 |
 Previous Article | Next Article 
The Journal of Neuroscience, December 1, 1998, 18(23):9822-9834
Glial Cell Line-Derived Neurotrophic Factor Requires Transforming
Growth Factor- for Exerting Its Full Neurotrophic Potential on
Peripheral and CNS Neurons
Kerstin
Krieglstein1,
Prisca
Henheik1,
Lilla
Farkas1,
Jozsef
Jaszai1,
Dagmar
Galter1,
Knut
Krohn2, and
Klaus
Unsicker1
1 Department of Neuroanatomy, University of Heidelberg,
D-69120 Heidelberg, Germany and 2 Center for Internal
Medicine, University of Leipzig, D-04103 Leipzig, Germany
 |
ABSTRACT |
Numerous studies have suggested that glial cell line-derived
neurotrophic factor (GDNF) is a potent neurotrophic molecule. We show
now on a variety of cultured neurons including peripheral autonomic,
sensory, and CNS dopaminergic neurons that GDNF is not trophically
active unless supplemented with TGF- . Immunoneutralization of
endogenous TGF- provided by serum or TGF--secreting cells, as
e.g., neurons, in culture abolishes the neurotrophic effect of GDNF.
The dose-response relationship required for the synergistic effect of
GDNF and TGF- identifies 60 pg/ml of either factor combined with 2 ng/ml of the other factor as the EC50. GDNF/TGF- signaling employs activation of phosphatidylinositol-3 (PI-3) kinase as an intermediate step as shown by the effect of the specific PI-3 kinase inhibitor wortmannin. The synergistic action of GDNF and
TGF- involves protection of glycosylphosphatidylinositol (GPI)-linked receptors as shown by the restoration of their
trophic effects after phosphatidylinositol-specific phospholipase
C-mediated hydrolysis of GPI-anchored GDNF family receptor  .
The biological significance of the trophic synergism of GDNF and
TGF- is underscored by colocalization of the receptors for TGF-
and GDNF on all investigated GDNF-responsive neuron populations
in vivo. Moreover, the in vivo relevance
of the TGF-/GDNF synergism is highlighted by the co-storage of
TGF- and GDNF in secretory vesicles of a model neuron, the chromaffin cell, and their activity-dependent release. Our results broaden the definition of a neurotrophic factor by incorporating the
possibility that two factors that lack a neurotrophic activity when
acting separately become neurotrophic when acting in concert. Moreover,
our data may have a substantial impact on the treatment of
neurodegenerative diseases.
Key words:
motoneurons; dopaminergic neurons; neurotrophic factors; exocytosis; chromaffin cells; neurodegenerative disease; signal
transduction
| |
INTRODUCTION |
The discovery of glial cell
line-derived neurotrophic factor (GDNF) as a neurotrophic factor for
midbrain dopaminergic neurons was a hallmark in the search for novel
molecules that may have relevance in the treatment of neurodegenerative
diseases, as e.g., Parkinson's disease (PD; Lin et al., 1993 ). The
significance of GDNF is underscored by its efficacy in several animal
models of PD, including nonhuman primates (Hoffer et al., 1994; Beck et al., 1995; Tomac et al., 1995a; Sauer et al., 1995; Gash et al., 1996),
ubiquitous expression in neurons of the CNS (Pochon et al., 1997), and
its widening spectrum of responsive neuron populations (Henderson et
al., 1994; Arenas et al., 1995; Buj-Bello et al., 1995; Trupp et al.,
1995; Farkas et al., 1997). GDNF signals via the tyrosine kinase
receptor c-Ret (Durbec et al., 1996; Trupp et al., 1996) in
cooperativity with a glycosylphosphatidylinositol (GPI)-linked
receptor, the GDNF family receptor (GFR) (Jing et
al., 1996; Treanor et al., 1996). GDNF is a member of the TGF- superfamily (Kingsley, 1994), its closest relative being neurturin (Kotzbauer et al., 1996). Targeted mutations of the GDNF (Moore et al.,
1996; Pichel et al., 1996; Sanchez et al., 1996) or c-Ret genes
(Schuchardt et al., 1994) have indicated that GDNF is essentially required for the development of the kidney, major portions of the
enteric nervous system, and the sympathetic superior cervical ganglion.
TGF-s are widely distributed and contextually acting cytokines with
prominent roles in development and cell cycle control (Roberts and
Sporn, 1990; Nathan and Sporn, 1991; Alexandrow and Moses, 1995;
Krieglstein et al., 1995a). TGF-s have been implicated in the
regulation of neuronal survival of, e.g., motoneurons (Martinou et al.,
1990), sensory neurons (Chalazonitis et al., 1992), and midbrain
dopaminergic neurons (Krieglstein and Unsicker, 1994; Poulsen et al.,
1994). It should be noted, however, that TGF- shows no or marginal
effects on highly enriched, serum-free neuron cultures, as e.g.,
sensory neurons (Krieglstein and Unsicker, 1996b), suggesting that
TGF- may require cooperating factors for eliciting its trophic effects.
Several lines of evidence suggest that GDNF similarly may require
cofactors for acting as a neurotrophic factor. First, GDNF does not
support the survival of most peripheral neurons in low-density dissociated cultures and defined media (Henderson et al., 1994; Krieglstein and Unsicker, 1996a). Second, follow-up experiments in
which GDNF was shown to promote the survival of enriched peripheral autonomic and sensory neurons (Buj-Bello et al., 1995; Trupp et al.,
1995) were all performed using serum throughout the whole culture
period. Third, the dopaminotrophic effect of GDNF was established in an
extremely complex culture system (Lin et al., 1993) in which its most
prominent effect did not become apparent until day 7 in culture.
