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First published online 14 February 2007
doi: 10.1242/dev.02808
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1 Department of Neuroscience, University of Geneva Medical School, CH-1211
Geneva, Switzerland.
2 Department of Anesthesiology, Pharmacology and Intensive Care, University
Hospital of Geneva, Geneva, Switzerland.
3 Laboratoire de Génétique et Physiologie du Développement,
CNRS 9943, Parc Scientifique de Luminy, Case 907, F-13288 Marseille Cedex 9,
France.
* Author for correspondence (e-mail: jozsef.kiss{at}medecine.unige.ch)
Accepted 5 January 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Neuronal survival, Neurotrophin, PSA-NCAM, p75 (Ngfr), Olfactory bulb, Neurogenesis
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Rousselot et al., 1995).
Polysialic acid (PSA) is a linear homopolymer of 
2,8-linked sialic acid
that is uniquely attached to the neural cell adhesion molecule (NCAM) in the
vertebrate brain (Kiss et al.,
2001; Rutishauser and
Landmesser, 1996). PSA-NCAM is abundantly expressed in the
developing central nervous system (CNS) where its presence is associated with
morphogenetic events, such as cell migration, axonal growth and synaptogenesis
(Edelman, 1986;
Kiss and Rougon, 1997). In the
postnatal period, the polysialic content of NCAM tends to decrease
(Rutishauser and Landmesser,
1996), but PSA-NCAM does persist in adult brain structures that
display a high degree of morphofunctional plasticity
(Durbec and Cremer, 2001).
The functional significance of PSA on NCAM at the cell surface of these
immature neurons is not completely understood. Genetic deletion of the NCAM
molecule (NCAM-/-) results in a 
30% decrease in the size of
the olfactory bulb (OB), and the overall brain size is reduced by about 10%
(Cremer et al., 1994;
Tomasiewicz et al., 1993).
These defects can be duplicated by the injection of Endo-N, an enzyme that
specifically removes the PSA moiety associated with NCAM, suggesting that the
observed phenotypical changes in the NCAM-/- animal are primarily
related to the absence of the PSA chain itself
(Ono et al., 1994). Parallel
to the reduction in OB size, an increased number of neuronal precursors are
observed in the subventricular zone and rostral migratory stream (SVZ-RMS) of
NCAM-/- animals as compared with wild-type (WT) littermates
(Chazal et al., 2000;
Ono et al., 1994). It has been
suggested that this accumulation of neuronal precursors in the SVZ-RMS is the
result of impaired chain migration of these cells toward the OB
(Hu et al., 1996;
Ono et al., 1994).
Although much has been learned about the molecular control of proliferation
and migration of SVZ-derived progenitors
(Alvarez-Buylla and Garcia-Verdugo,
2002), relatively little is known about factors that control their
survival. Sensory inputs (Miwa and Storm,
2005; Rochefort et al.,
2002), glutamate (Brazel et
al., 2005) and PTEN (Li et
al., 2002) have been shown to regulate survival of newly generated
neurons. In this study, we explored the possibility that PSA-NCAM is involved
in survival of postnatally generated, new neurons by focusing on the
SVZ-RMS-OB where PSA-NCAM expression is maintained at high levels throughout
life (Garcia-Verdugo et al.,
1998). We compared cell death in NCAM-/- mice and WT
animals and tested the effect of removing or blocking PSA-NCAM in a culture
model. Our results demonstrate that PSA-NCAM promotes neuronal survival by
regulating p75 (Ngfr - Mouse Genome Informatics) neurotrophin receptor
expression.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and
maintained in a C57/B6J background (five backcrosses).
Labeling of cells in the SVZ-RMS was performed by means of a lentivector
carrying GFP under the regulation of the ubiquitin promoter. Attenuated
lentivector particles were produced by transfection of 293T cells according to
standard protocols (Klages et al.,
2000). WT and NCAM-/- pups at postnatal day 7 (P7) were
anesthetized with 2% isofluran (Foren) in a 30%:70% mixture of
O2:air, and maintained in a stereotaxic frame. One microliter of a
suspension containing the lentivector at a concentration of
1x109 transducing units/ml was stereotactically injected
(coordinates from the bregma: 0 mm anterior, 0 mm posterior, 1 mm lateral)
with a Hamilton syringe at a depth of 1.5 mm from the surface of the brain.
Animals were sacrificed 4 days after the injection and brains processed for
immunofluorescence.
Cell culture and reagents
SVZ-derived cultures were prepared from newborn rats (P0) or P7 mice as
previously described (Gascon et al.,
2005). Briefly, the SVZ was dissected from coronal slices,
dissociated mechanically, trypsinized and purified using percoll gradient
centrifugation. Cells were plated onto polyornithine (Sigma, St Louis, MO)
-coated cell culture supports and allowed to grow in Neurobasal Medium (Gibco,
Paisley, UK) with 2% B27 supplement (Gibco), 2 mM L-glutamine
(Gibco) and 1 mM sodium pyruvate (Sigma).
For experiments with neurotrophins, recombinant human NGF and BDNF were
purchased from Regeneron Pharmaceuticals (Tarrytown, NY). The inhibitors
myriocin, fumonisin B1 and SP600125 were obtained from Biomol (Plymouth
Meeting, PA, USA) and K252a from Calbiochem (Merck Biosciences, Germany). To
remove PSA from cell surfaces, we used the enzyme Endo-N, purified from phage
K1 (Kiss et al., 1994). Endo-N
has been shown to rapidly and specifically degrade linear polymers of sialic
acid with 2,8-linkage, and with a minimum length of 7-9 residues
(Vimr et al., 1984).
Histology and immunofluorescence
To analyze the SVZ-RMS, P7 mice were sacrificed by decapitation. Brains
were removed and fixed overnight in 4% paraformaldehyde (PAF). Brains were
then frozen and cut using a cryostat (Leica, Germany). Sagittal slices of 20
µm were plated onto gelatin-coated slides and post-fixed for 30 minutes
with 4% PAF. To correlate the extent of the SVZ area with the number of
apoptotic cells, adjacent sections were stained by TUNEL labeling (see below)
and with Hematoxylin-Eosin (HE) following standard protocols.
For immunolabeling, sections were rinsed three times in PBS and then incubated with primary antibodies in 0.5% BSA (Sigma) 0.3% Triton X-100 in PBS for 48 hours at 4°C. Antibodies used were against GFAP (Dako, Dakopatts, Copenhagen, Denmark) 1/500, mCD24 (generous gift from G. Rougon, IBDML, Marseille, France) 1/200, GFP (Molecular Probes, Invitrogen) 1/2000, doublecortin (Santa Cruz Biotechnology, Santa Cruz, CA) 1/1000, NeuN (Chemicon) 1/500, and p75 (Promega, Madison, WI) 1/1000. After three washes with PBS, Alexa-conjugated secondary antibodies (Molecular Probes) diluted in PBS were applied for 90 minutes at room temperature.
