1 Center for Cancer Research, Massachusetts Institute of Technology, 40 Ames
Street, E17-227, Cambridge, MA 02139, USA
2 Medical Research Council and University of Edinburgh Center for Inflammation
Research, Teviot Place, Edinburgh EH8 9AG, UK
3 Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA
4 Windeyer Institute for Medical Sciences, University College London, 46
Cleveland Street, London W1T 4JF, UK
Author for correspondence (e-mail:
rohynes{at}mit.edu)
Accepted 28 October 2004
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SUMMARY |
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Key words: Cerebral hemorrhage, Spastic paraparesis, Ataxia, Mouse
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Introduction |
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v integrins are necessary for proper interactions between embryonic
cerebral blood vessels and brain parenchymal cells
(McCarty et al., 2002
). Mouse
embryos containing a null mutation in the
v gene, and thus lacking all
five
v integrin family members;
vß1,
vß3,
vß5,
vß6 and
vß8, exhibit normal vascular
morphogenesis except in the brain. These mutants develop intracerebral
hemorrhage and die shortly after birth. By contrast, deletion of both ß3
and ß5 integrin subunits has no effect on cerebral vascular development
(McCarty et al., 2002
);
however, deletion of the ß8 integrin gene leads to a cerebral hemorrhage
phenotype similar to that seen in the
v-null mice
(Zhu et al., 2002
). Thus, loss
of one specific integrin,
vß8, can account for the selective
cerebral blood vessel defects observed in the
v-nulls.
vß8
is a receptor for multiple extracellular matrix (ECM) proteins
(Milner et al., 1999
;
Mu et al., 2002
;
Nishimura et al., 1994
;
Venstrom and Reichardt, 1995
),
and is reportedly expressed on embryonic and postnatal glial cells and neurons
in the CNS (Nishimura et al.,
1998
). The molecular mechanisms underlying how
vß8
integrin functions to regulate cerebral blood vessel integrity remain to be
determined, although normal endothelial cell branching and sprouting, as well
as normal pericyte recruitment and apposition to the vasculature, all occur in
the brains of
v-null embryos (Bader
et al., 1998
; McCarty et al.,
2002
).
To circumvent the early neonatal lethality in the complete v-nulls
and to explore further the cellular basis for the cerebral vascular defects,
we have selectively ablated
v expression in cells of the embryonic and
postnatal CNS. Ablation of
v expression on CNS radial glial cells and
astrocytes leads to embryonic and neonatal cerebral hemorrhage. Deletion of
v expression on both CNS neurons and glia also leads to cerebral
hemorrhage, as well as additional neurological abnormalities, including
seizures, ataxia and loss of hindlimb coordination. This study proves that the
v integrin subunit expressed on neural cells, particularly glia, is
necessary for proper regulation of embryonic cerebral blood vessel
development. Additionally, we reveal a novel function for
v integrins
on postnatal CNS axons.
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Materials and methods |
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Mouse strains
Nestin-Cre mice have been described elsewhere
(Tronche et al., 1999) and
were purchased from Jackson Laboratories. The Tie2-Cre mice have been
previously described (Kisanuki et al.,
2001
). The hGFAPCre transgene contains a 2.2 kb fragment of the
human GFAP gene driving expression of Cre cDNA. Results presented here were
generated using one transgenic line containing 75-100 transgene copies.
Antibodies
The following antibodies were used for immunohistochemistry: anti-Nestin
and anti-RC2 (Developmental Studies Hybridoma Bank); anti-BLBP (Nathaniel
Heintz, Rockefeller University); anti-GFAP (DAKO); mouse anti-ß-tubulin
III (Sigma); mouse anti-Cre mAb (BabCo); mouse anti-PECAM (Pharmingen); and
mouse anti-Smooth Muscle -Actin (Sigma). The
anti-myelin/oligodendrocyte specific protein, anti-neurofilament, anti-NeuN
and anti-GFAP mAbs antibodies were purchased from Chemicon. Secondary
antibodies used for immunofluorescence were Alexa-conjugated goat anti-rabbit,
goat anti-mouse and goat anti-rat (Molecular Probes).