The present study identifies TGF- as the cotrophic factor required
to permit neurotrophic effects of GDNF.
| |
MATERIALS AND METHODS |
Cell cultures. Fertilized white Leghorn chicken eggs
were incubated in a humidified egg chamber at 37.8°C. Rat embryos
were obtained from Hanover-Wistar rats; the day of vaginal plug
identification was considered embryonic day 0 (E0). Embryonic chick
(E8, E10, and E12) ciliary, dorsal root, and sympathetic ganglia and
embryonic (E14) rat dorsal root ganglia were dissected, freed from
nerve roots and connective tissue, and collected in
Ca2+/Mg2+-free HBSS (CMF;
Sigma, St. Louis, MO). Ganglia were trypsinized, washed in CMF, and
triturated as described (Lachmund et al., 1994). Single cell
suspensions were seeded in poly-L-ornithine (PORN), laminin-coated microtiter plates (Costar, Cambridge, MA; A/2) at a
density of 1200 cells per well in DMEM supplemented with N1 additives
(transferrin, 6.25 × 10 8 M;
putrescine, 1 × 104 M;
progesterone, 2 × 108 M; and
selenium, 3 × 108 M; Sigma;
Bottenstein et al., 1980), 0.25% BSA (Sigma), 100 U/ml penicillin,
0.50 µg/ml streptomycin, 100 µg/ml neomycin (PSN) (Life
Technologies), and incubated at 37°C in a 5% CO2 and
95% air atmosphere (Lachmund et al., 1994).
Mesencephalic cell cultures from embryonic rat midbrain floor (E14)
were essentially established as described by Krieglstein et al.
(1995b). They were seeded at a density of 50,000 per
cm2, cultured under serum-free conditions, and
processed for immunocytochemistry on day 5 in vitro.
Dopaminergic neurons were visualized using a monoclonal antibody to rat
tyrosine hydroxylase (1:200, Boehringer Mannheim, Mannheim, Germany) as
described previously (Krieglstein et al., 1995b).
Embryonic rat motoneurons (Hanover-Wistar rats, E14) were prepared as
described by Camu and Henderson (1992) using a two-step purification
method: a density centrifugation using a metrizamide gradient followed
by a panning procedure using the monoclonal antibody MC-192 that
recognizes the low-affinity NGF receptor. The purified motoneurons were
seeded in 24-well plates (Nunc, Darmstadt, Germany; 1000 per well)
coated with PORN/laminin. After 15 hr all phase-bright cells
without vacuolar inclusions were counted to establish the 100% value,
and the neurotrophic factors were added. On day 4 in culture the
percentage of surviving motoneurons was established by a final counting.
Neurons were grown with or without different recombinant neurotrophic
factors [NGF, Boehringer Mannheim; CNTF, GDNF, FGF-2, and TGF-2, IC
Chemikalien; TGF-1 and TGF-3, a gift from Dr. M.B. Sporn; bone
morphogenetic proteins (BMPs) 2, 4, 6, 7, and 12, provided by
Dr. V. Rosen, Genetics Institute; protein lysate from bovine chromaffin
granules, Krieglstein and Unsicker, 1997], serum (horse serum, Life
Technologies; fetal calf serum, PAA laboratories, EuroBio, PAN
Systems), or neutralizing antibodies to TGF-1, TGF-2, and
TGF-3 (10 µg/ml; Genzyme, Boston, MA; catalog #1835-01) or to GDNF (20 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA). Chick
ciliary ganglia (CG) neurons were grown in the presence of
wortmannin (Calbiochem, La Jolla, CA) or were incubated for 1 hr at
37°C with 100 mU phosphatidylinositol-specific phospholipase C
(PIPLC) (Boehringer Mannheim) or 100 mU PIPLC plus 2 ng/ml
TGF-1, or they were incubated without enzyme before the
addition of growth factors. At appropriate times [CG, 24 hr; DRG, 48 hr, lumbar sympathetic ganglia (SG), 72 hr] cultures were fixed by
addition of 2.5% glutaraldehyde in PBS. Numbers of surviving
neurons were determined by direct counting of 30% of the surface area
using phase contrast microscopy.
The isolation of bovine adrenal medullary chromaffin cells was
performed by collagenase digestion and Percoll gradient centrifugation essentially as described by Unsicker et al. (1980). Chromaffin cells at
95% purity were seeded into 25 cm2 plastic culture
flasks (Falcon) at a density of 200,000 cells/cm2
and grown in 5 ml DMEM/N1. After 30 hr the cultures were washed three
times with medium and incubated for 15 min at 37°C in 2 ml prewarmed
DMEM/N1 with or without of the following secretagogues: carbachol (100 µM; Sigma), carbachol (100 µM) plus
verapamil (10 µM; Sigma), or the calcium ionophore A23187
(2 µM; Sigma).
B49, COS cells, and NIH 3T3 fibroblasts were grown in 10% FCS-DMEM
with PSN, and baby hamster kidney cells in DMEM-F12 with 5% FCS and PSN in plastic culture flasks (Falcon). Conditioned medium
of confluent cultures was collected over a period of 48 hr.