Immunocytochemistry in cultures was performed as previously described
(Gascon et al., 2005). Primary
antibodies used were: anti-ßIII-tubulin (Sigma) 1/500, anti-p75 (Promega)
1/1000, anti-activated caspase 3 (R&D systems, Avingdon, UK) 1/2000, Men B
IgM that recognizes 2,8-linked PSA of a chain length greater than 12
residues (Rougon et al., 1986)
1/500, and anti-GABA (Matute and Streit,
1986) 1/2000. For GABA, cells were fixed in 2% PAF, 2%
glutaraldehyde in phosphate buffer (pH 7.4) for 30 minutes followed by 1 hour
in 4% PAF. Before the incubation with the primary antibody, cells were rinsed
three times for 20 minutes each with PBS, and then incubated for 30 minutes in
a solution of 0.1 M NH4Cl.
TUNEL assay
Samples were rinsed with PBS and incubated for 15 minutes with the TUNEL
buffer (30 mM Tris, 140 mM sodium cacodylate and 1 mM cobalt chloride). Then,
0.3 U/µl terminal transferase (Roche, Germany) and 6 µM labeled dUTP
were applied for 90 minutes at room temperature. The reaction was stopped with
2x SSC (sodium citrate buffer) and the samples washed again with
PBS.
For brain slices, 7'-fluorescein dUTP (Roche, Germany) was chosen as a label. The fluorescent labeling enabled quantification of apoptotic cells under a fluorescent microscope (Axiophot, Zeiss, Germany), and evaluation of the colocalization of TUNEL-positive cells with GFAP, mCD24 or p75, under the confocal microscope (LSM 510, Zeiss, Germany).
Unless indicated otherwise, all treatments on cultured cells were performed 5 days after plating and lasted 20 hours. TUNEL cells were revealed using both 7'-fluorescein and 16'-biotin dUTP (Roche, Germany). For biotin detection, cells were incubated either with the ABC Kit (Vector Laboratories, Burlingame, CA) followed by the chromogenic substrate (diaminobencindine, DAB), or with avidin-Texas Red (1/1000) (Vector Laboratories).
SYBR green assay
Total RNA was extracted using the RNA-Easy Micro Kit (Qiagen, Hilden,
Germany) following the manufacturer's instructions. 50 ng of total RNA were
converted to cDNA using Sensiscript reverse transcriptase (Qiagen). Reverse
transcription was performed in a T3 thermocycler (Biometra, Göttingen,
Germany) for 1 hour at 37°C. Real-time RT-PCR was carried out in an ABI
Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA).
PCR reactions were performed in triplicate using SYBR Green PCR Master Mix
(Applied Biosystems). Each SYBR Green reaction (10 µl total volume)
contained 1 µl of cDNA as template and each primer at 0.3 µM. Controls
without template (water) or reverse transcriptase were always negative. The
oligonucleotide primers used for ß-actin, TrkB, TrkC and p75 have been
described elsewhere (Gascon et al.,
2005).
Western blotting
The RMS-OB and cortex were microdissected from P0 or P7 WT and
NCAM-/- mice (n=5). Samples (tissue or cultured cells)
were first homogenized in ice-cold disruption buffer (20 mM Tris-HCl, pH 7.4,
10 mM NaCl, 10 mM KCl, 3 mM MgCl2, 0.5% NP-40) containing a
protease inhibitor mix (Complete Mini Protease Inhibitors, Roche), and then
centrifuged for 10 minutes at 10,000xg at 4°C to
obtain a total cell lysate. The supernatant was removed and stored at
-80°C. 25 µg of protein were electrophoresed and then transferred to a
PVDF membrane in 25 mM Tris, 192 mM glycine and 20%
methanol. After blocking (for 1 hour at room temperature in PBS containing 5%
non-fat dried milk and 0.1% Tween-20), blots were incubated in primary mouse
monoclonal antibody anti--tubulin 1/8000 (Sigma), rabbit polyclonal
anti-p75 1/2000 (Promega), or the rabbit polyclonal anti-phospho Trk (Tyr 490)
1/1000 (Sigma), overnight at 4°C. Membranes were rinsed three times,
incubated with the appropriate horseradish peroxidase-linked secondary
antibody at 1/5000 (Biorad, Hercules, CA) for 2 hours at room temperature and
developed using ECL reagents (Amersham Biosciences, Little Chalfant, UK).
PSA and p75 quantification
PSA immunolabeling was performed on living cells. Briefly, medium was
harvested, cultures were rinsed in PBS and then incubated with a PSA antibody
(Ab 735, generous gift from Dr Gerardy-Schahn, Abteilung Zelluläre
Chemie, Medizinische Hochschule, Hannover, Germany) for 30 minutes at 4°C.
Cells were then fixed with 4% PAF and incubated with the secondary antibody
linked to rhodamine. Quantification of PSA surface fluorescence was carried
out using confocal microscopy (LSM 510) as previously described
(Kiss et al., 1994). Ninety
cells at each time point in three different experiments were randomly imaged.
Each captured field contained a single cell profile to be evaluated. All the
parameters were kept constant for each session of measurements. Fluorescence
was scaled between 0 (lowest intensity) and 255 (highest intensity). Levels of
PSA were estimated as the proportion of PSA-rich regions (characterized by
intensity values between 248 and 255) relative to the total area of the cell
(defined as the pixels whose intensity was higher than the background). For
p75, after background subtraction, the mean fluorescence intensity was
calculated for each cell. These values were grouped into 20 intensity
categories and the percentage of cells falling in each category was plotted
for the control and the Endo-N group.
Image acquisition and data analysis
The number of apoptotic cells along the SVZ-RMS and the striatum was
counted under a fluorescent microscope. The surface of these regions was
estimated with Scion Image software using Hoechst counterstaining on the
analyzed sections and HE staining on the next section. More than 10 sections
per brain (WT n=4; NCAM-/- n=4) were used to
calculate the density of apoptotic cells. For colocalization of TUNEL with
GFAP, mCD24 or p75, sections were examined under the confocal microscope (LSM
510, Zeiss). For this analysis, TUNEL-positive cells were examined along the
z-axis and scored as positive if they colocalized completely with the
other marker. Any doubtful cell was considered as negative. More than 200 (WT)
and 400 (NCAM-/-) TUNEL-positive cells were analyzed in six
different sections of at least three different brains.
