1 NeuroAdaptations Group, Max Planck Institute of Psychiatry, Kraepelinstrasse
2-10, D-80804 Munich, Germany
2 Neuroscience Group, Life and Health Sciences Research Institute (ICVS),
University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
* Author for correspondence (e-mail: osa{at}mpipsykl.mpg.de)
Accepted 9 May 2005
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SUMMARY |
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Key words: Neurogenesis, Apoptosis, Neuronal differentiation, Hippocampus, Cerebellum, TGFß2, BDNF, SMAD, Rat
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Introduction |
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Granule cells of the cerebellum and hippocampal dentate gyrus share several
morphological commonalities (Ramon y
Cajal, 1911); in addition, they both display dependencies on, or
expression of, a common set of growth factors and signaling pathways
(Dreyfus, 1998
). However,
cerebellar and hippocampal granule cells show distinct differences in their
repertoire of glutamate receptor subtypes
(Monyer et al., 1994
) and gene
expression profiles (Saito et al.,
2002
), features that most probably reflect their different
physiological roles. These similar, but also divergent, properties make these
two types of granule neuron interesting models for analyzing the intrinsic
factors responsible for the regulation of their proliferation and maturation.
Both dentate and cerebellar granule cells first appear late in embryogenesis,
with peak numbers appearing during the first postnatal week
(Altman, 1972
;
Altman and Bayer, 1990
;
Schlessinger et al., 1975
).
Hippocampal granule cells continue to proliferate throughout life, although
the rate of proliferation wanes with age
(Altman and Bayer, 1990
;
Kuhn et al., 1996
;
Cameron and McKay, 2001
). By
contrast, the genesis of cerebellar granule cells terminates within the first
two weeks of life (Altman,
1972
). Interestingly, gene profiling studies revealed that genes
involved in oncogenesis and ribosomal protein synthesis are most strongly
expressed at the peak of cerebellar granule cell production
(Saito et al., 2002
); of the
five gene clusters analyzed in that study, none showed any particular temporal
pattern of expression in the dentate gyrus.
To examine the hypothesis that tissue-specific factors may serve as `start'
and `stop' controls of proliferation in different brain areas, we here
measured neurogenesis by bromodeoxyuridine (BrdU) incorporation in
immunochemically characterized cells after exchanging conditioned medium (CM)
between rat postnatal day 4 (P4) hippocampal and P7 cerebellar granule cell
cultures; medium exchanges were performed over a period covering the
appearance of the subgranular (secondary germinative) layer of the hippocampus
(Altman and Bayer, 1990) and
the start of the disappearance of the external granule layer of the cerebellum
(Altman, 1972
). Our studies
reveal that TGFß2 and BDNF of cerebellar origin have strong
anti-proliferative and neuronal differentiating properties when applied to
mitotic hippocampal granule cells.
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Materials and methods |
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Slice culture
Cerebellar and hippocampal `interface' slice cultures were prepared from P7
Wistar rats based on a protocol published by Noraberg et al.
(Noraberg et al., 1999).
Briefly, hippocampal and cerebellar slices (400 µm) were placed on
Millicell semiporous membranes in six-well plates (Millipore). Slices from
each brain area were placed adjacent to each other in a single well and bathed
in 50% OPTIMEM/Dulbecco's modified Eagle's Medium (DMEM), including 10% fetal
bovine serum, 15% horse serum, 1 mM Glutamax and 0.1 mg/ml kanamycin in Hank's
buffered saline solution (all from Invitrogen). Co-cultures were maintained at
37°C (90% humidity) for 16 days, with medium changes every 3 days.
Cultures were treated with BrdU (20 µM, 24 hours) before fixation (4%
PFA).
HiB5 hippocampal cell line
Neural precursor SV40 T large antigen-immortalized HiB5 cells
(Renfranz et al., 1991)
(kindly provided by Dr Nina Rosenqvist, Lund, Sweden) were maintained in DMEM
containing 10% fetal calf serum and 1% kanamycin at the permissive temperature
(32°C) and a 5% CO2 environment.