Determination of TGF activity was performed by using mink lung
epithelial cells (MLEC) stably transfected with a luciferase reporter
gene under the control of a truncated PAI-1 promotor (Abe et al., 1994;
kindly provided by Dr. Rifkin, New York University) as described
earlier (Krieglstein and Unsicker, 1995). Transfected MLEC cultures
were plated in a 96-well microtiter plate (Costar) at a density of
1.6 × 104 cells per well in DMEM (high
glucose) with 10% FCS and geneticin (250 µg/ml) and allowed to
attach for 3 hr at 37°C in a 5% CO2 incubator. The
medium was then replaced with 100 µl of test sample and incubated
overnight at 37°C in a 5% CO2 incubator. Cells were washed twice with PBS and lysed by using 100 µl of lysis buffer (Promega, Madison, WI) for 2-3 hr at RT. To determine TGF-
activity, 80 µl of the lysates were transferred to a test tube and
analyzed using a Luminometer (EG&G Berthold, Bad Wildbach,
Germany) by 100 µl injections of luciferase reagent (Berthold
Detection Systems, Pfortzheim, Germany). Luciferase activity was
reported as relative light units (RLU), and all assays were performed
in triplicate.
RT-PCR. RT-PCR was used to determine the expression
pattern of TGF-2 and TGF-3 along with TR-II, GFR-1, and
c-ret (receptor components involved in TGF- or GDNF signaling) in
postnatal day 0 (P0) ventral mesencephalon, dorsal root ganglia, and
E14 purified motoneurons. Expression of TGF-1, TGF-2, TGF-3,
and GDNF was also determined in dissociated purified bovine adrenal
chromaffin cells. Total RNA was isolated by Trizol (Life Technologies)
according to the manufacturer's instruction and were treated with RQ1
DNase (Promega) to exclude amplification of contaminating genomic DNA in subsequent manipulations. Total RNA was subjected to reverse transcription. Total RNA (1 µg/reaction) was added to a "master mix" (24 µl) containing final concentrations of 0.5× first strand buffer (Life Technologies), 0.66 U/µl RNase inhibitor (MBI
Fermentas), 10 mM dithiothreitol (DTT), 1.5 µM oligo-dT primer (Life Technologies), and 0.3 mM deoxyribonucleoside triphosphates (dNTPs)
(Pharmacia, Freiburg, Germany). Sample mixtures were then heated to
65°C for 10 min. After cooling to 42°C, 200 U (5 µl) M-MLV
reverse transcriptase (Life Technologies) and 5× first strand buffer
to a final concentration of 1× were added. Reactions were incubated at
37°C for 60 min and were then stopped by incubation at 75°C for 10 min. After reverse transcription each cDNA sample was subjected to PCR
amplification using specific primers as follows: TGF-1 forward
5'-AAGAACTGCTGTGTTCGTCAGC-3' and reverse
5'-GATCATGTTGGACAACTGCTCC-3'; TGF-2 forward (bovine) 5'-TGGATGCCGCCTATTGCTTTAGG-3', forward (rat)
5'-CTACAGACTGGAGTCCCAGC-3', and reverse (bovine, rat)
5'-CATGTTGGAAAGTTGTTCGATC-3'; TGF-3 forward (bovine, rat)
5'-GGAAATCAAATTCAAAGGAGTGG-3' and reverse (bovine, rat)
5'-AGTTGGCATAGTAACCCTTAGG-3'; GDNF forward
5'-GGTCTACGGAGACCGGATCCGAGGTGC-3' and reverse
5'-TCTCTGGAGCCAGGGTCAGATACATC-3'; TR-II forward
5'-GCTGCATATCGTCCTGTG-3' and reverse 5'-CGTGGTAGGTGAGCTTGG-3'; GFR-1
(Baloh et al., 1997) forward 5'-GCACAGCTACGGGATGCTCTTCTG-3' and reverse
5'-GTAGTTGGGAGTCATGACTGTGCCAATC-3'; c-ret (Baloh et al., 1997)
forward 5'-TGGCACACCTCTGCTCTATG-3' and reverse
5'-TGTTCCCAGGAACTGTGGTC-3'; and S6 forward
5'-CTCCAAAGAAGATGATGTCC-3' and reverse
5'-TTTAGAAGTAGAAGCTCTCAG-3'. PCR reactions were performed in 0.2 ml
thin-walled reaction tubes using the "hot-start" method in a
Perkin-Elmer GeneAmp PCR system 9600. Reagents were assembled in a
final volume of 50 µl, and final concentrations of reagents were as
follows: 5 µl first strand cDNA, 1 µM forward primer, 1 µM reverse primer, 1× PCR buffer (Life Technologies), 1 mM MgCl2, 100 mM dNTPs
(Pharmacia) and RNase-free water to 50 µl. Samples were initially
denatured at 94°C for 4 min and 1.25 U recombinant Taq DNA polymerase (Life Technologies) was then added.
Thermocycling parameters were then 30 sec denaturation at 95°C, 30 sec annealing at 60°C, and 30 sec extension at 72°C repeated for 32 cycles with a final extension step at 72°C for 5 min. Aliquots (5 µl) of the PCR reaction were run on 0.2% agarose gels, and the size
of the reaction products was determined after ethidium bromide
staining. Identity of TGF- reaction products (TGF-1, TGF-2, and
TGF-3) was confirmed by Southern blotting and hybridization. Five
microliters of each TGF and S6 amplification products were run on
2% agarose gels and blotted to Hybond-N+ nylon
membranes (Amersham, Braunschweig, Germany). Membranes were probed
using internal oligonucleotide sequences (TGF-1
5'-TTGGGCTTGCGACCCACGTAGT-3'; TGF-2 5'-TGGATGCCGCCTATTGCTTTAGG-3';
TGF-3 5'-AGATGACCATGGCCGTGGAGACC-3'; and S6
5'-GGAGGCTGCAGAATATGCTAAACT-3') labeled with TdT (terminal deoxynucleotidyl transferase; AGS, Heidelberg, Germany) using 32P-gamma-ATP (DuPont NEN, Boston, MA). Signal intensities
were analyzed by ImageQuant/PhosphorImager (Molecular Dynamics).
