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* CNS Signaling Laboratory, Molecular Neuro-Oncology,
Stroke and Neurovascular Regulation, and
§ Developmental Neurobiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA
2Correspondence: CNS Signaling Laboratory, Molecular Neuro-Oncology, 149 13th St., Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA. E-mail: reeves{at}helix.mgh.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: protein tyrosine phosphatase • cell survival • cerebral ischemia • neuron
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5
). More recent studies have
demonstrated a role for SHP2 in signaling pathways initiated through
glycoprotein (gp)130, which is a common signal transducing subunit of
the ciliary neurotrophic factor (CNTF) family of cytokine receptors
(6)
. Cultured cell experiments using SHP2 dominant
negative mutants (1
, 7
8
9
10
11)
and antibody microinjection
approaches (1
, 12
, 13)
have established SHP2 as a required
positive component in RTK and gp130 pathways, acting upstream of
mitogen-activated protein kinase. In addition, SHP2 has been shown to
be a negative regulator of STAT3 activation during CNTF signaling
(6)
. In contrast to these cell culture studies, the
function of SHP2 during development is unclear, although SHP2 displays
a high degree of homology with the product of the Drosophila
corkscrew gene, which is required as a positive transducer downstream
of the Torso receptor tyrosine kinase for normal embryo differentiation
(14)
. Microinjection of a SHP2 dominant negative mutant
into Xenopus embryos has demonstrated that SHP2 is required
for bFGF induced mesoderm induction and completion of gastrulation
(3)
. In agreement with these findings, targeted disruption
of SHP2 in mice results in embryonic lethality during midgestation
(15
, 16)
, where the animals exhibit gastrulation defects.
Impaired bFGF and PDGF signaling is also observed in cultured embryonic
stem cells from shp2 null embryos (15)
. Thus,
SHP2 appears to be a critical regulator of growth factor-activated
signaling cascades in the developing embryo.
We have shown previously that SHP2 protein is present both in the
proliferating neuroepithelium and in the postmitotic marginal zones of
the mouse central nervous system (CNS) (17
, 18)
. However,
in the adult CNS, SHP2 is present only in a subset of neurons and in
astrocytes that have been made reactive due to ischemic injury
(17)
. We have also shown that SHP2 protein levels are
increased in dying neurons after cerebral ischemia (17)
.
These data and the fact that in a variety of cell types SHP2 has a
critical role in the activation of signal transduction cascades suggest
that SHP2 may play an important role during CNS development and in
mediating neuronal response to injury. In this report we investigated
the role of SHP2 in CNS development and neuronal response to injury by
using a transgenic mouse strategy that bypasses the early embryonic
lethality that occurs in shp2 null mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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into Bglll-HindIII-digested
PRK-NC1D (6)
. An EcoRI-HindIII
fragment containing the FLAG-tagged SHP2-CS mutant was then inserted
into a blunt-ended SpeI site of pBluescript II
KS/IRES-ßgeo. The rat nestin intron II and TK minimal promoter
(19
, 20)
was removed from gIITK (kindly provided by
Richard Josephson) by XhoI digestion and inserted into a
blunt-ended NotI site of the pBluescript II
KS/SHP2-CS-IRES-ßgeo. The nestin intron II and TK minimal
promoter/CS-SHP2 junction was sequenced to confirm that the orientation
of the insert was correct. The transgene was then removed from the
vector by PacI digestion, which had been inserted as linkers
at the 5' and 3' ends of the transgene. The resulting 8.9 kb fragment
was gel purified, phenol-chloroform extracted, and purified by Elutip
(Schleicher & Schuell, Keene, N.Y.), then used for injection of FVB/N
mouse zygotes. Embryo donors were superovulated females. Injected
embryos were implanted in the oviducts of day 1 pseudopregnant foster
females.
Southern blot analysis
Ten micrograms of genomic DNA extracted from mouse tails was
digested with EcoRV, separated on a 0.8% agarose gel, and
transferred to a positively charged nylon membrane (NEN Lifescience,
Boston, Mass.) by standard capillary blotting. A 1.1 Kb fragment from
lacZ cDNA or a 1.7 kb fragment from SHP2 cDNA was used as a
probe for hybridization. The blots were hybridized at 65°C overnight,
rinsed with 2 x SSC and 0.01% sodium dodecyl sulfate (SDS), and
washed in 0.2 x SSC and 0.01% SDS at 65°C for 30 min.