The anti-v antiserum (Bossy and
Reichardt, 1990
) was used at a 1:200 dilution. Prior to
application to sections, the diluted antiserum was pre-absorbed using
v-null acetone-extracted brain protein, prepared by standard methods
(Harlow and Lane, 1999). The antiserum was then applied to unfixed or
Carnoy's-fixed histological sections. The anti-ß8 antiserum was generated
using a synthetic peptide corresponding to the C-terminal tail of the human
ß8 cytoplasmic region, CTRAVTYRREKPEEIKMDISKLNAHETFRCNF. The specificity
of the anti-ß8 antibody was tested using COS cell lysates transiently
transfected with the pcDNA3.1 plasmid (Invitrogen) containing a human ß8
cDNA. The expressed ß8 protein contains a C-terminal V5 epitope tag.
Alternatively, cortical astrocytes cultured from
v+/- or
v-/- neonates were cell surface labeled with EZ-Link
Sulfo-NHS-LC-Biotin (Pierce). Lysates were immunoprecipitated with
anti-ß8, resolved by SDS-PAGE and immunoblotted with Streptavidin-HRP
(Vector Laboratories).
Histology and immunohistochemistry
Adult mice were anesthetized and fixed by perfusion with 4%
paraformaldehyde (PFA). For frozen embedding, adult brains were removed and
post-fixed for 12-16 hours. Embryonic and neonatal brains were dissected and
immersed in Carnoy's fixative or 4% PFA for 4 or 16 hours, respectively.
PFA-fixed tissue was cryopreserved in sucrose at 4°C and embedded in
Tissue Tek OCT (Miles). Alternatively, brains were dehydrated and processed
for standard paraffin embedding and basic histological analyses. Semi-thin (1
µm) plastic sections of embryonic brains were prepared according to
manufacturer's instructions (Polysciences).
Astrocyte culturing
Neonatal brains (P1-3) were removed and placed in sterile, ice-cold Hank's
Balanced Salt Solution (HBSS). Whole neocortices were dissected and the
hippocampus and internal structures were removed to leave only the cortical
sheets. The meninges were stripped away, and the cortical sheets were minced
with a razorblade and digested for 30 minutes at 37°C in Dulbecco's
modified essential media containing 150 units/ml collagenase (Worthington) and
40 µg/ml deoxyribonuclease (Sigma, St Louis, MO). The cortical tissue was
then triturated in DMEM containing 10% calf serum (Sigma, St Louis, MO) and
filtered through a 50 µm sterile mesh. The resulting single-cell suspension
was plated onto T-75 tissue culture flasks that had been pre-coated with 10
µg/ml mouse laminin (Sigma, St Louis, MO). Generally, cells from two or
three neonatal brains were plated per T-75 flask. After 7-10 days the
astroglial cells formed a confluent monolayer, with neurons, oligodendrocytes
and fibroblasts growing on top. These contaminating cells were removed by
rotary shaking the flasks overnight at 250 revolutions per minute. The
resulting cultures were composed of more than 95% astrocytes, as assessed by
anti-GFAP immunoreactivity. Immunostaining of cells was performed using
standard procedures. Briefly, astrocytes were trypsinized and replated onto
laminin-coated coverslips for 24 hours. Cells were fixed in 4% PFA,
permeabilized in 0.1% NP40/PBS, and immunostained with primary and secondary
antibodies.
Cell isolation
Adult brains were dissected and digested with collagenase and
deoxyribonuclease. Brains were then minced with a razorblade and triturated to
generate a cellular suspension. Brain endothelial cells were isolated using
magnetic beads (Dynal) that were coated with anti-PECAM and anti-Flk rat mAbs
(Pharmingen, Inc.) according to manufacturer's instructions.
