Muscular
Dystrophy Association. We gratefully acknowledge John Harding for his
excellent technical assistance and Dr. Judith Mosinger-Ogilvie for
valuable discussions of the results.
Correspondence should be addressed to Dr. William D. Snider, Center for
the Study of Nervous System Injury, Department of Neurology, Box 8111, Washington University School of Medicine, 600 South Euclid Avenue, St.
Louis, MO 63110.
| |
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R. Chiesa, P. Piccardo, S. Dossena, L. Nowoslawski, K. A. Roth, B. Ghetti, and D. A. Harris
Bax deletion prevents neuronal loss but not neurological symptoms in a transgenic model of inherited prion disease
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W. Sun, A. Winseck, S. Vinsant, O.-h. Park, H. Kim, and R. W. Oppenheim
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A. K. Wiggins, G. Wei, E. Doxakis, C. Wong, A. A. Tang, K. Zang, E. J. Luo, R. L. Neve, L. F. Reichardt, and E. J. Huang
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W. B. Jacobs, G. S. Walsh, and F. D. Miller
Neuronal Survival and p73/p63/p53: A Family Affair
Neuroscientist,
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[Abstract]
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N. G. Forger, G. J. Rosen, E. M. Waters, D. Jacob, R. B. Simerly, and G. J. de Vries
Deletion of Bax eliminates sex differences in the mouse forebrain
PNAS,
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L. Yang, D. Bula, J. G. Arroyo, and D. F. Chen
Preventing Retinal Detachment-Associated Photoreceptor Cell Loss in Bax-Deficient Mice
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H. Dong, A. Fazzaro, C. Xiang, S. J. Korsmeyer, M. F. Jacquin, and J. W. McDonald
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F. Wei, X.-M. Xia, J. Tang, H. Ao, S. Ko, J. Liauw, C.-S. Qiu, and M. Zhuo
Calmodulin Regulates Synaptic Plasticity in the Anterior Cingulate Cortex and Behavioral Responses: A Microelectroporation Study in Adult Rodents
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S. Gianino, J. R. Grider, J. Cresswell, H. Enomoto, and R. O. Heuckeroth
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C. Bechade, C. Mallecourt, F. Sedel, S. Vyas, and A. Triller
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H. R. Russell, Y. Lee, H. L. Miller, J. Zhao, and P. J. McKinnon
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D. A. Kerr, T. Larsen, S. H. Cook, Y.-R. Fannjiang, E. Choi, D. E. Griffin, J. M. Hardwick, and D. N. Irani
BCL-2 and BAX Protect Adult Mice from Lethal Sindbis Virus Infection but Do Not Protect Spinal Cord Motor Neurons or Prevent Paralysis
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R. A. Kirkland, J. A. Windelborn, J. M. Kasprzak, and J. L. Franklin
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W. P. Rakowicz, C. S. Staples, J. Milbrandt, J. E. Brunstrom, and E. M. Johnson Jr
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D. S. Papermaster
The Birth and Death of Photoreceptors : The Friedenwald Lecture
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L. D. Russell, H. Chiarini-Garcia, S. J. Korsmeyer, and C. M. Knudson
Bax-Dependent Spermatogonia Apoptosis Is Required for Testicular Development and Spermatogenesis
Biol Reprod,
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[Abstract]
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A. J. Windebank and E. Mcdonald
Book Review: Cell Death in the Peripheral Nervous System: Potential Rescue Strategies
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[Abstract]
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G. Middleton and A. M. Davies
Populations of NGF-dependent neurones differ in their requirement for BAX to undergo apoptosis in the absence of NGF/TrkA signalling in vivo
Development,
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[Abstract]
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S. Linnarsson, A. Mikaels, C. Baudet, and P. Ernfors
Activation by GDNF of a transcriptional program repressing neurite growth in dorsal root ganglia
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J.-B. Charrier, F. Lapointe, N. M. L. Douarin, and M.-A. Teillet
Anti-apoptotic role of Sonic hedgehog protein at the early stages of nervous system organogenesis
Development,
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[Abstract]
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R. Seidl, B. Bidmon, M. Bajo, B. C. Yoo, N. Cairns, E. C. LaCasse, and G. Lubec
Evidence for Apoptosis in the Fetal Down Syndrome Brain
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[Abstract]
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M. Vila, V. Jackson-Lewis, S. Vukosavic, R. Djaldetti, G. Liberatore, D. Offen, S. J. Korsmeyer, and S. Przedborski
Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease
PNAS,
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[Abstract]
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G Middleton, S Wyatt, N Ninkina, and A. Davies
Reciprocal developmental changes in the roles of Bcl-w and Bcl-x(L) in regulating sensory neuron survival
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[Abstract]
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S. Ghatan, S. Larner, Y. Kinoshita, M. Hetman, L. Patel, Z. Xia, R. J. Youle, and R. S. Morrison
p38 MAP Kinase Mediates Bax Translocation in Nitric Oxide-induced Apoptosis in Neurons
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F. Selimi, M. W. Vogel, and J. Mariani
Bax Inactivation in Lurcher Mutants Rescues Cerebellar Granule Cells But Not Purkinje Cells or Inferior Olivary Neurons
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G. V. Putcha, M. Deshmukh, and E. M. Johnson , Jr.
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M. L. Doughty, P. L. De Jager, S. J. Korsmeyer, and N. Heintz
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O. F. X. ALMEIDA, G. L. CONDÉ, C. CROCHEMORE, B. A. DEMENEIX, D. FISCHER, A. H. S. HASSAN, M. MEYER, F. HOLSBOER, and T. M. MICHAELIDIS
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M. J. Chong, M. R. Murray, E. C. Gosink, H. R. C. Russell, A. Srinivasan, M. Kapsetaki, S. J. Korsmeyer, and P. J. McKinnon
Atm and Bax cooperate in ionizing radiation-induced apoptosis in the central nervous system
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K. A. Roth, C.-Y. Kuan, T. F. Haydar, C. D'Sa-Eipper, K. S. Shindler, T. S. Zheng, K. Kuida, R. A. Flavell, and P. Rakic
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C. Raoul, C. E. Henderson, and B. Pettmann
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G. V. Putcha, M. Deshmukh, and E. M. Johnson Jr
BAX Translocation Is a Critical Event in Neuronal Apoptosis: Regulation by Neuroprotectants, BCL-2, and Caspases
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S. I. Lentz, C. M. Knudson, S. J. Korsmeyer, and W. D. Snider
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[Abstract]
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K. L. Moulder, O. Onodera, J. R. Burke, W. J. Strittmatter, and E. M. Johnson Jr
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R. Wetts and J. E. Vaughn
Peripheral and Central Target Requirements for Survival of Embryonic Rat Dorsal Root Ganglion Neurons in Slice Cultures
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M. Vila, V. Jackson-Lewis, S. Vukosavic, R. Djaldetti, G. Liberatore, D. Offen, S. J. Korsmeyer, and S. Przedborski
Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease
PNAS,
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S. Linnarsson, A. Mikaels, C. Baudet, and P. Ernfors
Activation by GDNF of a transcriptional program repressing neurite growth in dorsal root ganglia
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N. Orike, G. Middleton, E. Borthwick, V. Buchman, T. Cowen, and A. M. Davies
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Top
Abstract
Introduction