Cultured cells were analyzed under a light microscope (Eclipse TE2000-U, Nikon, Zurich, Switzerland). For quantification of cell death rate, half of the culture medium was replaced by fresh medium containing the appropriate treatment. 20 hours later, dying cells were revealed using TUNEL or caspase 3. Positive cells were counted on 40 random fields (0.064 mm2) in at least two sister cultures and expressed as a percentage of the total cell number. More than 300 cells per condition were considered in each experiment. Results were expressed as mean±s.d. of at least three independent experiments.
For statistical analysis, one-way ANOVA followed by a two-tailed unpaired t-test or Holm-Sidak test were performed. Statistical differences were set for P<0.05.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Ono et al., 1994), a larger
SVZ-RMS and a smaller OB were observed in the NCAM-/- mice
(Fig. 1A). Using the TUNEL
assay, we observed that dying cells were present in all brain regions but
their number was particularly elevated in the SVZ-RMS
(Fig. 1B,C). Quantitative
analysis revealed a nearly threefold increase in the density of TUNEL-positive
cells in the SVZ-RMS of the NCAM-/- animals as compared with WT
(Fig. 1B,C). No significant
differences were found in the OB or in other brain regions such as the
striatum (Fig. 1C), confirming
the specificity of the observed increase in the SVZ-RMS system.
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).
We found that less than 5% of TUNEL profiles colocalized with GFAP (Glial
Fibrillary Acidic Protein), a marker of astrocytes. In addition, there was no
difference in the percentage of double-labeled cells (TUNEL+
GFAP+) between WT and NCAM-/- mice (5.03±2.8% in
WT versus 4.03±1.07% in NCAM-/-). We then examined whether
TUNEL-positive cells displayed neuronal markers. For this purpose, we used
mCD24 (Cd24a), a cell surface protein whose expression largely overlaps with
PSA-NCAM (Calaora et al.,
1996). We found that many TUNEL-positive nuclei colocalized with
mCD24 in the WT animal. However, the proportion of these double-labeled cells
was significantly higher in the mutant SVZ-RMS (43.97±8.5% in WT versus
61.05±5.8% in NCAM-/-, P<0.05). Thus, the
absence of NCAM/PSA-NCAM leads to enhanced apoptosis specifically in the
population of newly generated neurons.
Removal or inactivation of the PSA moiety on NCAM modifies the survival of immature SVZ-derived neurons in culture
The increased cell death in the SVZ-RMS of the NCAM-/- mice
could be related to a direct effect of the absence of PSA-NCAM on cell
survival, or to an indirect effect due to impaired migration and accumulation
of newly generated neurons in the RMS
(Chazal et al., 2000;
Ono et al., 1994). To address
this issue, we took advantage of an in vitro model system, where neurons from
the postnatal SVZ are purified and developed as olfactory interneurons
(Gascon et al., 2005). This
model enables the evaluation of cell survival independently of migration. As
seen in Fig. 2, neurons in
these low-density cultures survive and display a GABAergic phenotype. PSA-NCAM
and NCAM were expressed in cells from WT animals, whereas immunostaining for
these molecules was completely absent in NCAM-/- cultures
(Fig. 2). Under basal
conditions, few cells underwent apoptosis by the end of the fourth day in
vitro (DIV), as revealed by caspase 3 immunostaining and TUNEL assay
(Fig. 3A). TUNEL labeling was
significantly increased in WT cultures when the PSA moiety was removed from
NCAM by the enzyme Endo-N (PSA- NCAM+)
(Fig. 3B). Enhanced apoptosis
was also observed when cells were exposed to the PSA function-blocking
antibody m735 (Frosch et al.,
1985) (Fig. 3B).
Neither Endo-N nor the m735 antibody affected cell survival of neurons
isolated from the NCAM-/- mice, arguing against a possible
non-specific toxic effect of these treatments (not shown). These data suggests
that PSA-NCAM plays a role in the survival of SVZ-derived neurons in
culture.
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).
Accordingly, it has been shown that administration of exogenous neurotrophins
in vivo promotes survival of newly generated neurons in the SVZ-RMS-OB
(Zigova et al., 1998). To
better understand the role of PSA-NCAM in neuronal survival of SVZ-derived
neurons, we exposed WT, Endo-N-treated WT and NCAM-/- cells to
exogenous BDNF and NGF (Brain Derived Neurotrophic Factor and Nerve Growth
Factor, respectively) and assessed the effect of these neurotrophins on cell
death rate. As shown in Fig.
3C, BDNF significantly decreased apoptotic cell death in WT
cultures and this effect was reduced by Endo-N treatment. BDNF did not reduce
cell death, even at high doses, in cultures isolated from NCAM-/-
animals (Fig. 3C). The effects
of NGF on neuronal survival were very different from those of BDNF. NGF did
not modify cell survival in WT cultures; however, it increased cell death in
NCAM-/- as well as in Endo-N-treated cultures
(Fig. 3C). Thus, PSA seems to
modulate the response to neurotrophins in SVZ-derived neurons. It should be
emphasized here that the increase in apoptotic rate determined in our
short-term experiments led to massive cell loss at longer exposure times.
Thus, 3 days of Endo-N treatment, alone or with NGF (50 ng/ml), reduced the
number of neurons by 30% or 60%, respectively.
We then took advantage of the fact that, along with maturation, neurons
spontaneously downregulate PSA expression
(Kiss and Rougon, 1997;
Rutishauser and Landmesser,
1996). In agreement with these reports, we observed a
time-dependent decrease of PSA-NCAM immunostaining in cultured SVZ-derived
neurons. Our quantitative analysis (see Materials and methods) revealed that
the intensity of PSA staining decreased nearly fivefold between 3 and 9 DIV
(Fig. 4A,B). Consistent with
this observation, we found that addition of NGF to 9-DIV cultures (low PSA
levels) triggered a significant increase in cell death rate compared with
basal conditions (sister cultures treated with the equivalent amount of
control medium) (Fig. 4C). By
contrast, this effect was not detected in cells challenged with NGF at 3 or 6
DIV (high PSA levels) (Fig.
4C). When the same experiments were repeated with
NCAM-/- cells, we found a similar increase in cell death in
response to NGF at all time points (increase in cell death, NGF versus
control: at 3 DIV, 94.64±29.22%; at 6 DIV, 100.35±36.6%; at 9
DIV, 68.95±36.37%). Thus, the increased sensitivity of WT cells to NGF
at mature stages seems to be related to their low PSA content and confirms our
observations obtained using Endo-N and the m735 antibody.