Mouse behavioral analyses
A Rotarod device (Ugo Basile) was used to measure motor coordination and
strength. The times at which mice fell from the rod were recorded. Mice were
allowed to stay on the rod for a maximum of 360 seconds (6 minutes). Six
separate trials were performed over a 2-day period (three trials per day).
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Results |
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|
Tie2-Cre+; vflox/- mutants were born in
expected Mendelian ratios and at birth displayed no phenotypic defects. Gross
analyses of neonatal (Fig. 1K),
as well as embryonic and adult mutant brains (data not shown) revealed no
obvious cerebral vascular defects. Furthermore, microscopic analysis of mutant
brains showed intact cerebral blood vessels with no indication of dilation or
hemorrhage (Fig. 1L). Thus,
loss of
v expression in vascular endothelium does not account for the
cerebral hemorrhage phenotype observed in the complete
v knockouts.
vß8 integrin protein is expressed on neuroepithelial processes in the embryonic brain
Previous papers using different anti-v integrin antibodies have
reported
v protein expression on embryonic neuroepithelial and radial
glial cells (Anton et al.,
1999
; Hirsch et al.,
1994
). To confirm these results, we used these same anti-
v
antibodies to immunostain E13.5
v+/- and
v-/- brain sections. Surprisingly, we observed a
neuroepithelial expression pattern in both
v+/- and
v-/- brain sections (data not shown). To eliminate this
non-specific
v immunoreactivity, we pre-absorbed anti-
v
antiserum (Bossy and Reichardt,
1990
) using acetone-extracts prepared from
v-null embryos.
Immunostaining
v+/- and
v-/- brain
sections revealed
v-specific neuroepithelial immunoreactivity, as
determined by co-localization with nestin
(Fig. 2A-C). The anti-
v
immunoreactivity did not colocalize with markers for newly differentiated
neurons, such as ß-tubulin III or MAP2 (see Fig. S1 in the supplementary
material and data not shown).
|
Neural cell deletion of v integrin leads to cerebral hemorrhage
We attempted to ablate v expression selectively on embryonic and
postnatal glial cells using Cre under the control of the human GFAP promoter.
In most mammalian species, GFAP is expressed primarily by postnatal
astrocytes; however, in primates, GFAP is also expressed by embryonic radial
glia and independently generated hGFAP-Cre transgenes show Cre expression in
both embryonic and post-natal glial cells
(Bajenaru et al., 2002
;
Malatesta et al., 2003
;
Marino et al., 2000
;
Zhuo et al., 2001
).
We used an anti-Cre antibody to monitor Cre protein expression in our
hGFAP-Cre transgenics. Nuclear Cre protein was most prominent in cells
adjacent to, and occasionally within, the ventricular zone of the neocortex
(Fig. 3A,B) and ganglionic
eminence (data not shown) as early as E15. Based on this pattern, we analyzed
whether Cre protein was present in radial glial cells. Using antibodies
recognizing the brain lipid-binding protein (BLBP) and the astrocyte-specific
glutamate transporter (GLAST) (Anthony et
al., 2004; Malatesta et al.,
2003
), we showed Cre protein in many cortical radial glial cells
in E15 embryos (Fig. 3A,B). By
contrast, at E15, no co-localization of Cre and ß-tubulin III (a marker
for neurons) was detected in the neocortex
(Fig. 3C) or ganglionic
eminence (data not shown). Cre protein was also expressed by many
GFAP-positive Bergmann glia in the cerebellum as well as astrocytes in the
neonatal cerebral cortex (Fig.