Immunohistochemistry, immunoprecipitation, and Western
blotting. Immunohistochemistry was essentially performed as
described previously (Krieglstein et al., 1996). White Leghorn chick
embryos (E8) were fixed by immersion in ice-cold Bouin solution for 4 hr (Krieglstein et al., 1998). Pregnant Wistar rats were killed by
CO2 aspiration, and embryos (E21) were removed and fixed by transcardial perfusion with 4% paraformaldehyde buffered in PBS. The
spinal cord of newborn (P0) Wistar rats was removed after transcardial
perfusion with 10% neutral buffered formol and post-fixed in Bouin
solution. Tissue pieces were dehydrated in increasing concentrations of
ethanol and embedded in paraffin wax. Sections (10 µm) from E21 rat
adrenal glands were deparaffinized and incubated for 30 min in 10%
horse serum (HS), 1% BSA, and 0.1% Triton X-100. Sections were then
exposed to either anti-chromogranin A antibody (rabbit polyclonal
1:200, Dakopatts, Copenhagen, Denmark; or mouse monoclonal 1:100,
kindly provided by Dr. W. Huttner, University of Heidelberg) at 4°C
overnight, followed by incubation with anti-GDNF antibody (rabbit
polyclonal 1:200, Santa Cruz Biotechnology; or mouse monoclonal 1:100,
R & D Systems, Minneapolis, MN). As a specificity control, consecutive
rather than identical sections were incubated with just one primary
antibody at a time and processed similarly. Specificity of the
stainings was verified by omitting the first antibody, by preabsorbing
with an excess of antigen (rhGDNF, bovine chromaffin granule lysates
containing chromogranin A), and by Western blotting of bovine
chromaffin cell and granule lysates (see this study and Huttner et al.,
1991; Bieger et al., 1995; Krieglstein et al., 1996). Cultured bovine
chromaffin cells were processed similarly after a 10 min fixation using
4% paraformaldehyde buffered with PBS. Paraffin sections (5-10 µm)
from rat spinal cord (P0) or chicken DRG (E8) were rehydrated, and
antigen unmasking was performed by microwave treatment in 10 mM citrate buffer. After blocking with serum, sections were
immunostained for TGF-2, TGF-3, or TR-II (TGF-2, sc-90;
TGF-3, sc-82; and TR-II, sc-400, Santa Cruz Biotechnology) using
the isoform-specific antibodies at dilutions of 1:200-1:500. The
immunostaining was visualized using the streptavidin-Cy3 method as
previously described (Krieglstein et al., 1996). Experimental controls
were performed by replacing the primary antibody with normal rabbit
serum, preincubation with the corresponding antigen, and by
immunoprecipitation (data not shown, Unsicker et al., 1996; Krieglstein
et al., 1998).
For immunoprecipitation of GDNF or TGF-1, samples were incubated
with 10 µl/ml of the appropriate agarose-conjugated antibody (polyclonal rabbit anti-GDNF or anti-TGF-1, Santa Cruz
Biotechnology) at 4°C overnight under mild agitation. Samples were
washed with PBS and then resuspended in 50 µl of electrophoresis
sample buffer and boiled for 2 min. Samples were separated by
electrophoresis on a 12.5% SDS-polyacrylamide gel and
transferred to nitrocellulose membrane (Hybond; Amersham) as described
earlier (Bieger et al., 1995). For dot blot analysis 2 µl (40 ng) of each growth factor sample was loaded onto nitrocellulose
membrane. The membranes were blocked with 3% low fat milk powder and
0.1% BSA in Tris-buffered saline (TBS), pH 7.3, incubated with primary
antibody (1:200 in 0.1% BSA-TBS) overnight at 4°C followed by
peroxidase conjugated anti-rabbit antibody (1:2000 in 0.1% BSA-TBS).
Finally, the membrane was developed using the Amersham enhanced
chemiluminescence (ECL) detection system.
| |
RESULTS |
The neurotrophic action of GDNF on several populations of
peripheral and CNS neurons essentially requires TGF-
GDNF has been proposed as a potent neurotrophic factor for a
variety of cultured CNS and peripheral neurons. All of these cultures
share significant cellular complexity and/or the use of serum resulting
in uncontrolled trophic conditions. A well known constituent of almost
every cell type as well as serum is TGF- (Roberts and Sporn, 1990).
Quantitative determinations of TGF- (biological activity) in
cultures of B49 (from which GDNF was originally isolated), BHK, COS
cells, and 3T3 fibroblasts (frequently used for transfection
experiments) revealed that these cells secrete within 48 hr 0.2 to 0.4 ng/ml TGF- into their culture medium (data not shown; cf. Abe et
al., 1994; Krieglstein and Unsicker, 1995). Different batches of fetal
calf and horse sera contain varying, but significant amounts of TGF-
(0.1-0.2 ng/ml in culture media with 10% serum; data not shown).
Using chick ciliary ganglionic neurons as an example, Figure
1A demonstrates that
GDNF supplemented with 10% horse serum maintains neurons over a 24 hr
period as effectively as a saturating concentration of CNTF (5 ng/ml).