Together, these results support the hypothesis that the presence of PSA at the cell surface influences the response of immature SVZ-derived neurons towards neurotrophins and is essential for their proper survival.
Inhibitors of p75 block the effect of PSA removal on cell survival
Neurotrophins could exert their biological effects through Trk receptors
and/or the low-affinity p75 receptor
(Miller and Kaplan, 2001). We
have previously shown that SVZ-derived neurons in vitro express neurotrophin
receptors p75, TrkB and TrkC, but not TrkA (Ngfr, Ntrk2, Ntrk3 and Ntrk1,
respectively, Mouse Genome Informatics)
(Gascon et al., 2005). Our
results, showing that BDNF-induced cell survival was substantially reduced in
the absence of PSA and that NGF further increased cell death under these
conditions raised the possibility that the presence or absence of PSA might
modify cell survival independently of Trk signaling pathways. To test this
hypothesis, we used the pan-Trk inhibitor K252a (400 nM), which is known to
block signaling through all Trk receptors
(Koizumi et al., 1988). Using
western blots, we confirmed that K252a effectively blocks phosphorylation of
Trk receptors in response to BDNF (Fig.
5A). We also found that in the presence of K252a
(Fig. 5B), the number of
apoptotic cells was still significantly increased in NCAM-/-
cultures as well as after Endo-N treatment, as compared with control WT
cultures. Most importantly, in the absence of both Trk signaling and PSA-NCAM
at the cell surface, treatment either with BDNF or NGF significantly increased
apoptotic cell death (Fig. 5B),
indicating that Trk receptors do not mediate this effect.
We also examined whether blocking p75 signaling might rescue cells from
death after PSA removal. We inhibited the proapoptotic cascades activated
through p75 by exposing cells to fumonisin B1 (10 µM) or myriocin (50 nM).
Both molecules are known to block p75-mediated accumulation of the second
messenger ceramide by depleting cells of its sphingolipidic precursor
(Dobrowsky et al., 1994).
Under basal conditions, fumonisin B1 and myriocin did not affect neuronal
survival (Fig. 5C). However,
cell death induced in the presence of Endo-N was significantly reduced by
these inhibitors (Fig. 5C).
|
; Friedman,
2000). Blockade of this enzyme by its selective antagonist
SP600125 also reversed the effect of Endo-N
(Fig. 5C). These experiments
suggest that the p75 signaling pathway plays an essential role in enhanced
apoptosis observed after removal or inactivation of PSA-NCAM.
Increased expression of p75 in the absence of PSA in vitro
Since the above experiments indicated an augmented p75 receptor activation
following PSA removal, we explored the possibility that p75 expression was
increased under these conditions. First, we assessed the levels of p75 mRNA
after Endo-N treatment using realtime RT-PCR. We found that treatment with
Endo-N for 20 and 48 hours was sufficient to double the levels of p75
transcripts compared with the vehicle-treated cultures
(Fig. 6A). No later time points
were considered because of the progressive cell loss in the Endo-N-treated
cultures. Importantly, TrkB and TrkC mRNA levels were not modified upon Endo-N
administration, arguing against a non-specific effect of Endo-N
(Fig. 6A). These findings are
consistent with our earlier report demonstrating that the maturation of these
cells (and thus the downregulation of PSA) is accompanied by a progressive
increase in p75 expression (Gascon et al.,
2005).
We next examined whether PSA inactivation triggers an increase in p75 at the protein level. As illustrated in Fig. 6B, treatment with Endo-N for 20 hours resulted in a dramatic augmentation of p75 immunoreactivity compared with vehicle-treated cells. Quantification of p75 intensity using confocal microscopy (see Materials and methods) revealed that Endo-N produced a significant shift in the intensity of p75 immunoreactivity (Fig. 6C), thus confirming our real-time RT-PCR results. Together, these observations suggest that the presence of polysialylated NCAM at the cell surface may closely regulate the expression of p75 receptor.
Absence of NCAM in vivo leads to an increased expression of p75 in the SVZ-RMS that is accompanied by an early maturation of newborn neurons
We then examined whether p75 expression was also modified in vivo in the
absence of PSA-NCAM. Western blot analysis of p75 in RMS-OB or the cerebral
cortex from NCAM-/- and WT animals revealed that p75 was
downregulated with age in both strains
(Fig. 7A), as previously
described (Roux and Barker,
2002). More importantly, we found a striking increase in p75
protein levels in NCAM-/- RMS-OB, but not in the cortex, when
compared with WT (Fig. 7A). The
increase was more prominent in newborn animals, most likely because of the low
proportion of cells that expressed p75 at later time points. These results
were confirmed by immunofluorescence. In WT animals, we observed a few
p75-positive cells scattered along the SVZ-RMS. They displayed a round
morphology and occasionally formed clusters
(Fig. 7B). In agreement with a
previous report (Giuliani et al.,
2004), these cells were not associated with doublecortin staining
(Fig. 7B), suggesting that they
were immature progenitors. In the NCAM-/- mice, p75 labeling was
substantially increased in the SVZ-RMS compared with WT animals. Although
small round cells similar to those observed in WT animals were found, the
majority of p75-positive cells in the NCAM-/- SVZ-RMS were
process-bearing cells exhibiting a more complex morphology and colocalized
with doublecortin (Fig.
7C).
To further explore the origin and phenotype of this latter cell population
in the SVZ-RMS, a lentivector encoding GFP was unilaterally injected into the
lateral ventricle of WT and NCAM-/- animals as previously described
(Gascon et al., 2006). We used
a low number of vector particles in order to transduce a limited number of
cells in the SVZ-RMS so as to allow a morphological analysis under the
confocal microscope. As illustrated in Fig.
8A, 4 days after vector injection, most cells in the SVZ-RMS of WT
animals displayed the typical features of migrating neurons, i.e. an elongated
cell body and a leading process oriented towards the OB. In addition, these
cells were immunopositive for doublecortin
(Fig. 8A). By contrast, in
NCAM-/- mice, only a fraction of GFP-expressing cells exhibited
this morphology. In agreement with a previous report
(Petridis et al., 2004), we
found that many of these cells displayed a more complex and branched
morphology reminiscent of maturating neurons
(Fig. 8A). These cells had a
neuronal phenotype as they colocalized with markers such as doublecortin
(Fig. 8A). These results raised
the possibility that new neurons abnormally maturate in the SVZ-RMS of
NCAM-/- animals. To explore this hypothesis, we performed
immunostaining with NeuN (Neuna60 - Mouse Genome Informatics), a marker known
to detect neurons at more mature stages than doublecortin. A significant
number of NeuN+ profiles were found in the mutant SVZ-RMS, whereas
no labeling was detected in WT animals
(Fig. 8B). Interestingly, 4
days after viral injection, most GFP-expressing cells in the
NCAM-/- SVZ-RMS were doublecortin+, and only a few of
them colocalized with NeuN. These data suggest that the lack of PSA-NCAM
results in an early neuronal maturation in the SVZ-RMS that is accompanied by
increased p75 expression in these cells.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
reasons for this discrepancy between the present and earlier reports might be
methodological. Thus, to quantify cell death, Ono et al.