3D,E). hGFAP-Cre transgene expression in astrocytes and neurons
was also observed in vivo using the Rosa26-LoxSTOPLox-lacZ reporter
strain (Soriano, 1999
) to
monitor hGFAP-driven Cre activity (data not shown). Owing to their
multi-potential nature, CNS radial glial cells give rise to neurons throughout
the brain (Anthony et al.,
2004
; Malatesta et al.,
2003
). Therefore, it is likely that in our hGFAP-Cre strain, some
embryonic and postnatal neurons may lack
v expression. Any
contributions that these neurons normally make to cerebral blood vessel
function would also be defective in the hGFAPCre conditional
v
mutants.
|
hGFAP-Cre+; v+/- progeny were mated with mice
containing a loxP-flanked (floxed)
v allele (Lacy-Hulbert et al.,
submitted). Thus, the resulting mutant progeny are hemizygous for the
hGFAP-Cre transgene, and carry one
v-flox and one
v-null allele.
Littermate controls were hemizygous for the hGFAP-Cre transgene, and carry one
vflox and one
v-wild type allele.
hGFAP-Cre+; vflox/- mutants were born in
expected Mendelian ratios (18 mutants from 68 total F1 progeny, or 26%).
Analysis of hGFAP-Cre conditional
v mutant brains revealed that
intracerebral hemorrhage occurred with 100% penetrance. P5 mutant neonates
(Fig. 3K) or E18 embryos (data
not shown) developed obvious punctate regions of microhemorrhage in the
cerebral cortex, mid-brain and occasionally in the cerebellum
(Fig. 3M; data not shown).
Brains from older neonates between P7 and P14 revealed progressive reduction
in cerebral microhemorrhage (data not shown), and gross and microscopic
analyses of adult mutant brains revealed no obvious signs of hemorrhage or
edema (Fig. 3O,Q).
Immunolabeling with anti-laminin antibody revealed intact intracerebral blood
vessels with no indications of distension or rupture (data not shown).
Furthermore, perfusion of a small molecular weight reactive biotin tracer,
revealed no obvious blood-brain barrier abnormalities in adult hGFAP-Cre
conditional
v mutants (see Fig. S2 in the supplementary material).
Deletion of v integrin in both neurons and glia also leads to cerebral vascular defects
We hypothesized that the significantly less severe hemorrhage observed in
the hGFAP-Cre conditional v mutants (as compared with complete
v-nulls) was connected to the late onset (
E15) of Cre expression.
To ablate
v expression earlier during CNS development we used a
Nestin-Cre transgenic mouse strain which expresses Cre under control of a
Nestin promoter (Graus-Porta et al.,
2001
; Haigh et al.,
2003
; Tronche et al.,
1999
). The Nestin-Cre transgene drives Cre expression primarily in
CNS neural precursor cells that give rise to both neurons and glia
(Haigh et al., 2003
).
Nestin-Cre+; v+/- mice were mated with mice containing a
conditional (floxed)
v allele. The resulting mutant progeny were
hemizygous for the Nestin-Cre transgene, and carried one
v-flox and one
v-null allele. Littermate controls were hemizygous for the Nestin-Cre
transgene, and carried one
vflox allele and one
v wild-type
allele. Cre-mediated recombination of the
v-flox allele was confirmed
by PCR analysis of DNA from adult mutant brains. DNA isolated from the tail
yielded PCR products of 350 and 250 bp, indicating no detectable Cre-mediated
recombination (Fig. 4A,B). DNA
isolated from adult brain tissue, however, showed a dramatic reduction in the
350 bp product (
v-flox), owing to Cre-mediated recombination of the
v-flox allele. The low level of 350 bp band
(Fig. 4B) is probably due to
the absence of Cre-mediated recombination in non-neural cells, e.g.
circulating blood cells, fibroblasts, etc. Additionally, immunoblotting
lysates prepared from adult cerebral cortex and cerebellum with anti-
v
antibody revealed marked reduction in
v protein in Nestin-Cre
conditional
v mutants (Fig.
4C). The protein band at
80 kDa
(Fig. 4C) is nonspecific. It is
not a fragment of full-length
v, as it is observed in
v-null
cell and tissue lysates (data not shown).
|
Central nervous system-specific deletion of v integrin leads to behavioral abnormalities
Nestin-Cre conditional v mutants survived beyond the neonatal period
and were behaviorally normal until 2-3 weeks after birth. At this point,
30% (13 of 39 mutants identified so far) began to display obvious signs
of motor dysfunction (Fig. 5B).