Immunocytochemistry
Slice and dispersed cell cultures were fixed in 4% paraformaldehyde,
permeabilized (0.3% Triton-X100/PBS) and incubated in 3% donkey serum/0.3%
Triton (30 minutes) before incubation (1 hour; room temperature) with primary
antibodies diluted 1:500 in 3% donkey serum/0.3% Triton X-100 in PBS:
anti-BrdU (DAKO), anti-Nestin (Chemicon), anti-TuJ1 (Babco), anti-MAP2
(Sigma), anti-doublecortin (Santa Cruz Biotechnology), anti-GFAP (Sigma),
anti-Mash1 and anti-
Math1 (kind gifts from Dr Jane Johnson,
Dallas, TX). After washing in PBS, cells and slices were incubated (30
minutes, room temperature) with biotinylated anti-mouse or anti-rabbit
secondary antibody (1:500; Sigma), washed and incubated (30 minutes) with
FITC- or horseradish peroxidase-conjugated Avidin (1:500; Sigma). HRP was
developed with diaminobenzidine. In some instances, nuclear staining was
achieved using Hoechst 33342 (1:1000 in PBS; 15 minutes; Roche). Cells
staining positive for BrdU or one of the various neural markers were counted
with respect to the total number of cells in five randomly chosen microscopic
fields (0.072 mm2; 400 x magnification) across the long axis
of each object; an average of 1000 cells were sampled on each coverslip and
the results shown represent values from 6-10 coverslips per treatment.
Cell death assay
Cell death was examined in 4% PFA-fixed cells by TUNEL histochemistry
(Almeida et al., 2000) or
Hoechst 33342 staining. Apoptotic cells were identified as dark-brown nuclear
staining showing DNA fragmentation without plasma membrane damage. The
relative number of apoptotic versus total number of cells was measured in at
least five randomly chosen microscopic fields (400 x magnification).
Western blotting
Cells were harvested in lysis buffer, briefly sonicated (on ice). Lysates
were cleared by centrifugation, and proteins were electrophoretically resolved
on 10 or 8% SDS polyacrylamide gels before transfer onto nitrocellulose
membranes. Membranes were blocked (5% non-fat milk and 0.2% Tween-20 in PBS),
and incubated with specific primary antibodies (anti-MAP2a/b, Sigma, 1:5000;
anti-synapsin, Chemicon, 1:400; anti-p21, Pharmingen, 1:500; anti-p27, Santa
Cruz, 1:200). Antigens were revealed by enhanced chemoluminescence (Amersham
Biosciences) after incubation with appropriate horseradish peroxidase-IgG
conjugates (Amersham).
Concentration and purification of conditioned medium from cerebellar cultures (CMCerebellum)
A total of 1 L of CMCerebellum was collected from cultures
between 8 and 14 days in vitro (d.i.v.). A 100-fold concentrate, containing
peptides with a Mr greater than 6 kDa, was prepared using
Vivaspin columns (Vivascience) before running through Q-ion exchange columns
(Vivapure 20; Vivascience) and elution with a sequential salt gradient buffer.
Bio-active fractions were further separated on Affigel blue columns (BioRad)
and analyzed for their proliferative, differentiating and apoptotic properties
(see above).
Heat lability test
Concentrated (100x) CMCerebellum was boiled for 15 minutes
before addition to hippocampal cultures and measurement of bioactivity (cell
proliferation and neuronal markers).
Immunoneutralization
Antibodies against brain-derived neurotrophic factor (BDNF) and nerve
growth factor (NGF) were purchased from Santa Cruz Biotechnology; antibodies
against TGFß2 were from R&D Systems. All antisera were purified IgG,
and species-matched purified IgG preparations (Chemicon) were used in
controls. CMCerebellum was adsorbed with these antisera for 1 hour
(room temperature) before being added to cell cultures at dilutions ranging
from 1:10 to 1:10,000, after which the biological activity of the
CMCerebellum was assessed (see above).
BDNF studies
Results from the BDNF immunoneutralization experiments were confirmed by
adding BDNF (hBDNF, 10-100 ng/ml; Alomone Laboratories) to hippocampal
cultures at 7 d.i.v., and monitoring for BrdU incorporation and MAP2a/b
expression after 24 hours.