(Ono et al., 1994) used the
pyknotic index, a detection method less sensitive than the TUNEL assay used in
our study (Clarke, 1990;
McCloskey et al., 1998). The
enhanced rate of apoptotic cell death we document here in NCAM-/-
animals is cell-specific because it occurred in the population of migrating
neuroblasts (PSA+ NCAM+) but not in GFAP-positive
astrocytes (PSA-NCAM+), supporting the hypothesis that
PSA-NCAM and not NCAM is important for cell survival. In addition, we observed
increased TUNEL staining only in the SVZ and the RMS of NCAM-deficient brains
but not in other brain regions. However, in view of the rapid clearing of
apoptotic cells (Ferrer et al.,
1990; Savill,
1998) in vivo, we cannot exclude the possibility that enhanced,
albeit low levels of apoptosis may occur elsewhere.
The regional and cell-specific occurrence of enhanced TUNEL labeling in the
NCAM-/- animal strongly suggests the direct involvement of PSA-NCAM
in the adequate survival of newly generated neurons. This hypothesis receives
further support from our in vitro experiments: (1) enzymatic removal or
antibody blocking of PSA produce a significant increase in TUNEL labeling; (2)
in the absence of PSA, levels of apoptosis are higher than in control cultures
in response to neurotrophins; and (3) spontaneous downregulation of PSA
associated with maturation mimics the effects of genetic or enzymatic
elimination of PSA. These results are also consistent with the recent
observation that the lack of PSA-NCAM increases cell death of newly generated
neurons in cortical cultures (Vutskits et
al., 2006). Relevant to the enhanced apoptosis shown in this study
in the absence of PSA-NCAM is the observation that the OB as well the whole
brain of NCAM-/- animals are significantly smaller than in
heterozygous or WT animals (Cremer et al.,
1994).
Given the net increase of apoptosis in the SVZ-RMS of NCAM-/-
animals, the lack of similarly increased cell death in cultures prepared from
NCAM-deficient mice under basal conditions was surprising. This might reflect
the existence of compensatory mechanisms in mutant cells that could counteract
the effects of the absence of NCAM under basal conditions, but not when cells
are challenged in a more complex in vivo environment. Since exogenous
application of NGF in culture induced significantly more apoptosis in
NCAM-/- cultures than in WT, one possibility is that cells could be
exposed to this neurotrophin in the SVZ-RMS in vivo and that this may explain
the increased apoptotic cell death. The influence of NGF on SVZ neural
precursors has been demonstrated under pathological conditions such as
experimental allergic encephalitis (Triaca
et al., 2005). It should be noted that in contrast to
NCAM-/- cultures, removal of PSA by Endo-N increased apoptosis even
under basal in vitro conditions. This raises the possibility that putative
compensatory mechanisms might not be operational when PSA is acutely removed,
and/or NCAM without PSA may promote cell death at this developmental stage of
neuronal precursors. Further studies should clarify these issues.
|
;
Vutskits et al., 2001). The
results presented here give support to this observation, because the
survival-promoting effect of BDNF was significantly weaker in Endo-N-treated
and NCAM-/- cultures than in control preparations. Moreover, our
observations point to the involvement of p75 receptor in cell death observed
in the absence of PSA-NCAM. In view of the evidence that SVZ-derived neurons
in culture express p75, TrkB and TrkC, but not TrkA
(Gascon et al., 2005), our
finding that NGF significantly increased apoptosis in cells lacking PSA
suggests that this effect is mediated through p75. In agreement with these
results, pharmacological blockade of p75, but not of Trk receptors, prevented
neuronal cell death induced by the removal of PSA. We demonstrate that the
inhibition of two well-established pro-apoptotic cascades downstream of p75,
ceramide and c-Jun N-terminal kinase
(Barrett et al., 1998;
Casaccia-Bonnefil et al., 1996;
Dobrowsky et al., 1994;
Frago et al., 1998;
Hirata et al., 2001),
completely prevented neuronal cell death induced by the absence of PSA in
control as well as NGF-treated cultures. Together, these data raised the
possibility that the removal of PSA from NCAM induced an enhanced activation
of p75 signaling pathways. In agreement with this idea, we found that both in
vivo and in vitro, immature neurons lacking PSA-NCAM express significantly
higher levels of p75 than control cells. However, definitive evidence for this
hypothesis would require additional experiments involving the use of
p75-knockout animals and function-blocking antibodies. We also demonstrated
that PSA removal does not affect TrkB or TrkC expression, thus illustrating
the specificity of this effect. The current study is, to our knowledge, the
first indication of a role of PSA-NCAM in the regulation of p75 receptor
expression. Interestingly, p75 expression in Schwann cells is tightly
regulated by Egr1 (Zif268) (Nikam et al.,
1995), a transcription factor known to be activated downstream of
the FGF receptor (Midgley and Khachigian,
2004; Santiago et al.,
1999; Wang et al.,
1997). Given the fact that NCAM at the cell membrane can interact
with and activate FGF receptors (Doherty
and Walsh, 1996), this signaling pathway would be an interesting
candidate to modulate p75 expression and cell survival. Further studies are
required to address this hypothesis.