These conditional
v mutants also developed spontaneous convulsions, and
ultimately died by 4 weeks of age (data not shown). Histological analyses of
CNS tissue isolated from mutants revealed focal regions of microhemorrhage
throughout the brain and spinal cord (Fig.
5D,F). As hemorrhage begins in utero and is present prior to the
onset of apparent seizure (Fig.
4E,G,I), it is very likely that the cerebral hemorrhage causes the
seizures and premature death. The incomplete penetrance associated with the
seizures and early death may be related to genetic heterogeneity, as the mice
used in this study were a mixture of C57BL/6, 129S4 and FVB genetic
backgrounds.
|
Nestin-Cre conditional v mutants develop motor deficits due to axonal degeneration in the spinal cord and cerebellum
We initially expected the hindlimb dysfunction in the adult mutants to be
linked to neurological damage because of persistent cerebral hemorrhage and
stroke. However, gross and microscopic examination of adult mutant brains
revealed no pathological indication of hemorrhage (see Fig. S3 in the
supplementary material). CNS deletion of the ß1 integrin subunit also
leads to ataxia and premature death, largely owing to abnormal anchorage of
glial end-feet to marginal zone basement membranes in the cortex and
cerebellum (Graus-Porta et al.,
2001). Thus, it was possible that selective loss of
vß1 could account for the neurological defects observed in the
Nestin-Cre conditional
v mutants. However, immunofluorescence analysis
of Nestin-Cre conditional
v mutants revealed normal glial end-feet
attachments in the cortex and cerebellum (see Fig. S3 in the supplementary
material). Histological analysis of brains from neonatal and adult Nestin-Cre
mutants, both by Hematoxylin and Eosin staining, as well as
immunohistochemistry using an antibody directed against the neuronal nuclear
marker NeuN, revealed relatively normal neuronal patterning in the cerebral
cortex and cerebellum (see Fig. S3 in the supplementary material; data not
shown). Additionally, silver staining to label axonal neurofilaments, or
immunohistochemistry using an anti-MAP2 antibody to label dendrites, revealed
normal neuronal arborizations (see Fig. S3 in the supplementary material; data
not shown).
We also suspected that the onset of spastic paraparesis in the Nestin-Cre
conditional v mutants might be due to neuromuscular defects. However,
examination of mutant quadriceps showed no indications of muscular atrophy or
wasting (see Fig. S4 in the supplementary material). Furthermore, inspection
of hindlimb peripheral nerves as well as peripheral nerves connecting to
thoracic and lumbar spinal cord dorsal root ganglia revealed no obvious
pathological indication of peripheral axonal degeneration or demyelination
(see Fig. S4 in the supplementary material; data not shown). This is unlike
the paralysis and demyelination defects observed in mutant mice where ß1
integrin is selectively ablated in peripheral nervous system Schwann cells
(Feltri et al., 2002
).
Analyses of grey matter spinal cord regions from controls and Nestin-Cre
conditional v mutants did not reveal motor neuron dystrophy or
degeneration (Fig. 6C,D).
However, analysis of spinal cord white matter revealed demyelination
(Fig. 6F) and axonal dystrophy
(data not shown) within the fasciculus gracilis, an axonal tract that
regulates hind limb proprioception and discriminative touch. Additionally, an
obvious macrophage infiltrate was present, with many macrophages containing
myelin fragments (Fig. 6F) as
well as axonal fragments (data not shown), suggesting both glial and axonal
degeneration.
|
vß8 integrin is expressed on axons in the cerebellum
Ablation of the ß8 gene leads to a cerebral hemorrhage phenotype
(Zhu et al., 2002), which is
essentially identical to that observed in the complete
v-nulls
(Bader et al., 1998
). With this
knowledge, we hypothesized that the neurological defects observed in the
Nestin-Cre conditional
v mutants were probably due to the selective
loss of
vß8 function. To determine the expression pattern for
vß8 protein in the adult cerebellum, we used an antibody directed
against the ß8 integrin subunit (Fig.