Further verification of BDNF effects was obtained by transiently transfecting primary hippocampal cultures with a BDNF expression vector (pcDNA3-BDNF-Citron, kindly provided by Dr Oliver Griesbeck, Martinsried, Germany) or pEGFP (as control). Transfection was carried out using 1 µg DNA/well and Lipofectamine 2000 (Invitrogen) under serum-free conditions. Twenty-four hours after transfection, media were exchanged between BDNF- and EGFP-transfected cells, and BrdU (20 µM) was added to the cultures. BrdU incorporation was assessed after a further 24 hours.
MAP kinase mediation of the pro-mitotic actions of BDNF was examined by assaying BrdU retention and MAP2a/b expression after treating cultures with the MEK inhibitor PD98059 (0.1 µM; Calbiochem). The ability of BDNF (100 ng/ml) to stimulate cytoplasm-to-nucleus translocation of key TGFß signaling partners was analyzed by transiently transfecting hippocampal cultures with fluorescence-tagged Smad2 and Smad4 (see below) and microscopic examination.
Cellular contents of TGFß2 after BDNF or CMCerebellum
treatment were measured by semi-quantitative immunocytochemistry, using
recombinant TGFß2 standards and ABTS [2,2'-azino-di-(3
ethylbenzthia-zoline sulfonic acid)] as chromogen. Attempts were made to
measure secreted TGFß using either a commercial ELISA kit (Promega) or
the plasminogen activator inhibitor 1 (PA1)-luciferase assay described by Abe
et al. (Abe et al., 1994). For
the latter assay, mink lung epithelial cells (MLEC, clone 32) stably
transfected with PA1-luciferase, were kindly provided by Dr D. Rifkin (New
York, NY). Neither assay proved to be sufficiently sensitive to measure
secreted TGFß2.
Nuclear translocation of SMADs
To study nuclear translocation of SMAD, primary hippocampal neurons (7
d.i.v.), were transfected with pEGFP-SMAD2 or pEGFP-SMAD4 (kindly provided by
Dr Kelly Mayo, Evanston, IL). Transfection was carried out using 1 µg
DNA/well and Lipofectamine 2000. Transfection efficiency, judged in control
transfections with pEGFP, was 10%. Following transfection, cells were
returned (3 hours) to standard growing medium or CMCerebellum or
exposed to TGFß2 (1 ng/ml) or BDNF (100 ng/ml), stained with Hoechst
33342 and examined.
Receptor signaling
Analysis of BDNF (TRKB) and TGFß receptor (TGFß-RII) signaling
was studied in primary hippocampal cells after transfection (see above) with 1
µg/well of the following plasmids: dominant-negative TGFßRII
(pRK5-TßRII-DN-F; generously provided by Dr R. Derynck, San Francisco,
CA, USA) and TRKB dominant-negative (pEF/BOS-TRKB.T1-Flag; kind gift from Dr
Eero Castren, Helsinki, Finland); pEGFP was used as an internal control.
Twenty-four hours after transfection and exposure to either control or
CMCerebellum, the number of BrdU-positive or MAP2a/b-positive cells
was counted as a proportion of all cells (stained with Hoechst 33342).
Transfection efficiency was approximately 10%.
SMAD signaling
Analysis of SMAD signaling was studied in primary hippocampal cells after
transfection (see above) with 1 µg/well of either dominant-negative SMAD3
[pCS2-FLAG-SMAD3(3S-A)] or dominant-negative SMAD4 [pCMV5-FLAG-DPC4(1-514)]
(provided by Dr Joan Massague, New York, NY); pEGFP was used as an internal
control. Transfection efficiency was 10%. Twenty-four hours after
transfection and exposure to either control or CMCerebellum, the
number of BrdU-positive or MAP2a/b-positive cells were assessed with respect
to the number of Hoechst 33342-stained cells.