It has been proposed that p75 receptors function both as a dependence
receptor and a death receptor depending on the cellular context, including
associated Trk expression, ligand presentation and expression of
p75-interacting molecules (Bredesen et al.,
2005). In the first case, the expression of p75 creates states of
dependence on its ligands and activates death following the withdrawal of
neurotrophic factors (Barrett and Bartlett,
1994; Rabizadeh et al.,
1993). In the second, p75 may mediate the cellular response to a
mismatched neurotrophin (e.g. exposure of a neuron-expressing TrkB and p75 to
NGF, which binds TrkA and p75) through its function as a death receptor
(Aloyz et al., 1998;
Casaccia-Bonnefil et al., 1996;
Frade et al., 1996). In both
situations, p75-mediated apoptosis may serve to eliminate cells when they
experience declining or inappropriate trophic support. One potential
implication of our findings is that by limiting p75 expression, PSA-NCAM may
protect newborn neurons from being dependent on trophic support before
integration into olfactory circuits. This may ensure that enough cells arrive
and compete in the OB. Neurons having reached their appropriate place in the
granule/glomerular layer, and having established synaptic connections, would
be coupled to network activity that is crucial for their long-term survival
(Miwa and Storm, 2005;
Rochefort et al., 2002). The
progressive downregulation of PSA-NCAM and the increase in p75 expression
during maturation would contribute to the elimination of non-integrated and/or
misplaced cells. This mechanism may become operational precociously in the RMS
of the NCAM-/- animal. Our studies revealed a large number of
disoriented, process-bearing neurons in the RMS of the NCAM-/-
animals that were also immunopositive for p75. These results confirm and
extend previous reports demonstrating that, in the absence of PSA, chain
migration is disrupted (Chazal et al.,
2000; Ono et al.,
1994) and neuronal precursors start differentiating
(Petridis et al., 2004).
Whether this precocious differentiation is the cause or the effect of altered
migration remains to be determined.
|
; Giehl et al.,
2001; Kokaia et al.,
1998; Rende et al.,
1993; Roux et al.,
1999). Remarkably, blockade or enzymatic disruption of PSA leads
to an impaired repair process (Bonfanti et
al., 1996; Daniloff et al.,
1986) and the NCAM-knockout mice exhibit deficient recovery after
cortical lesions (Troncoso et al.,
2004). It has been reported that, in a mice model of amyotrophic
lateral sclerosis, surviving motoneurons in transgenic animals express
PSA-NCAM (Warita et al.,
2001), raising the possibility that PSA re-expression protects
undamaged and functional neurons from p75-mediated apoptosis. Together, the
data presented here provide a rational for the potential application of NCAMs
as targets for therapy. |
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Aloyz, R. S., Bamji, S. X., Pozniak, C. D., Toma, J. G., Atwal,
J., Kaplan, D. R. and Miller, F. D. (1998). p53 is essential
for developmental neuron death as regulated by the TrkA and p75 neurotrophin
receptors. J. Cell Biol.
143,1691
-1703.
Alvarez-Buylla, A. and Garcia-Verdugo, J. M.
(2002). Neurogenesis in adult subventricular zone. J.
Neurosci. 22,629
-634.
Barrett, G. L. and Bartlett, P. F. (1994). The
p75 nerve growth factor receptor mediates survival or death depending on the
stage of sensory neuron development. Proc. Natl. Acad. Sci.
USA 91,6501
-6505.
Barrett, G. L., Georgiou, A., Reid, K., Bartlett, P. F. and
Leung, D. (1998). Rescue of dorsal root sensory neurons by
nerve growth factor and neurotrophin-3, but not brain-derived neurotrophic
factor or neurotrophin-4, is dependent on the level of the p75 neurotrophin
receptor. Neuroscience
85,1321
-1328.[CrossRef][Medline]
Bonfanti, L., Merighi, A. and Theodosis, D. T.
(1996). Dorsal rhizotomy induces transient expression of the
highly sialylated isoform of the neural cell adhesion molecule in neurons and
astrocytes of the adult rat spinal cord. Neuroscience
74,619
-623.[CrossRef][Medline]
Brazel, C. Y., Nunez, J. L., Yang, Z. and Levison, S. W.
(2005). Glutamate enhances survival and proliferation of neural
progenitors derived from the subventricular zone.
Neuroscience 131,55
-65.[CrossRef][Medline]
Bredesen, D. E., Mehlen, P. and Rabizadeh, S.
(2005). Receptors that mediate cellular dependence.
Cell Death Differ. 12,1031
-1043.[CrossRef][Medline]
Calaora, V., Chazal, G., Nielsen, P. J., Rougon, G. and Moreau,
H. (1996). mCD24 expression in the developing mouse brain and
in zones of secondary neurogenesis in the adult.
Neuroscience 73,581
-594.[CrossRef][Medline]
Casaccia-Bonnefil, P., Carter, B. D., Dobrowsky, R. T. and Chao,
M. V. (1996). Death of oligodendrocytes mediated by the
interaction of nerve growth factor with its receptor p75.
Nature 383,716
-719.[CrossRef][Medline]
Chazal, G., Durbec, P., Jankovski, A., Rougon, G. and Cremer,
H. (2000). Consequences of neural cell adhesion molecule
deficiency on cell migration in the rostral migratory stream of the mouse.
J. Neurosci. 20,1446
-1457.
Clarke, P. G. (1990). Developmental cell death:
morphological diversity and multiple mechanisms. Anat.
Embryol. 181,195
-213.[Medline]
Cremer, H., Lange, R., Christoph, A., Plomann, M., Vopper, G.,
Roes, J., Brown, R., Baldwin, S., Kraemer, P., Scheff, S. et al.
(1994). Inactivation of the N-CAM gene in mice results in size
reduction of the olfactory bulb and deficits in spatial learning.
Nature 367,455
-459.[CrossRef][Medline]
Daniloff, J. K., Levi, G., Grumet, M., Rieger, F. and Edelman,
G. M. (1986). Altered expression of neuronal cell adhesion
molecules induced by nerve injury and repair. J. Cell
Biol. 103,929
-945.
Dobrowsky, R. T., Werner, M. H., Castellino, A. M., Chao, M. V.
and Hannun, Y. A. (1994). Activation of the sphingomyelin
cycle through the low-affinity neurotrophin receptor.
Science 265,1596
-1599.
Doherty, P. and Walsh, F. S. (1996). CAM-FGF
receptor interactions: a model for axonal growth. Mol. Cell.
Neurosci. 8,99
-111.[CrossRef][Medline]
Durbec, P. and Cremer, H. (2001). Revisiting
the function of PSA-NCAM in the nervous system. Mol.
Neurobiol. 24,53
-64.[CrossRef][Medline]
Edelman, G. M. (1986). Cell adhesion molecules
in neural histogenesis. Annu. Rev. Physiol.
48,417
-430.[CrossRef][Medline]
Ferrer, I., Bernet, E., Soriano, E., del Rio, T. and Fonseca,
M. (1990). Naturally occurring cell death in the cerebral
cortex of the rat and removal of dead cells by transitory phagocytes.
Neuroscience 39,451
-458.[CrossRef][Medline]
Frade, J. M., Rodriguez-Tebar, A. and Barde, Y. A.