7A,D,G).
vß8 was expressed on axons in the caudal
cerebellum, as determined by co-localization with an anti-neurofilament
antibody (Fig. 7C). However,
vß8 was not detected on cerebellar white matter astrocytes
(Fig. 7F) or oligodendrocytes
(Fig. 7I).
vß8
protein was also expressed on spinal cord axons, including the fasciculus
gracilis (data not shown). Interestingly,
vß8 was also expressed
on axons in white matter regions of the cerebral cortex (data not shown),
although no pathological lesions were observed in these regions at the time of
death.
|
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Discussion |
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vß8 integrin on embryonic CNS neural cells is necessary for proper cerebral blood vessel development
Cerebral blood vessels are composed of endothelial cells and pericytes
surrounded by a vascular basement membrane. The close juxtaposition between
cerebral blood vessels and surrounding neural cells, particularly glia, evokes
an attractive model, whereby vß8 integrin on the glial processes
mediates interactions with blood vessels via adhesion to ECM ligands within
the vascular basement membrane. Neural cell adhesion via
vß8 may
provide physical support that maintains proper blood vessel morphology.
Alternatively,
vß8 may play a more dynamic role in mediating the
adhesion and communication between neural cells and blood vessels. We propose
a model where
vß8 integrin is necessary for the initiation and
maintenance of neural cell-blood vessel adhesion and communication, perhaps
including proper assembly of the basement membrane. A wave of secondary events
would then serve to affect proper neural cell-blood vessel juxtaposition. We
are currently identifying and characterizing the signal transduction pathways
activated by
vß8 adhesion in an attempt to understand better
neural cell-mediated regulation of cerebral blood vessel development.
Differences between conditional v-null and complete
v-null phenotypes
Why do 100% of complete v-nulls die by the first day of birth,
whereas the majority of the conditional
v mutants survive for several
months? First, conditional
v mutants do not develop other non-CNS
abnormalities that occur with variable penetrance in complete
v-nulls
(Bader et al., 1998
). Second,
the hemorrhage in the conditional
v knockout mice is significantly less
severe than that in the complete
v-nulls, most probably because of
differences in Cre spatial expression. Additionally, there may be a temporal
delay from the onset of Cre expression (and
v-flox deletion) to the
point where
v integrin mRNA and protein are completely absent from the
cells. Last, epigenetic events may preclude all glial cells from efficiently
recombining the
v-flox allele, leading to a mosaic pattern of Cre
expression. Such mosaic Cre expression would decrease the overall proportion
of
v-null glial cells, thus leading to a reduction in the severity of
hemorrhage. Alternatively, it is possible that loss of
v expression on
multiple CNS cell types is necessary to recapitulate fully the severe cerebral
hemorrhage observed in the complete
v-nulls.
vß8 integrin mediates long-term axonal survival
We initially hypothesized that the neurological phenotypes observed in the
adult Nestin-Cre conditional v mutants were the result of CNS trauma
caused by the early embryonic and neonatal hemorrhage. Indeed, this is the
case for humans with various forms of cerebral palsy, where localized CNS
trauma during prenatal or neonatal periods leads to neuronal cell death and
the unfortunate symptoms associated with this disease
(Ozduman et al., 2004
).
However, the hemorrhage in the Nestin-Cre conditional
v mutants occurs
throughout the embryonic and neonatal brain, whereas the neurological lesions
first develop in specific regions of the adult spinal cord and cerebellum,
suggesting a more localized defect responsible for the neurological
phenotype.
Alternatively, the neurological lesions may result from progressive
hydrocephaly resulting from intraventricular hemorrhage. Indeed, complete
v-null mutants develop intraventricular hemorrhage as early as E12.5,
and display severe hydrocephaly at birth
(Bader et al., 1998
).