ß-Galactosidase staining of whole-mount embryos
For analysis of transgene expression, transgenic founder males
or males born from founder females (F1) were crossed with wild-type
FVB/N females, and E12 mice were analyzed by X-gal staining
(21)
. Briefly, embryos were fixed in 1% formaldehyde,
0.2% glutaraldehyde, 2 mM MgCl2, 5 mM EGTA, and
0.02% Nonidet P-40 for 60 min at room temperature, and then washed
three times in phosphate-buffered saline (PBS) containing 0.02%
Nonidet P-40. Finally, ß-galactosidase activity was visualized by
submerging the embryos in X-gal buffer [5 mM
K3Fe(CN)6, 5 mM
K4Fe(CN)6, 2 mM
MgCl2, 0.01% Na-deoxycholate, 0.02% Nonidet
P-40, and 0.5 mg/ml 5bromo-4-chloro-3-indole-ß-D-galactoside
(X-gal)] for overnight. After whole mount X-gal staining, brains were
cut (50–80 mm) using a Vibratome and mounted on slides.
ß-Galactosidase staining of adult brains and
immunohistochemistry for cell type-specific markers
Animals were anesthetized by an intraperitoneal (i.p.) injection
of a mixture of ketamine (10 mg/kg body weight) and xylazine (10 mg/kg
body weight) and killed by intracardiac perfusion with 3%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.35. The brains were
removed, fixed for 5 h, and transferred to PBS. Brain sections
were prepared on a Vibratome at 50–80 mm thickness in the sagittal or
coronal plane. For X-gal staining, floating sections were incubated in
X-gal buffer for 5 h at 37°C. Sections were washed three times
in PBS and either mounted on glass slides with DAPI mounting media
(Vector Laboratories) or used for immunohistochemical staining. For
immunohistochemistry, Vibratome sections were incubated with 3% normal
donkey serum, 0.04% Tween-20 in PBS for 45 min at room temperature and
then incubated with primary antibody for overnight at 4°C. The
polyclonal anti-GFAP (Dako, Carpinteria, Calif.), monoclonal anti-NeuN
(Chemicon, El Segundo, Calif.), and polyclonal anti-nestin (gift from
Dr. R. J. D. Mckay, NINDS) antibodies were used to identify
astrocytes, neurons, and neuroepithelial stem cells, respectively.
Cy3-conjugated donkey anti-rabbit or goat anti-mouse IgG antibodies
(Jackson Immunoresearch, West Grove, Pa.) were used as secondary
antibodies. The sections were observed under a fluorescence microscope.
Immunoprecipitation and Western blotting
Frozen brains were homogenized in lysis buffer (50 mM Tris HCl,
pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 0.25%
Na-deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 1 mM sodium
fluoride, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml
pepstatin, 50 µg/ml phenylmethanesulfonyl fluoride). Tissue lysates
were clarified using microcentrifugation at 10,000 g for 30
min at 4°C and immunoprecipitated with anti-FLAG epitope monoclonal
antibody (M2, Kodak). Immunoprecipitates were fractionated on a 8%
SDS-polyacrylamide gel and subjected to Western blotting analysis using
anti-FLAG antibody (M2). Filters were stripped and probed with
anti-SHP2 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz,
Calif.).
BrdU labeling of embryos for cell proliferation studies
Wild-type FVB/N females were mated with transgenic line 306
males. At embryonic day 12 (E12), pregnant females were injected with
BrdU (bromodeoxyuridine, Sigma Chemical Co., St. Louis, Mo.) at a dose
of 50 mg/g body weight (22)
and killed 1 h later.
Embryos were removed and their heads were fixed overnight in 70%
ethanol. The remaining part of the body was used for genomic DNA
isolation and genotyping by Southern blotting. Fixed heads were
dehydrated in graded ethanol solutions and embedded in paraffin.