3TP-Lux reporter assay
The p3TP-Lux reporter gene, containing a known TGFß-inducible
plasminogen activator inhibitor promoter
(Wrana et al., 1994) (provided
by Dr J. Massague), was transfected into HiB5 cells seeded in 24-well plates
(4 x104 cells per well), together with dominant-negative
SMADs, dominant-negative TRKB and TGFßRII, or wild-type BDNF. Plasmid
(625 ng of total DNA) was introduced using Lipofectamine 2000 into cells
maintained in Neurobasal A medium/B27 supplement. Twenty-four hours after
transfection (efficiency
10%), cells were treated with
CMCerebellum (10 µl/ml). Cells were lysed after 24 hours in 100
µl of 1 x lysis buffer (Promega), cleared by centrifugation and
assayed for ß-gal and luciferase activity. For ß-gal detection, 10
µl of cellular extract was mixed with 100 µl of ß-gal buffer (60 mM
Na2HPO4, 40 mM NaH2PO4, 10 mM KCl,
1 mM MgCl2, 2 mM ß-mercaptoethanol) and 20 µl of
O-nitrophenyl-ß-D-galactopyranoside (Sigma). The reaction was terminated
with 50 µl of Na2CO3 (1 M) and luciferase activity
was measured by mixing 30 µl of cellular extract with 50 µl of a buffer
containing 75 mM Tris-HCl and 1 mM MgCl2 (pH 8). Substrate
D-() Luciferin (1 mM) was automatically injected and light emission
(410 nm) was measured over 20 seconds in a luminometer.
Statistical analysis
All data are depicted as mean±s.d. and represent the observations
from three to five independent experiments, with three or four replicates for
each data point. Data were analyzed for statistical significance using ANOVA
and appropriate post-hoc tests (Student-Newman-Keuls or Kruskal-Wallis
multiple comparison procedures) in which P<0.05 was set as the
minimum level of significance.
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Results |
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Cerebellar `stop' signals vs. hippocampal `go' signals
BrdU incorporation by hippocampal cultures maintained in conditioned medium
derived from cerebellar cultures (CMCerebellum) was used to test
the hypothesis that cerebellar cells secrete factors that can serve as an
`instructive' micro-environment. As shown in
Fig. 1B, exposure of
hippocampal cultures to CMCerebellum resulted in a significant
reduction in BrdU incorporation (P<0.001); the converse experiment
(cerebellar cultures treated with CMHippocampus) resulted in a
significant increase of BrdU-positive cerebellar cells (P<0.01)
(Fig. 1C). Similar results were
obtained when hippocampal and cerebellar slices (donor age: 7 days) were
co-cultured for 7 days. Monitoring of BrdU retention (20 µM, final 24
hours; Fig. 1D) showed that
cell proliferation in hippocampal slices was significantly reduced in the
presence of cerebellar slices when compared with when hippocampal slices were
grown alone (P<0.01); by contrast, cell proliferation in
cerebellar slices was slightly, but not significantly, increased in the
presence of hippocampal tissue.
The above results indicate that soluble factors with mitotic and anti-proliferative properties are secreted into CMHippocampus and CMCerebellum, respectively; the remainder of our investigations focused on the anti-proliferative activity of CMCerebellum on hippocampal cells. Incubation of hippocampal cell cultures with varying volumes of 100-fold concentrated CMCerebellum established that its putative anti-proliferative activity dose-dependently reduces BrdU incorporation in primary hippocampal cells (Fig. 1E) and in a hippocampus-derived cell line, HiB5 (Fig. 1F). An insight into the physicochemical nature of the putative anti-proliferative factor(s) was gained by assaying the effects of boiled 100-fold concentrated CMCerebellum on BrdU incorporation by recipient hippocampal cells. As Fig. 1E (last column) shows, the anti-mitotic activity of CMCerebellum was abolished by boiling for 15 minutes, pointing to the peptidergic/proteinaceous nature of the anti-proliferative moieties.
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In accordance with our earlier results that showed that CMCerebellum concomitantly blocks proliferation of hippocampal cells while promoting their maturation (Fig. 2), immunoneutralization against TGFß2 (antibody concentrations that proved efficient at reversing the anti-mitotic effects) significantly reduced MAP2a/b expression (P<0.01; Fig. 3B). By contrast, anti-NGF (which did not influence proliferation) and anti-BDNF did not alter the relative number of MAP2a/b-immunoreactive cells, when compared with CMCerebellum that had not been pre-adsorbed with these antisera (Fig. 3B).