However, administering culture medium that had been preincubated with
neutralizing antibodies to TGF- (10 µg/ml, known to neutralize
>95% of 1 ng/ml of TGF- isoforms TGF-1, TGF-2, and TGF-3;
for specificity of the antibody, see Fig. 1B)
significantly reduced the effect of GDNF, suggesting that GDNF requires
TGF- for displaying its trophic effect. Consistent with this notion,
if a serum-containing culture medium was replaced with a fully defined
culture medium, both GDNF and TGF- alone (each at the saturating
amount of 2 ng/ml) showed only marginal survival-promoting
effects. However, when combined, the two factors promoted
neurons as effectively as CNTF. To determine dose-response relationships required for the synergistic effect of GDNF and TGF-,
each single factor at a concentration of 2 ng/ml was titrated in
combination with serial dilutions of the other one. As shown in Figure
1C, 60 pg/ml of either factor combined with 2 ng/ml of the
other factor represented the EC50. The combination of 0.25 ng/ml and 2 ng/ml already elicited saturating effects. All isoforms of
TGF- (TGF-1, TGF-2, and TGF-3) were consistently equipotent under the conditions used (data not shown). TGF- could not be replaced by BMP2, 4, 7, or 12 (data not shown). We conclude that the
combination of purified recombinant TGF- and GDNF promotes ciliary
ganglionic neuron survival to the same extent as CNTF, whereas each
single factor is virtually ineffective. The combinatorial effect is
specific in that TGF- cannot be replaced by other members of the
TGF- superfamily.
View larger version (41K):
[in this window]
[in a new window]
|
Figure 1.
Survival of peripheral autonomic and
sensory neurons by the synergistic action of GDNF and TGF-.
A, Neurons from CG, DRG, and SG ganglia were isolated at
E8 and grown under the indicated conditions. Neurons were maintained in
serum-containing medium (10% HS) (gray bars) or
in serum-free medium (white bars). In the presence of
serum, addition of a saturating concentration of GDNF promoted survival
of each of the three neuron populations at levels identical to those
achieved by addition of the respective neurotrophic factor (CNTF for
CG, NGF for DRG and SG neurons; 10 ng/ml each) to serum-free culture
media. Addition of a neutralizing antibody to TGF-1, TGF-2, and
TGF-3 (a-TGF-, 10 µg/ml) reduced neuron survival to levels seen
with the addition of serum alone, suggesting that GDNF required TGF-
in the serum to achieve its survival-promoting effect. In serum-free
conditions, GDNF and TGF--1 (2 ng/ml), when added separately had
virtually no survival-promoting effect. However, when combined at
optimal concentrations, both factors permitted neuron survival at
levels identical to those achieved with the established neurotrophic
factors CNTF and NGF, respectively. Data are given as mean ± SEM
(n = 6), p values derived from
Student's t test are ***p < 0.001 for increased survival as compared with single factors and
+++p < 0.001 for decreased survival after antibody
treatment. B, Dot blot showing that the anti-TGF-
antibody recognizes TGF-1 and TGF-3, but not any other TGF-
superfamily or neurotrophin family member tested. C,
Dose-response curve for the combined action of GDNF and TGF-
on chick ciliary neurons. Squares represent neuron
survival achieved in the presence of a constant amount of GDNF (2 ng/ml) plus the indicated amounts of TGF-1. Circles
represent neuron survival achieved in the presence of a constant amount
of TGF-1 (2 ng/ml) plus the indicated amounts of GDNF.
|
|
To investigate whether the synergistic effect of GDNF and TGF- also
applied to other populations of peripheral neurons, identical experiments were performed using chick sensory (DRG) and paravertebral sympathetic neurons isolated from E8 embryos. As shown in Figure 1,
D and E, GDNF and TGF-, when coadministered to
serum-free cultures, maintained sensory and sympathetic neurons,
respectively, as supported by a saturating concentration of NGF (5 ng/ml). Again, 10% horse serum substituted for TGF-.
To exclude that the above effects are restricted to a brief
developmental time window, the same set of experiments was performed on
ciliary, sensory DRG and sympathetic neurons from E10 and E12 chick
embryos. Figure
2A,B
shows that coadministration of GDNF and TGF- mimicked the
survival-promoting effect of CNTF or NGF, respectively, at all ages and
on all neuron populations studied.
View larger version (55K):
[in this window]
[in a new window]
|
Figure 2.
Assay as performed in Figure
1A using neurons from the respective ganglia of
chick E10 (A) and E12 (B)
embryos. Data indicate that the GDNF/TGF- synergism also applies to
neurons at more advanced stages of development. Data are given as
mean ± SEM (n = 6), p values
derived from Student's t test are
***p < 0.001 for increased survival as compared
with single factors and +++p < 0.001 for decreased
survival after antibody treatment.
|
|
To establish that neurons from chick as well as mammals responded in a
similar manner to GDNF and TGF-, lumbar DRG neurons from E14 rat
embryos were subject to the same treatments as above. Figure
3A shows that, in contrast to
NGF, the GDNF-mediated survival-promoting effect in the presence of
10% horse serum was abolished by a saturating amount (10 µg/ml) of
neutralizing antibodies to TGF-.
View larger version (24K):
[in this window]
[in a new window]
|
Figure 3.