Embedded heads were sectioned in the coronal plane at 4 µm. BrdU
immunohistochemistry was performed as described previously (22
, 23)
. Anti-BrdU monoclonal antibody (Becton Dickinson,
Rutherford, N.J.) was used at a 1:75 dilution at room temperature for
30 min. Staining was developed using the Vecstatin ABC kit. Sections
were counterstained with basic Fuchsin as described previously
(22)
.
Transient focal cerebral ischemia
Adult transgenic mice and wild-type littermates (4–5 wk of age,
18–24 g) were initially anesthetized with 2% halothane in 70%
N20 and 30% O2 and then
maintained on 1% halothane in a similar gaseous mixture. Core body
temperature was monitored rectally (YSI, Yellow Springs, Ohio) and
maintained normothermic (36.5°°C–37.5°C) by an electric blanket.
Focal cerebral ischemia was induced by the occlusion of the middle
cerebral artery (MCA) using the intraluminal filament technique
(24)
. Briefly, The left common and external carotid
arteries were isolated and ligated through a ventral midline incision.
A microvascular clip (Zen temporary clip, Ohwa Tsusho, Tokyo, Japan)
was temporarily placed on the internal carotid artery and the
pterygopalatine artery. An 8–0 nylon monofilament (Ethicon,
Somerville, N.J.) coated with silicone was introduced into the internal
carotid artery via the external carotid artery and then advanced 10 mm
distal to the carotid bifurcation so as to occlude the MCA. The wound
was sutured and the animals returned to their cages. Two hours
postocclusion, the animals were briefly anesthetized as described above
and the filament was withdrawn to initiate reperfusion.
Determination of infarct volume and infarct area
Six days after the MCA occlusion, animals were anesthetized by
an i.p. injection of a mixture of ketamine and xylazine and killed by
perfusion via an intracardiac route as described above. After
perfusion, brains were immersed in the same fixative for overnight at
4°C. The brains were immersed in 15% sucrose overnight, then in 30%
sucrose overnight, and frozen on dry ice. Coronal 20 mm sections were
collected at 2 mm intervals and used for hematoxylin-eosin staining.
The infarction area was measured using an image analysis system (M4,
Imaging Research, St. Catherine’s, Ontario, Canada) and infarction
volume was calculated by summing the infarct volumes of sequential
sections. To eliminate the effects of edema, infarct size was also
calculated as a percentage of infarction by the formula: (contralateral
hemisphere-ipsilateral nonischemic hemisphere)/contralateral
hemisphere x 100%, as described previously (25)
.
Measurement of cerebral blood flow (CBF)
Successful occlusion of the middle cerebral artery was
verified by laser Doppler flowmetry (Model PF 2B, Perimed, Stockholm,
Sweden) and recorded on a MacLab/8 data acquisition system (AD
Instruments, Milford, Mass.). Cortical CBF was recorded from a
fiberoptic probe affixed to the skull (2 mm posterior and 6 mm lateral
to bregma) in an area known to correspond to the ischemic core.
Baseline CBF was measured before MCA occlusion and recorded
continuously during and after the ischemic period. The resulting
changes in CBF were expressed as a percentage relative to the baseline
values.
An indicator fractionation technique using
14C-iodoamphetamine (26
, 27)
was
used to determine whether mutant and wild-type mice differed in the
absolute value of their resting cerebral blood flow. Briefly, animals
were anesthetized as described above. The right femoral artery and
jugular vein were cannulated with PE-10 polyethylene tubing. After
determining MABP and blood gases, arterial blood was withdrawn
continuously from the femoral artery at a rate of 0.3 ml/min. One
microcurie of N-isopropyl-[methyl
1,3-14C]-p-iodoamphetamine (American
Radiolabeled Chemicals Inc., St. Louis, Mo.) dissolved in 0.1
ml saline was injected into the jugular vein as a bolus (<1 s). Twenty
seconds after injection, the animal was decapitated and the blood
withdrawal terminated simultaneously. The brain was removed and quickly
frozen in isopentane solution, chilled with ice, and then dissected
into right and left hemispheres. After adding scintigest (Fisher
Scientific, Pittsburgh, Pa.) and incubating (50°C for 6 h),
scintillation fluid and
H2O2 were added. Twelve
hours after shaking, radioactivity in the brain and blood were measured
by liquid scintillation spectrometry and CBF was calculated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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Transgene expression in E12 nestin-SHP2-CS mice
The expression pattern of the nestin-SHP2-CS transgene was
analyzed in F1 (from male founders) and F2 (from female founders)
offspring from two male (302 and 305) and five female (306, 307, 308, 309, and 310) transgenic lines. The nestin intron II has
been shown previously to direct expression of a transgene to the
neuroepithelium of the embryo, which is detectable between E9.5 and
E17.5 (19
, 20)
. Whole-mount X-gal staining of E12 mice
from lines 305, 306, and 310 showed strong staining in the
neuroepithelia of the forebrain, midbrain, hindbrain, and spinal cord.