As immunoneutralization experiments provide only qualitative information and may be compromised by factors such as antibody affinity and purity, we next examined hippocampal cell proliferation and maturation after addition of either BDNF or TGFß2 to normal medium; for comparison, hippocampal cultures were grown in CMCerebellum. Significant reductions in BrdU incorporation were observed after treatment with either BDNF at 50 ng/ml (P<0.05) and 100 ng/ml (P<0.001) (Fig. 3C) or TGFß2 at 1 ng/ml (P<0.01) and 10 ng/ml (P<0.01) (Fig. 3D). The results obtained with exogenous BDNF peptide were confirmed when hippocampal cells were transfected with a BDNF expression plasmid (P<0.001, compared with cells transfected with pEGFP; inset, Fig. 3C) and when medium from pBDNF-transfected cells (24 hours) was added to pEGFP-transfected cells (third bar in inset, Fig. 3C; P<0.01).
In addition to inhibiting cell proliferation, both exogenous BDNF and TGFß2 were found to promote neuronal maturation, evidenced by significant increases (P<0.01 and 0.05, respectively) in the number of MAP2a/b-immunopositive cells after addition of purified forms of each peptide (Fig. 3E). These findings indicate that BDNF and TGFß2 account for a large part of the pro-neuronal properties of CMCerebellum. Using another indicator of pro-neuronal activity, namely neurite extension (`long neurites' defined as neurites with lengths that were more than twice the diameter of the cell body), we observed that both BDNF (at 10-100 ng/ml; P<0.05) and TGFß2 (at 1-10 ng/ml; P<0.05) stimulated neurite growth in a manner comparable with that observed with CMCerebellum (Fig. 3E).
The temporal patterns of BDNF and TGFß2 expression in hippocampal and cerebellar cultures were examined using immunocytochemistry after varying numbers of days in vitro. Generally, the intensity of BDNF and TGFß2 staining was stronger in cerebellar versus hippocampal cultures (data not shown). When compared with hippocampal cells, a significantly larger number of cerebellar cells expressed BDNF on 3 and 6 d.i.v. (P<0.001 and P<0.05; data not shown). At 9 d.i.v. (peak), more cerebellar cells than hippocampal cells were immunoreactive for TGFß2 (P<0.05; data not shown).
Anti-proliferative and differentiating effects of CMCerebellum are mediated by TRKB and TGFß-RII receptors
CMCerebellum failed to inhibit proliferation in primary
hippocampal cells expressing dominant-negative forms of the BDNF receptor TRKB
(pEF/BOS-TRKB.T1-flag) or the TGFß receptor, TGFß-RII
(pRK5-TßRII-DN-F) (Fig.
4A). In addition, hippocampal neuronal maturation induced by
CMCerebellum, as measured by the number of MAP2a/b-immunoreactive
cells, was significantly reduced in cells expressing dominant-negative forms
of TRKB and TGFß-RII (Fig.
4B). These results add further support for the roles of BDNF and
TGFß-2 as the anti-proliferative and pro-differentiation moieties in
CMCerebellum.
Mediation of CMCerebellum, BDNF and TGFß2 actions by SMAD pathways
TGFß2 exerts its biological actions through the mediation of SMAD
proteins. SMAD2 and SMAD3 specifically transduce TGFß signals after
dimerizing with co-SMAD4, translocating the resulting complex to the nucleus
and modulating the transcriptional machinery
(Attisano and Wrana, 2002). The
data depicted in Fig. 4C show
that pEGFP-SMAD2 and pEGFP-SMAD4 are translocated to the nucleus after
exposure of primary hippocampal neurons to CMCerebellum, as well as
to purified TGFß2 and BDNF.
We showed in Fig. 1F that, as in primary hippocampal cells, CMCerebellum exerts anti-mitotic actions in the hippocampus-derived HiB5 cell line. CMCerebellum failed to inhibit proliferation or to stimulate differentiation in hippocampal cells expressing dominant-negative forms of SMAD3 (pCS2-FLAG-SMAD3-3SA) or co-SMAD4 (pCMV-FLAG-DPC4(1-514)) (Fig. 4D,E).