Survival-promoting effect of GDNF and TGF- on
rat DRG neurons (A), rat mesencephalic
dopaminergic neurons (B), and rat motoneurons
(C). A, Dissociated DRG (E14)
cultures were treated with GDNF (10 ng/ml), NGF (10 ng/ml),
anti-TGF- antibodies (10 µg/ml), or NGF or GDNF plus anti-TGF-
antibodies in the presence of 10% horse serum. Data are given as
mean ± SEM (n = 6), p values
derived from Student's t test are
***p < 0.001 for increased survival as compared
with untreated controls and +++p < 0.001 for
decreased survival after antibody treatment. B,
Dissociated mesencephalic dopaminergic neurons (E14) were cultured at a
density of 50,000 cells/cm2 in serum-free medium and
treated with GDNF (2 ng/ml), TGF-3 (2 ng/ml), GDNF plus TGF-3, or
GDNF plus anti-TGF- antibodies (10 µg/ml). Data are given as
mean ± SEM (n = 4), p values
derived from Student's t test are
+++p < 0.001 for decreased survival after antibody
treatment. C, Dissociated purified motoneurons (E14)
were cultured for 4 d using serum-free medium and treated with
GDNF in the absence or presence, respectively, of different amounts of
TGF-1 or anti-TGF- antibodies (10 µg/ml). Data are given as
mean ± SEM (n = 4) and expressed in percent
of cells present at day 1.
|
|
As GDNF was first characterized by its pronounced trophic effects on
midbrain dopaminergic neurons, we asked whether this effect was
dependent on or independent from TGF-. As shown in Figure
3B, GDNF and TGF- each promoted the survival of
dopaminergic neurons under serum-free conditions approximately twofold
as compared with untreated control cultures. Combinations of the
factors further enhanced survival (threefold as compared with
controls). Addition of neutralizing antibodies to TGF- (10 µg/ml)
abolished the trophic effect of GDNF.
Motoneurons have been the only neuron population studied so far that
showed a convincing neurotrophic response to recombinant GDNF (cf.
Henderson et al., 1994). Even so, as shown in Figure 3C,
TGF- significantly enhanced the trophic effect of GDNF. In contrast
to dopaminergic neurons, however, antibodies to TGF- (10 µg/ml)
failed to reduce survival in the absence of exogenous factors.
In summary, the above data indicate that TGF- is required to have
GDNF exert its full neurotrophic potential on both peripheral and CNS neurons.
TGF- synergizes with GDNF by protecting
GPI-linked receptors
To begin to characterize details of the specific signal
transduction pathway used by GDNF to cooperate with TGF-, we
investigated whether activation of PI-3 kinase that has been shown as
an early event in GDNF/c-ret-mediated signal transduction (van Weering and Bos, 1997) was involved. Figure
4A shows that the
specific PI-3 kinase inhibitor wortmannin at a concentration of 0.25 µM completely abolished the survival-promoting effect of
GDNF in conjunction with TGF- on cultured ciliary ganglionic
neurons. Wortmannin did not interfere with the survival-promoting
effect of CNTF, indicating that activation of PI-3 kinase is an
essential event in mediating the survival-promoting effect of
GDNF/TGF-, but not that of CNTF. Furthermore, this result indicates
that wortmannin did not unspecifically compromise survival.
View larger version (31K):
[in this window]
[in a new window]
|
Figure 4.
Mechanisms underlying the synergistic actions of
GDNF and TGF-. A, Chick ciliary ganglionic neurons
(E8) were treated with GDNF (2 ng/ml), TGF-1 (2 ng/ml), GDNF plus
TGF-1 (2 ng/ml each), and GDNF plus TGF-1 plus wortmannin (0.25 µM), a specific inhibitor of PI-3 kinase. Wortmannin
abolishes GDNF/TGF--mediated survival of ciliary ganglionic neurons,
indicating that PI-3 kinase is an essential mediator in signal
transduction of the combined action of GDNF and TGF-. Data are given
as mean ± SEM (n = 6), p
values derived from Student's t test are
+++p < 0.001 for decreased survival after
wortmannin treatment. B, Chick ciliary ganglionic
neurons were treated with CNTF (2 ng/ml), FGF-2 (10 ng/ml), and GDNF
plus TGF-1 (2 ng/ml each; white bars) after
pretreatment with PIPLC (100 mU, 1 hr), which liberates GPI-anchored
cytokine receptors from the plasma membrane. PIPLC interferes, as
expected, with the survival-promoting effect of CNTF but not that of
FGF-2. Pretreatment of isolated ciliary neurons with PIPLC
significantly reduces the survival-promoting effect of GDNF and TGF-
consistent with the essential role of a GPI-linked GFR in GDNF
signal transduction. Addition of both PIPLC and TGF- to isolated
ciliary neurons protects the GPI-linked -receptors, suggesting that
the synergistic neurotrophic action of GDNF and TGF- may be caused
by a protective action of TGF- on the GFR. Data are given as
mean ± SEM (n = 6), p values
derived from Student's t test are
+++p < 0.001 for decreased survival after PIPLC
treatment and ***p < 0.001 for increased survival
in the presence of TGF-1.
|
|
We next asked whether TGF- might be involved in the stabilization
and recruitment of the GPI-linked GFR. PIPLC at a concentration of
0.1 U/ml was used to hydrolyze the GPI-anchored receptors on dissociated ciliary neurons before plating. This procedure effectively reduced the survival-promoting effect of CNTF (which utilizes the
GPI-anchored GPAR; Heller et al., 1995) without affecting the
survival-promoting effect of FGF-2 (Fig. 4B).
Hydrolysis of GPI-linked receptors significantly reduced the
survival-promoting effect of GDNF in its combination with TGF-.
However, when the PIPLC pretreatment was conducted in the presence of
TGF- (2 ng/ml TGF-1) the survival-promoting effects of GDNF and
CNTF were maintained (Fig. 4B). These data suggest an
essential involvement of a GPI-linked receptor component in the
neurotrophic effect of GDNF and a possible role of TGF- in the
stabilization and/or recruitment of the receptor component.