(1996). Induction of cell death by endogenous nerve growth factor
through its p75 receptor. Nature
383,166
-168.[CrossRef][Medline]
Frago, L. M., Leon, Y., de la Rosa, E. J., Gomez-Munoz, A. and
Varela-Nieto, I. (1998). Nerve growth factor and ceramides
modulate cell death in the early developing inner ear. J. Cell
Sci. 111,549
-556.[Abstract]
Friedman, W. J. (2000). Neurotrophins induce
death of hippocampal neurons via the p75 receptor. J.
Neurosci. 20,6340
-6346.
Frosch, M., Gorgen, I., Boulnois, G. J., Timmis, K. N. and
Bitter-Suermann, D. (1985). NZB mouse system for production
of monoclonal antibodies to weak bacterial antigens: isolation of an IgG
antibody to the polysaccharide capsules of Escherichia coli K1 and group B
meningococci. Proc. Natl. Acad. Sci. USA
82,1194
-1198.
Gage, F. H., Batchelor, P., Chen, K. S., Chin, D., Higgins, G.
A., Koh, S., Deputy, S., Rosenberg, M. B., Fischer, W. and Bjorklund, A.
(1989). NGF receptor reexpression and NGF-mediated cholinergic
neuronal hypertrophy in the damaged adult neostriatum.
Neuron 2,1177
-1184.[CrossRef][Medline]
Garcia-Verdugo, J. M., Doetsch, F., Wichterle, H., Lim, D. A.
and Alvarez-Buylla, A. (1998). Architecture and cell types of
the adult subventricular zone: in search of the stem cells. J.
Neurobiol. 36,234
-248.[CrossRef][Medline]
Gascon, E., Vutskits, L., Zhang, H., Barral-Moran, M. J., Kiss,
P. J., Mas, C. and Kiss, J. Z. (2005). Sequential activation
of p75 and TrkB is involved in dendritic development of subventricular
zone-derived neuronal progenitors in vitro. Eur. J.
Neurosci. 21,69
-80.[CrossRef][Medline]
Gascon, E., Dayer, A. G., Sauvain, M. O., Potter, G., Jenny, B.,
De Roo, M., Zgraggen, E., Demaurex, N., Muller, D. and Kiss, J. Z.
(2006). GABA regulates dendritic growth by stabilizing
lamellipodia in newly generated interneurons of the olfactory bulb.
J. Neurosci. 26,12956
-12966.
Giehl, K. M., Rohrig, S., Bonatz, H., Gutjahr, M., Leiner, B.,
Bartke, I., Yan, Q., Reichardt, L. F., Backus, C., Welcher, A. A. et al.
(2001). Endogenous brain-derived neurotrophic factor and
neurotrophin-3 antagonistically regulate survival of axotomized corticospinal
neurons in vivo. J. Neurosci.
21,3492
-3502.
Giuliani, A., D'Intino, G., Paradisi, M., Giardino, L. and
Calza, L. (2004). p75(NTR)-immunoreactivity in the
subventricular zone of adult male rats: expression by cycling cells.
J. Mol. Histol. 35,749
-758.[CrossRef][Medline]
Hirata, H., Hibasami, H., Yoshida, T., Ogawa, M., Matsumoto, M.,
Morita, A. and Uchida, A. (2001). Nerve growth factor
signaling of p75 induces differentiation and ceramide-mediated apoptosis in
Schwann cells cultured from degenerating nerves. Glia
36,245
-258.[CrossRef][Medline]
Hu, H., Tomasiewicz, H., Magnuson, T. and Rutishauser, U.
(1996). The role of polysialic acid in migration of olfactory
bulb interneuron precursors in the subventricular zone.
Neuron 16,735
-743.[CrossRef][Medline]
Kiss, J. Z. and Rougon, G. (1997). Cell biology
of polysialic acid. Curr. Opin. Neurobiol.
7, 640-646.[CrossRef][Medline]
Kiss, J. Z., Wang, C., Olive, S., Rougon, G., Lang, J., Baetens,
D., Harry, D. and Pralong, W. F. (1994). Activity-dependent
mobilization of the adhesion molecule polysialic NCAM to the cell surface of
neurons and endocrine cells. EMBO J.
13,5284
-5292.[Medline]
Kiss, J. Z., Troncoso, E., Djebbara, Z., Vutskits, L. and
Muller, D. (2001). The role of neural cell adhesion molecules
in plasticity and repair. Brain Res. Brain Res. Rev.
36,175
-184.[CrossRef][Medline]
Klages, N., Zufferey, R. and Trono, D. (2000).
A stable system for the high-titer production of multiply attenuated
lentiviral vectors. Mol. Ther.
2, 170-176.[CrossRef][Medline]
Koizumi, S., Contreras, M. L., Matsuda, Y., Hama, T.,
Lazarovici, P. and Guroff, G. (1988). K-252a: a specific
inhibitor of the action of nerve growth factor on PC 12 cells. J.
Neurosci. 8,715
-721.[Abstract]
Kokaia, Z., Andsberg, G., Martinez-Serrano, A. and Lindvall,
O. (1998). Focal cerebral ischemia in rats induces expression
of P75 neurotrophin receptor in resistant striatal cholinergic neurons.
Neuroscience 84,1113
-1125.[CrossRef][Medline]
Li, L., Liu, F., Salmonsen, R. A., Turner, T. K., Litofsky, N.
S., Di Cristofano, A., Pandolfi, P. P., Jones, S. N., Recht, L. D. and Ross,
A. H. (2002). PTEN in neural precursor cells: regulation of
migration, apoptosis, and proliferation. Mol. Cell.
Neurosci. 20,21
-29.[CrossRef][Medline]
Matute, C. and Streit, P. (1986). Monoclonal
antibodies demonstrating GABA-like immunoreactivity.
Histochemistry 86,147
-157.[CrossRef][Medline]
McCloskey, T. W., Chavan, S., Lakshmi Tamma, S. M. and Pahwa,
S. (1998). Comparison of seven quantitative assays to assess
lymphocyte cell death during HIV infection: measurement of induced apoptosis
in anti-Fas-treated Jurkat cells and spontaneous apoptosis in peripheral blood
mononuclear cells from children infected with HIV. AIDS Res. Hum.
Retroviruses 14,1413
-1422.[Medline]
Midgley, V. C. and Khachigian, L. M. (2004).
Fibroblast growth factor-2 induction of platelet-derived growth factor-C chain
transcription in vascular smooth muscle cells is ERK-dependent but not
JNK-dependent and mediated by Egr-1. J. Biol. Chem.
279,40289
-40295.
Miller, F. D. and Kaplan, D. R. (2001).
Neurotrophin signalling pathways regulating neuronal apoptosis.
Cell. Mol. Life Sci. 58,1045
-1053.[CrossRef][Medline]
Miwa, N. and Storm, D. R. (2005).