Interestingly, severe hydrocephaly in humans can cause pathological lesions in
the long white matter tracts of the cerebral cortex and cerebellum, leading to
symptoms such as seizures, ataxia, lower limb spasticity and urinary
dysfunction (Silverberg,
2004
). However, we have yet to observe intraventricular hemorrhage
or obvious hydrocephalus in the Nestin-Cre conditional
v mutants.
Nonetheless, it remains possible that subtle changes in ventricle volume may
account for some of the neurological abnormalities.
A more likely explanation for the neurological phenotypes, and one
supported by our experimental data, is that the adult-onset defects are
unrelated to the embryonic and neonatal hemorrhage. Indeed, to date, no
neurological defects or premature death are observed in the hGFAP-Cre
conditional v mutants, which also develop embryonic and neonatal
cerebral hemorrhage, albeit less severe than we see in the Nestin-Cre
conditional
v mutants. Additionally, the progressive neurological
phenotypes observed in the Nestin-Cre conditional
v mutants develop
over a period of several months, well beyond the time of embryonic and
neonatal hemorrhage. Last,
vß8 integrin protein is expressed on
axons in the spinal cord and cerebellum where pathologic lesions develop in
the Nestin-Cre conditional
v mutants. These data collectively suggest
that the CNS abnormalities observed in the Nestin-Cre conditional
v
mutants are likely to be related to the specific loss of
vß8, as
they do not occur in ß3/ß5 integrin double knockout mice
(McCarty et al., 2002
), or
ß6 integrin knockouts (Huang et al.,
1996
), and are distinct from the neurological defects in mice
lacking ß1 integrins in the CNS or PNS
(Graus-Porta et al., 2001
;
Feltri et al., 2002
). The
confirmation of this hypothesis awaits generation and analysis of conditional
ß8 knockout mice.
Interestingly, in the Nestin-Cre conditional v mutants, we observe
not only spinocerebellar axonal degeneration, but also significant
demyelination. Thus, analogous to its role in regulating glia-blood vessel
juxtaposition, axonally expressed
vß8 may also mediate proper
axonal-glial interactions. CNS axons intimately associate with myelinating
oligodendrocytes via direct cell-cell contacts, as well as cell-ECM adhesion.
Recent evidence also shows cooperative signaling between integrins and a
variety of axonal guidance and survival factors, including the Ephs/ephrins
(Zou et al., 1999
),
semaphorins (Pasterkamp et al.,
2003
), netrins (Yebra et al.,
2003
) and slits (Stevens and
Jacobs, 2002
). It will be interesting to determine whether
vß8 integrin signaling events `crosstalk' with these other
pathways, and how these events may regulate axonal survival.
v mutants and human genetic diseases
The cerebral hemorrhage in the conditional v mutants is very similar
to pathologies associated with the human genetic disorder cerebral cavernous
malformation (CCM). Individuals with CCM develop CNS hemorrhage characterized
by tortuous blood vessels devoid of surrounding brain parenchyma, leading to
clinical symptoms ranging from mild headaches to sporadic seizures and loss of
motor function (Marchuk et al.,
2003
). Mutations in the KRIT1 gene have been linked to
50% of all familial CCM cases (Gunel
et al., 2002
; Laberge-le
Couteulx et al., 1999
). We are currently investigating whether
vß8 integrin plays a role in regulating Krit1 function and how
these events may be connected to development of CCM.
Finally, the neurological abnormalities observed in the adult Nestin-Cre
conditional v mutants are similar to a human genetic disorder known as
hereditary spastic paraparesis (HSP)
(Kobayashi et al., 1996
;
McDermott et al., 2000
).
Individuals with HSP develop progressive lower limb spasticity partly because
of deterioration of spinocerebellar axons. It will be interesting to determine
if the Nestin-Cre conditional
v mutants might serve as a useful mouse
model for studying the pathogenesis and treatment of symptoms related to
HSP.
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Supplementary material |
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ACKNOWLEDGMENTS |
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Footnotes |
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