Expression of TGF-s and TGF- receptor (TR-II) in
GDNF-responsive neuron populations in vivo
To investigate whether neuron populations known to be responsive
to GDNF coexpress TGF- receptor and its ligands in vivo, we studied expression of both mRNA and protein in select neurons of the
CNS and peripheral nervous system. As shown in Figure
5 motoneurons purified from E14 rat
spinal cord, DRG, and ventral mesencephalon from newborn rat expressed
mRNA signals for TGF-2, TGF-3, and TR-II along with the GDNF
receptors GFR-1 and c-ret. Figure 6
demonstrates the cellular localization of TGF-2, TGF-3, and
TR-II immunoreactivities in DRG (E8 chick) and spinal motoneurons (neonatal rat). These data are supplemented by previously published results compiled in Table 1, showing that
the investigated neuron populations are well equipped with receptors
for GDNF and TGF- in vivo. Moreover, these neurons also
express the ligand TGF-, whereas GDNF is consistently found in
neuronal target areas. Together, these data make a compelling case for
an in vivo scenario of GDNF and TGF- cooperativity.
View larger version (36K):
[in this window]
[in a new window]
|
Figure 5.
RT-PCR (30 cycles) of P0 rat tissues and E14
purified motoneurons with primers specific for TGF-2 (355 bp),
TGF-3 (290 bp), TR-II (296 bp), GFR-1 (286 bp), and c-ret (185 bp). PCR products obtained from rat ventral mesencephalon, DRG and
purified motoneurons, and a 100 bp ladder are shown.
Arrows indicate the size of the PCR products.
|
|
View larger version (135K):
[in this window]
[in a new window]
|
Figure 6.
Immunohistochemistry showing localization of
TGF-2 (A, B), TGF-3
(C, D), and TR-II (E,
F) in chicken DRG (E8; A,
C, E) and in rat spinal cord motoneurons
(E14; B, D, F).
Note strong immunoreactivities in the cell bodies of DRG and
motoneurons. Asterisks mark the entrance of dorsal root
fibers into the ganglion. Scale bars, 50 µm.
|
|
Co-release of GDNF and TGF- from a model neuron, the
chromaffin cell
To further investigate the biological significance of the
cotrophic action of GDNF and TGF- we studied their putative
co-release. The best-established model system for studying secretion
from a neuron is the chromaffin cell (Neher and Marty, 1982; Burgoyne et al., 1989; Huttner et al., 1991; Unsicker, 1993;
Ferro-Novick and Jahn, 1994; Jahn and Südhoff, 1994). Chromaffin
cells are known to store within their secretory granules ("chromaffin
granules") and release after stimulation with a cholinergic agonist a
neurotrophic activity, which has been shown to promote in
vitro the survival of a number of neuron populations, including
chick ciliary ganglionic neurons (Fig. 7;
Unsicker and Lietzke, 1987; Lachmund et al., 1994; Unsicker and
Krieglstein, 1996). Few protein growth factors have been
identified in the past that can promote the survival of chick ciliary
ganglionic neurons in vitro in the absence of serum. These
are the ciliary neurotrophic factor (CNTF; Barbin et al., 1984),
growth-promoting activity (GPA; Eckenstein et al., 1990), possibly the
chick homolog of CNTF, and basic fibroblast growth factor (FGF-2;
Unsicker et al., 1987). Although CNTF and FGF-2 are synthesized by
chromaffin cells (Bieger et al., 1995; K. Krohn, personal
communication), previous investigations and the present study
reveal that both FGF-2 (Stachowiak et al., 1994; Bieger et al., 1995)
and CNTF (see below) are excluded from chromaffin granules. This
observation is consistent with the lack of a conventional signal
peptide in the FGF-2 and CNTF molecules that would target these
proteins to the rough endoplasmic reticulum and Golgi network for
secretion. Moreover, chromaffin granules do not contain any detectable
CNTF, as assayable using a highly sensitive CNTF-ELISA technique with a
detection limit of <0.3 ng/ml (Maysinger et al., 1996; data not
shown).
View larger version (33K):
[in this window]
[in a new window]
|
Figure 7.
The soluble proteins of chromaffin granules
(VP) promote the survival of chick ciliary ganglionic
neurons at a level identical to that achieved with a saturating
concentration of CNTF (10 ng/ml). Addition of neutralizing antibodies
to either GDNF (20 µg/ml) or the TGF-s TGF-1, TGF-2, and
TGF-3 (10 µg/ml) significantly reduces the promoting effect of VP.
Addition of both antibodies completely abolishes the neurotrophic
effect of VP (0.5 mg/ml), suggesting that GDNF and TGF- are the
long-sought ciliary neurotrophic proteins contained in VP. Data are
given as mean ± SEM (n = 6), p
values derived from Student's t test are
***p < 0.001 for decreased survival after antibody
treatment.
|
|
Having shown that (1) chromaffin granule lysates promote ciliary
ganglionic neuron survival, and (2) GDNF and TGF- can mimic the
neurotrophic effects of CNTF or FGF-2, respectively, on these neurons,
we asked whether GDNF and TGF- are synthesized, stored, and released
from chromaffin cells. Figure 8 shows
that transcripts for GDNF, TGF-1, TGF-2, and TGF-3 can be
detected by RT-PCR in bovine chromaffin cells. We next asked whether
GDNF was localized within chromaffin granules. Figure
9 shows immunolocalizations of the
chromaffin granule-specific marker chromogranin A (Winkler and
Fischer-Colbrie, 1992) and GDNF in rat adrenal chromaffin tissue (Fig.
9A) and cultured bovine chromaffin cells (Fig.