Another set of experiments in HiB5 cells showed that
CMCerebellum dose-dependently transactivates the specific SMAD
reporter gene 3TP-Lux (Fig.
4F). Stimulation of reporter gene activity was abolished in the
presence of dominant-negative forms of SMAD3 (pCS2-FLAG-SMAD3-3SA) or co-SMAD4
(pCMV-FLAG-DPC4(1-514) (Fig.
4G), again pointing to the involvement of SMAD signaling in the
mediation of CMCerebellum actions. These and the other
above-reported data do not, however, completely rule out the participation of
other SMAD-linked factors, as co-SMAD4 complexes with other members of the
SMAD family, independently of TGFß; indeed, as functional inhibition of
SMAD4 resulted in stronger (P<0.01) inhibition of the
anti-proliferative and differentiating effects of CMCerebellum
(Fig. 4D,E), when compared with
those observed after SMAD3 inhibition, activation of other SMAD pathways by
non-TGFß ligands is highly plausible (see
Attisano and Wrana, 2002).
Last, as shown in Fig. 4H, CMCerebellum transactivation of the 3TP-Lux SMAD reporter gene was significantly attenuated (P<0.01) when HiB5 cells were transfected with plasmids expressing the dominant-negative forms of TGFßRII or TRKB. These data demonstrate the essential role of TGFßRII or TRKB receptors in coupling CMCerebellum-initiated signals (putatively, through TGFß2 and BDNF, respectively) with the SMAD signaling cascade.
BDNF actions involve convergence of MAPK and SMAD signaling pathways
Adding to the evidence that TGFßRII are involved in mediating the
biological actions of BDNF, we observed that BDNF failed to exert
anti-proliferative effects when it was applied to primary hippocampal cells
that were transiently transfected with a dominant-negative form of
TGFßRII (Fig. 5A).
As BDNF signaling pathways reportedly converge on those triggered by
TGFß2 following the activation of ERK1/2 by the neurotrophin
(Segal and Greenberg, 1996;
Pera et al., 2003
), we
pharmacologically tested the involvement of these kinases using the MEK
inhibitor PD98059. This drug significantly abrogated the inhibitory effects of
CMCerebellum on BrdU incorporation (P<0.001;
Fig. 5B). PD98059 also
abolished the pro-differentiating potential of CMCerebellum
(P<0.001; Fig. 5C).
Together, these observations indicate that CMCerebellum-induced
effects on proliferation and differentiation are mediated through MAP kinases
and, in accordance with earlier reports
(Marshall, 1995
;
Du et al., 2003
), they hint at
the involvement of BDNF in these processes.
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Discussion |
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In an analogous approach to those used previously in animals
(Renfranz et al., 1991;
Vicario-Abejón et al.,
1995
), the different developmental profiles in the hippocampus and
cerebellum were exploited in the present study to identify factors
contributing to the regulation of hippocampal granule cell proliferation and
differentiation. By potential (BrdU incorporation) in hippocampal and
cerebellar cultures from animals of a given developmental age, we confirmed
that, when compared with hippocampal cultures, cerebellar cultures were more
mature and mainly postmitotic after 14 d.i.v. Next, cultures of one type were
treated with conditioned medium from the other (CMCerebellum and
CMHippocampus). Analysis of BrdU uptake revealed that whereas
CMHippocampus stimulated proliferation in cerebellar cultures,
CMCerebellum treatment inhibited cell proliferation and accelerated
neuronal maturation in hippocampal cultures in a dose-dependent manner. The
anti-proliferative/pro-differentiating effects of CMCerebellum
coincided with increased expression of p21 and p27, two cell cycle
arrest-related molecules. Similar results were obtained after treating
hippocampus-cerebellum slice co-cultures or a hippocampus-derived cell line
(HiB5) with CMCerebellum. Together, these results indicate that
cerebellar and hippocampal cells secrete cell type-specific factors in a
temporally coordinated manner, and that these factors exert distinct
influences on neurogenesis and differentiation.