An example of this pattern of developmental expression from line 306 is
shown in Fig. 2A
, B
, C
and a summary of staining of all
transgenic animal lines is shown in Fig. 2E
. The overall
staining was stronger in lines 306 and 310 than in line 305. Line 309
showed strong staining in the ventricular zone of the forebrain and
midbrain, but weak staining in the hindbrain and none in the spinal
cord. Line 308 showed weak staining only in the forebrain. Lines 302
and 307 did not show detectable transgene expression (data not shown).
To demonstrate that the SHP2-CS mutant is cotranslated with ßgeo, we
immunoprecipitated the FLAG epitope-tagged SHP2-CS mutant from total
head lysates derived from nestin-SHP2-CS line 306 and wild-type E12
embryos. Probing the filters with either anti-FLAG or anti-SHP2
antibodies confirmed that the SHP2-CS mutant is cotranslated with
ßgeo (Fig. 2F
).
|
Transgene expression in the adult brain
To examine regional and cell-type specific expression of the
nestin-SHP2-CS transgene in the adult brain, we performed X-gal
staining on sections of brains from 2 month-old F1 offspring from line
306 (Fig. 3A
, B
, C
, D
, E
, F
). Staining was seen in the
subventricular zone (SVZ), rostal migratory stream (RMS), dentate gyrus
of the hippocampus, and cerebellum, which is in agreement with where
nestin immunoreactivity has been reported previously (19
, 20
, 28
29
30
31)
(see Fig. 4A
, B
for an example of costaining for X-gal and nestin in the
SVZ). Thus, the transgene appears to be localized to regions of the
adult brain that are known to contain nestin-positive proliferating
progenitor cells. In previous studies nestin immunoreactivity has not
been detectable in the midbrain and the cerebral cortex, although some
immunoreactivity has been seen in blood vessels (32)
.
Therefore, the nestin-SHP2-CS transgene also is expressed in cells not
previously known to be nestin positive. A summary of transgene
expression in all adult transgenic lines is shown in Fig. 3F
. Because line 306 showed the strongest level of transgene
expression, this line was used for further analysis.
|
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To determine whether the transgene expressing cells were neurons or
glia, we performed immunohistochemistry on brain sections from line 306
using antibodies to nestin, NeuN and GFAP, which are markers for
progenitor cells, neurons and astrocytes respectively (Fig. 4E
). None of the X-gal-labeled cells were colabeled for
GFAP; however, some cells in the cerebral cortex, midbrain, and
hippocampal pyramidal cell layer were colabeled for NeuN (see Fig. 4C
, D
). In summary, nestin-SHP2-CS transgene expression is
directed to cells in the SVZ, RMS, dentate gyrus, and cerebellum. Some
neurons in the cerebral cortex and midbrain and pyramidal cells in the
hippocampus also express the transgene.