Odorant-induced activation of extracellular signal-regulated
kinase/mitogen-activated protein kinase in the olfactory bulb promotes
survival of newly formed granule cells. J. Neurosci.
25,5404
-5412.
Muller, D., Djebbara-Hannas, Z., Jourdain, P., Vutskits, L.,
Durbec, P., Rougon, G. and Kiss, J. Z. (2000). Brain-derived
neurotrophic factor restores long-term potentiation in polysialic acid-neural
cell adhesion molecule-deficient hippocampus. Proc. Natl. Acad.
Sci. USA 97,4315
-4320.
Nikam, S. S., Tennekoon, G. I., Christy, B. A., Yoshino, J. E.
and Rutkowski, J. L. (1995). The zinc finger transcription
factor Zif268/Egr-1 is essential for Schwann cell expression of the p75 NGF
receptor. Mol. Cell. Neurosci.
6, 337-348.[CrossRef][Medline]
Ono, K., Tomasiewicz, H., Magnuson, T. and Rutishauser, U.
(1994). N-CAM mutation inhibits tangential neuronal migration and
is phenocopied by enzymatic removal of polysialic acid.
Neuron 13,595
-609.[CrossRef][Medline]
Petridis, A. K., El-Maarouf, A. and Rutishauser, U.
(2004). Polysialic acid regulates cell contact-dependent neuronal
differentiation of progenitor cells from the subventricular zone.
Dev. Dyn. 230,675
-684.[CrossRef][Medline]
Rabizadeh, S., Oh, J., Zhong, L. T., Yang, J., Bitler, C. M.,
Butcher, L. L. and Bredesen, D. E. (1993). Induction of
apoptosis by the low-affinity NGF receptor. Science
261,345
-348.
Rende, M., Provenzano, C. and Tonali, P.
(1993). Modulation of low-affinity nerve growth factor receptor
in injured adult rat spinal cord motoneurons. J. Comp.
Neurol. 338,560
-574.[CrossRef][Medline]
Rochefort, C., Gheusi, G., Vincent, J. D. and Lledo, P. M.
(2002). Enriched odor exposure increases the number of newborn
neurons in the adult olfactory bulb and improves odor memory. J.
Neurosci. 22,2679
-2689.
Rougon, G., Dubois, C., Buckley, N., Magnani, J. L. and
Zollinger, W. (1986). A monoclonal antibody against
meningococcus group B polysaccharides distinguishes embryonic from adult
N-CAM. J. Cell Biol.
103,2429
-2437.
Rousselot, P., Lois, C. and Alvarez-Buylla, A.
(1995). Embryonic (PSA) N-CAM reveals chains of migrating
neuroblasts between the lateral ventricle and the olfactory bulb of adult
mice. J. Comp. Neurol.
351, 51-61.[CrossRef][Medline]
Roux, P. P. and Barker, P. A. (2002).
Neurotrophin signaling through the p75 neurotrophin receptor. Prog.
Neurobiol. 67,203
-233.[CrossRef][Medline]
Roux, P. P., Colicos, M. A., Barker, P. A. and Kennedy, T.
E. (1999). p75 neurotrophin receptor expression is induced in
apoptotic neurons after seizure. J. Neurosci.
19,6887
-6896.
Rutishauser, U. and Landmesser, L. (1996).
Polysialic acid in the vertebrate nervous system: a promoter of plasticity in
cell-cell interactions. Trends Neurosci.
19,422
-427.[Medline]
Santiago, F. S., Lowe, H. C., Day, F. L., Chesterman, C. N. and
Khachigian, L. M. (1999). Early growth response factor-1
induction by injury is triggered by release and paracrine activation by
fibroblast growth factor-2. Am. J. Pathol.
154,937
-944.
Savill, J. (1998). Apoptosis. Phagocytic
docking without shocking. Nature
392,442
-443.[CrossRef][Medline]
Tomasiewicz, H., Ono, K., Yee, D., Thompson, C., Goridis, C.,
Rutishauser, U. and Magnuson, T. (1993). Genetic deletion of
a neural cell adhesion molecule variant (N-CAM-180) produces distinct defects
in the central nervous system. Neuron
11,1163
-1174.[CrossRef][Medline]
Triaca, V., Tirassa, P. and Aloe, L. (2005).
Presence of nerve growth factor and TrkA expression in the SVZ of EAE rats:
evidence for a possible functional significance. Exp.
Neurol. 191,53
-64.[CrossRef][Medline]
Troncoso, E., Muller, D., Korodi, K., Steimer, T., Welker, E.
and Kiss, J. Z. (2004). Recovery of evoked potentials,
metabolic activity and behavior in a mouse model of somatosensory cortex
lesion: role of the neural cell adhesion molecule (NCAM). Cereb.
Cortex 14,332
-341.
Vimr, E. R., McCoy, R. D., Vollger, H. F., Wilkison, N. C. and
Troy, F. A. (1984). Use of prokaryotic-derived probes to
identify poly(sialic acid) in neonatal neuronal membranes. Proc.
Natl. Acad. Sci. USA 81,1971
-1975.
Vutskits, L., Djebbara-Hannas, Z., Zhang, H., Paccaud, J. P.,
Durbec, P., Rougon, G., Muller, D. and Kiss, J. Z. (2001).
PSA-NCAM modulates BDNF-dependent survival and differentiation of cortical
neurons. Eur. J. Neurosci.
13,1391
-1402.[CrossRef][Medline]
Vutskits, L., Gascon, E., Zgraggen, E. and Kiss, J. Z.
(2006). The polysialylated neural cell adhesion molecule promotes
neurogenesis in vitro. Neurochem. Res.
31,215
-225.[CrossRef][Medline]
Wang, D., Mayo, M. W. and Baldwin, A. S., Jr
(1997). Basic fibroblast growth factor transcriptional
autoregulation requires EGR-1. Oncogene
14,2291
-2299.[CrossRef][Medline]
Warita, H., Murakami, T., Manabe, Y., Sato, K., Hayashi, T.,
Seki, T. and Abe, K. (2001). Induction of polysialic
acid-neural cell adhesion molecule in surviving motoneurons of transgenic
amyotrophic lateral sclerosis mice. Neurosci. Lett.
300, 75-78.[CrossRef][Medline]
Zigova, T., Pencea, V., Wiegand, S. J. and Luskin, M. B.
(1998). Intraventricular administration of BDNF increases the
number of newly generated neurons in the adult olfactory bulb. Mol.
Cell. Neurosci. 11,234
-245.[CrossRef][Medline]
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