The loss of biological activity after boiling CMCerebellum
hinted at the polypeptide nature of its anti-proliferative/pro-differentiating
activities. Importantly, biological potency was retained in
CMCerebellum that was subjected to ion exchange chromatography, but
the anti-mitotic, apoptotic and differentiating activities eluted at different
ionic strengths. Immunoneutralization was used as an approach to identify the
active moieties in CMCerebellum; the selection of candidates was
based on reports that TGFß2, NGF and BDNF are differentially expressed in
hippocampal and cerebellar tissues during development
(Unsicker et al., 1991;
Sakamoto et al., 1998
;
Dieni and Rees, 2002
). We
found that immunoneutralization of BDNF and TGFß2, but not NGF, abrogated
the proliferative actions of CMCerebellum. At the same time,
pre-adsorption of CMCerebellum with anti-TGFß2 (but not
anti-BDNF or anti-NGF) inhibited the pro-differentiating actions of
CMCerebellum.
Further studies focused on verifying the roles of TGFß2 and BDNF in the observed CMCerebellum-induced effects on hippocampal cell development. Both TGFß2 and BDNF were found to be more strongly expressed in age-matched cerebellar versus hippocampal cultures (data not shown), and treatment of hippocampal cultures with either cytokine resulted in reduced BrdU incorporation and increased signs of neuronal differentiation.
Three separate genes encode three isoforms of TGFß: TGFß1
(normally restricted to the choroid plexus); and the neuron- and
glia-expressed isoforms TGFß2 and TGFß3
(Unsicker et al., 1991;
Pratt and McPherson, 1997
).
TGFß1 and TGFß3 have been implicated in neuroprotection, while
neurotrophic functions have been ascribed to TGFß2 and TGFß3
(Finch et al., 1993
;
Böttner et al., 2000
;
Pratt and McPherson, 1997
).
The latter include stimulation
(Mahanthappa and Schwarting,
1993
) or inhibition (Constam
et al., 1994
) of neurogenesis, or both
(Kane et al., 1996
), as well
as the regulation of neuronal differentiation
(Ishihara et al., 1994
;
Abe et al., 1996
;
Cameron et al., 1998
).
TGFß2, the isoform focused on in this work, is expressed in the external
granular (neurogenic) layer and in Purkinje and radial glia of the cerebellum
according to a strict temporal pattern and, interestingly, appreciable levels
of TGFß2 are not seen in other brain sites of neuronal proliferation
(Flanders et al., 1991
;
Constam et al., 1994
;
Unsicker and Strelau,
2000
).
Members of the TGFß superfamily signal by sequentially binding to two
TGFß receptors (TGFß-R) that are transmembrane protein
serine/threonine kinases; binding of TGFß ligand to TGFß-RII
activates TGFß-RI (expressed in the developing and adult rat hippocampus)
(Böttner et al., 1996)
and its substrates, the receptor-regulated SMAD proteins (R-SMADs). Upon
phosphorylation, the latter bind co-SMAD4 and translocate to the nucleus where
they form a transcriptionally active complex after association with
DNA-binding partner(s). This complex binds to promoter elements of target
genes whose functions include regulation of the cell cycle and differentiation
(Moustakas et al., 2001
;
Chang et al., 2002
;
Shi and Massagué,
2003
). For example, cell cycle arrest by TGFß involves
suppression of the oncogene Myc, a repressor of the CDK inhibitors p21 and p27
(Seoane et al., 2002
;
Gartel and Shchors, 2003
).
Supporting the view that TGFß2 may be responsible for at least some of
the anti-mitogenic activity of CMCerebellum we here observed an
upregulation of p21 and p27 after CMCerebellum treatment of
proliferating hippocampal neurons.