CNS development is normal in nestin-SHP2-CS mice
In the developing mouse, CNS postmitotic cells exit the
proliferative neuroepithelium lining the ventricular cavities and
migrate along radial glial fibers to reach the marginal zones where
they differentiate and extend axonal processes. SHP2 may have a role in
progenitor cell proliferation as well as neuronal migration and
differentiation. To examine whether removal of SHP2 function during
development disrupts progenitor cell proliferation, pregnant females
were pulsed for 1 h with BrdU at two developmental time points
(E12 and E16). No significant difference in the BrdU labeling index
(number of BrdU-labeled nuclei/total nuclei per unit area of the
proliferative zone) was observed between transgenic mice and wild-type
littermates in the neocortical or striatal proliferative zones in the
forebrain at either age (Table 1
). These results suggest that removal of SHP2 does not affect cell cycle
kinetics of neuroepithelial progenitor cells, at least in the
neocortical and striatal primordia. To examine whether SHP2 removal
disrupts progenitor cell migration, we compared cresyl violet or basic
Fuchsin-stained sections of brains from E12, E17, and 2 month-old
nestin-SHP2-CS mice and wild-type littermates. No differences in brain
size, cortical thickness, lamination, or other histological features of
the cerebral cortex, hippocampus, or cerebellum were observed between
transgenic and wild-type mice (data not shown). We also stained
sections of the brain from 2 month-old nestin-SHP2-CS and wild-type
mice with antibodies to GFAP, nestin, and NeuN. Again, there was no
difference in the pattern of staining for these markers (data not
shown). These results indicate that in the nestin-SHP2-CS mice there
are no obvious abnormalities in the histology of the brain during
embryonic development or at maturity.
|
The nestin promoter-driven SHP2-CS transgene is induced within
6 h postcerebral ischemia
Induction of the endogenous nestin gene has been observed in
neurons and reactive astrocytes after transient MCA occlusion-induced
cerebral ischemia in rodents (33
34
35)
. To examine
induction of the nestin promoter-driven transgene after cerebral
ischemia, nestin-SHP2-CS mice were subjected to transient MCA occlusion
and processed 6 h later for X-gal staining. Expression was
measured at this early postischemic phase because infiltration of
reactive astrocytes, microglia, and proliferation of endothelial cells
is minimal. The region affected by the transient ischemic involves the
ventral and lateral cerebral cortex, neostriatum, and the amygdala. On
the contralateral side some transgene expressing cells were present;
however, on the ischemic side, there was a significant increase in the
number and intensity of transgene expressing cells throughout the
ischemic region. Induction of transgene expression was seen in the
caudate putamen and outer layer of the piriform cortex and was
especially evident in the amygdala (Fig. 5A
, B
). Sections from this region were stained with
X-gal and NeuN or GFAP antibodies to identify specific cell types that
expressed the transgene (Fig. 5C
, D
). Sections
from the ischemic side showed a substantial decrease in NeuN
immunoreactivity compared to the contralateral side, indicating that
considerable neuronal death had occurred during this early postischemic
phase. Some of the remaining NeuN-positive cells were costained with
X-gal. Sections were not stained with GFAP antibody confirming reactive
astrocytes were not present. These results indicate that during the
early postischemic phase, transgene expression is induced in neurons
and/or microglia within the infarct region.
|
Ischemic damage is increased in nestin-SHP2-CS mice
We have shown in previous studies that in the uninjured brain, low
levels of SHP2 protein are detectable in a variety of neuronal cell
types, including large motor neurons to smaller local circuit
interneurons, but not in nonreactive glial cell types
(17)
; however, SHP2 protein levels are strikingly
increased in dying neurons and reactive astrocytes after focal cerebral
ischemia (17)
. Because transgene expression is also
induced after cerebral ischemia in nestin-SHP2-CS mice, we used these
animals to examine the affect the SHP2-CS dominant negative mutant has
on ischemia-induced damage. To this end, focal ischemia was induced in
the forebrain of nestin-SHP2-CS mice and nontransgenic littermates by
transient MCA occlusion and infarct volume and infarct percentage were
measured 6 days after reperfusion. We evaluated the consequences of
nestin-SHP2-CS expression at this late postischemia time because it
allows sufficient time for reactive gliosis to occur and therefore the
potential impact the mutant has not only on neuronal survival signaling
pathways but also the cellular response to the damage (i.e., reactive
gliosis). Mice overexpressing the SHP2-CS mutant sustained larger
infarction volumes than nontransgenic controls, 39.16 ± 1.8
mm3 (n=9) and 29.9 ± 2.6
mm3 (n=9), respectively (Fig. 6
). This result could not be attributed to enhanced tissue swelling in
the mutant animals since the difference in the lesion size remained
after a correction for edema (percentage of infarction: 28.0±2.9 in
transgenic and 18.6±2.8 in wild-type mice, P<0.05). Laser
Doppler flowmetry did not reveal a significant difference in the
reduction of cortical blood flow after the occlusion of the middle
cerebral artery in both mutant (22±6% of baseline, n=9)
and wild-type (23±4% of baseline, n=9, P>0.05)
mice. Moreover, the resting absolute cerebral blood flow did not
significantly differ between the two groups [220 ± 18 ml/100
g/min (n=6) and 220 ± 17 ml/100 g/min (n=6)
in transgenic and wild-type mice, respectively]. Last, carbon
black perfusion (36)
did not reveal any qualitative
differences in the cerebrovascular anatomy, with respect to the circle
of Willis and its major tributaries, between mutant and wild-type mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cell
culture studies have indicated that these nervous system progenitor
cells require mitogens that include bFGF and EGF for renewal and
lineage restriction, and neurotrophic factors such as CNTF for
differentiation (37)
. Significantly, an ample body of
evidence based on cell culture data has indicated that SHP2 has a
critical role in cell signaling pathways initiated by these growth and
differentiation factors (38
, 39)
. Thus, a
priori, it would be expected that removal of endogenous SHP2
function in nervous system progenitor cells would affect nervous system
development. However, nestin-SHP2-CS transgenic mice are viable, appear
to develop normally, and do not show any obvious histological
abnormalities in the embryonic and adult brain. One explanation for
these results could be that SHP2 function is redundant during
development of the nervous system. The distribution and function of the
related protein tyrosine phosphatase SHP1 (reviewed in refs 38
, 39
) has not been extensively examined in the nervous system in
this regard. Alternatively, the level of expression of the SHP2-CS
mutant in the progenitor cells of the developing neuroepithelium may
not be high enough to attenuate endogenous SHP2 activity.
In contrast to the lack of an obvious developmental defect in
nestin-SHP2-CS animals, the neuronal response to ischemic injury was
clearly compromised in the transgenic mice. This was evident in the MCA
occlusion experiments, where the ischemia-induced infarct size in
nestin-SHP2-CS mice was 34% larger than that observed in wild-type
littermates. Significant differences in physiological parameters, such
as MABP, arterial blood gases and rectal temperature, were not observed
between nestin-SHP2-CS and wild-type mice (data not shown). Moreover,
transgenic mice did not differ from wild-type littermates
hemodynamically or in their cerebrovascular anatomy, indicating that
the enhanced severity of the ischemic outcome in the mutants was a
result of the overexpression of the dominant negative form of SHP2.
These results indicate that SHP2 is a crucial component in cell
signaling important in neuroprotection during ischemia. Several studies
have shown that ischemic damage leads to differential regulation of
mRNAs or proteins for neurotrophic factors and growth factors; for
example, induction of NGF and BDNF (40
, 41)
; bFGF
(42
43
44)
, IGF (40)
, TGF
(45)
,
CNTF (46)
and reduction of NT-3 (41
, 47)
.
Furthermore, intraventricular injection of BDNF (48)
,
IGF-1 (49
, 50)
, bFGF (51)
, and NT-4/6
(52)
or overexpression of NGF in transgenic mice
(53)
results in a reduced infarct size after cerebral
ischemia. These results indicate that a number of neurotrophic and
mitogenic growth factors can provide neuroprotection after cerebral
ischemia, raising the possibility that SHP2 may have a role in one or
more of the signaling cascades activated by these factors (17
, 38
, 39)
.
Although our studies demonstrate that SHP2 is important for preventing or limiting the amount of cell death that occurs after ischemia-induced brain damage, it still remains to be determined whether the increased sensitivity to cell death in nestin-SHP2-CS animals is due to a defect in a survival pathway in the hypoxic neurons or to a defect in a protective cellular response to the damage. Both possibilities should be considered either separately or jointly, and studies are under way to elaborate on this. Indeed, preliminary experiments with neuronal cultures derived from wild-type or nestin-SHP2-CS animals indicate that SHP2-CS mutant neurons display an increased sensitivity to hypoxia-induced apoptotic death.
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ACKNOWLEDGMENTS |
|---|
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FOOTNOTES |
|---|
Received for publication March 1, 2000.
Revision received April 19, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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