Additional evidence for a key role of TGFß2 in the hippocampal cell
fate-determining actions of CMCerebellum was obtained by studying
TGFß signal-propagating SMAD proteins. Of the various members of the SMAD
system, SMAD2 and SMAD3 mediate TGFß signals. SMAD4 is a requisite
partner for transcriptional activity of all SMADs, including SMAD2 and SMAD3;
the generation of specific downstream responses is presumed to depend on the
formation of specific R-SMAD-SMAD4 complexes that then recruit different
sequence-specific DNA-binding factors
(Massagué and Wotton,
2000). We demonstrated that CMCerebellum treatment can
induce nuclear translocation of EGFP-SMAD2 and EGFP-SMAD4. Essential roles for
SMAD3 and SMAD4 were demonstrated insofar that transient expression of the
dominant-negative forms of either of these molecules in hippocampal cells
prevented CMCerebellum-induced transactivation of the TGFß
reporter gene (3TP-Lux) and abrogated the anti-proliferative and
pro-differentiating effects of CMCerebellum. Further support for
the view that TGFß2, at least partially, accounts for the
anti-proliferative activity present in CMCerebellum is provided by
the observation that expression of a vector containing a dominant-negative
form of TGFßRII in either primary hippocampal cells or a
hippocampus-derived cell line (Hib5) abolishes CMCerebellum-induced
effects on BrdU incorporation and 3TP-Lux reporter activity.
As already mentioned, hippocampal cells responded to exogenous BDNF (or a
BDNF-expressing plasmid) with an inhibition of BrdU uptake, and an increase in
the number of MAP2a/b neurons and neuritic lengths; the effects of this
neurotrophin therefore closely resembled those of TGFß2. BDNF effects on
neuronal differentiation are mediated through TRKB receptors
(Klein et al., 1991) and BDNF
can either promote or inhibit neuronal proliferation by activating the
TRK-MAPK-ERK pathway (Marshall,
1995
; Du et al.,
2003
), thus raising the issue of whether ERK signaling is involved
in the biological actions of CMCerebellum. We found that upstream
(MEK) inhibition of this pathway using PD98059 negates the proliferative and
differentiating actions of CMCerebellum. Furthermore, we observed
that transient expression of a dominant-negative form of TRKB in hippocampal
cells abolishes CMCerebellum-induced effects on proliferation and
differentiation, while, at the same time, abolishing the ability of
CMCerebellum to induce nuclear translocation of SMAD2 and SMAD4,
and TGFß 3TP-Lux transactivation. These findings are consistent with
previous reports that suggested crosstalk
(Lutz et al., 2004
) or
interdependence/synergism (Unsicker and
Strelau, 2000
) between these trophic factors and their signal
transduction pathways. Additional support for this interpretation is provided
by our observation that BDNF cannot stimulate 3TP-Lux reporter activity after
functional blockade of SMAD4 and TGFßRII expression, and that
expression of a dominant-negative form of TGFßRII attenuates the
anti-proliferative activity of BDNF. In an initial analysis of the upstream
mechanisms that might be responsible for crosstalk between the BDNF and
TGFß pathways, we found that BDNF can stimulate the cellular content of
TGFß2 in hippocampal cells; unfortunately, limited assay sensitivity
precluded information on whether BDNF can also stimulate TGFß2 secretion.
Seoane et al. (Seoane et al.,
2002
) showed that TGFß can induce cell cycle arrest by
activating cdk inhibitors such as p21 and p27. The present study shows that
both these latter proteins are upregulated in hippocampal cells after exposure
to CMCerebellum, raising the following questions for future
research. Do BDNF-activated TRKB receptors induce cell cycle arrest? If so, is
the TGFß/SMAD pathway involved?
In summary, we have demonstrated that BDNF and TGFß2 and their
respective signaling machineries, by acting in a dynamic, but strictly
coordinated, spatiotemporal fashion, play a decisive role in determining
hippocampal cell fate by inhibiting cell proliferation and promoting neuronal
differentiation. We have also shown that BDNF, better known for TRK
receptor-mediated promotion of neurogenesis and differentiation
(Klein et al., 1991;
Gao et al., 1995
;
Pencea et al., 2001
), can
exert anti-proliferative and pro-differentiating effects on hippocampal
granule cells by activating MAPK and, subsequently, TGFß signaling
pathways; the latter is a novel observation and provides a mechanism through
which diverse cytokine signals can converge on a common signaling `hub' to
direct neuronal development.
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ACKNOWLEDGMENTS |
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