9B), suggesting that both immunoreactivities are colocalized
in chromaffin granules. Western blotting confirmed that protein lysates
from bovine chromaffin granules contained immunoreactive GDNF and
TGF-1 at their correct sizes (Fig.
10). We next analyzed supernatants from
cultured bovine chromaffin cells for the release of immunoreactive GDNF
and TGF- using immunopreciptation and Western blotting. As shown in
Figure 10, stimulation with the cholinergic agonist carbachol
(105 M, 15 min) induced release into
the culture supernatant of immunoprecipitable TGF-1 as well as GDNF,
suggesting their activity-dependent co-release from chromaffin
granules. Stimulation with carbachol in the presence of the
Ca2+ channel blocker verapamil reduced amounts of
TGF-1 and GDNF in culture supernatants by ~50% as determined by
densitometric evaluation of Western blots. Likewise, exposure of
chromaffin cells to the calcium ionophore A 23187 resulted in the
accumulation of TGF-1 and GDNF in culture supernatants. These data
suggest a regulated activity-dependent release of both TGF-1 and
GDNF from a neuronal secretory organelle, the chromaffin granule.
View larger version (84K):
[in this window]
[in a new window]
|
Figure 8.
RT-PCR analysis of bovine chromaffin cells
(lanes 3 and 4) using primers
specific for ribosomal S6 (293 bp), GDNF (415 bp), TGF-1 (279 bp),
TGF-2 (359 bp), and TGF-3 (291 bp). Bovine chromaffin cells were
analyzed after 18 hr of culture with lanes 3 and
4, representing two different RNA preparations. In
lane 2 RNA from cultured cortical astrocytes is
amplified. Lane 1 contains a negative control with water
instead of cDNA.
|
|
View larger version (27K):
[in this window]
[in a new window]
|
Figure 9.
Immunohistochemistry showing granular
localization of GDNF in rat adrenal (A) and
cultured bovine chromaffin (B) cells.
A, Confocal fluorescence microscopy of anti-chromogranin
A (monoclonal, green), anti-GDNF (polyclonal,
red), and its double detection
(yellow) using paraffin sections of embryonic rat
adrenals (E21). B, Fluorescence microscopy of
anti-chromogranin A (polyclonal, red), anti-GDNF
(monoclonal, green), and its double detection
(yellow) of bovine chromaffin cells after 6 d in culture.
|
|
View larger version (44K):
[in this window]
[in a new window]
|
Figure 10.
GDNF as well as TGF-1 are released from bovine
chromaffin cells after cholinergic stimulation. A,
Western blot showing immunoprecipitated TGF-1 from supernatants of
bovine chromaffin cells after a 15 min stimulation with medium only,
with a calcium ionophore A23187, carbachol plus verapamil, or carbachol
alone. For comparison, rhTGF-1 and protein extracts from isolated
bovine chromaffin granules (VP) were used. The
asterisks indicate the position of the primary antibody
used for immunoprecipitation. B, Western blot showing
immunoprecipitated GDNF from the same supernatants of bovine chromaffin
cells as used for A. For comparison, rhGDNF and VP were
used. Bands immunopositive for GDNF were quantified densitometrically
and given as arbitrary units.
|
|
Having shown that GDNF and TGF- are co-stored in and co-released
from chromaffin granules, we tested whether neutralizing antibodies to
either GDNF or TGF- would block the trophic activity in chromaffin
granule lysates for ciliary ganglionic neurons. Figure 7 provides
evidence that both antibodies when applied separately significantly
reduced the survival-promoting effect of chromaffin granule protein on
cultured ciliary ganglionic neurons. Neutralization of both GDNF and
TGF- activity completely abolished the activity. These results
suggest that GDNF and TGF- are the active "ciliary" neurotrophic
components stored in chromaffin granules. More importantly, they
underscore the presence, availability, and potency of the neurotrophic
activity resulting from the GDNF/TGF- synergism.
| |
DISCUSSION |
The novel scenario of GDNF-mediated neurotrophic actions
The present study identifies TGF-, which is widely expressed in
the developing and adult nervous system (Flanders et al., 1991;
Unsicker et al., 1991) as an essential component in GDNF-mediated neurotrophic actions. Initial studies with GDNF had suggested that the
protein fails to elicit neurotrophic effects on most highly purified
neuron populations in vitro (Henderson et al., 1994) but
promotes neuron survival if administered to complex culture systems
(Lin et al., 1993; Ebendal et al., 1995; Buj-Bello et al., 1995; Trupp
et al., 1995). Our findings broaden the definition of a neurotrophic
factor by incorporating the possibility that two factors that lack a
neurotrophic activity when acting separately become neurotrophic when
acting in concert. Our findings may also imply that activities of
established neurotrophic factors might result, entirely or in part,
from endogenously present cofactors. Previous studies have illustrated
that the magnitude of effects elicited by neurotrophic factors may be
substantially affected by endogenous factors (Krieglstein and Unsicker,
1996b).
The contextual actions of TGF-
TGF- is a member of a still growing superfamily of
multifunctional cytokines with prominent roles in development,
differentiation, and cell cycle control (for review, see Roberts and
Sporn, 1990; Alexandrow and Moses, 1995; Hogan, 1996). Three isoforms,
TGF-1, TGF-2, and TGF-3 have been identified in mammalian
tissues. In the developing and unlesioned adult nervous system TGF-1
expression seems to be restricted to meninges and choroid plexus,
whereas TGF-2 and TGF-3 are coexpressed in astroglial and Schwann
cells as well as in many populations of central and peripheral neurons, especially in long-range projecting neurons (for review, see
Krieglstein et al., 1995a). Current knowledge on functions of TGF-
in the nervous system is deduced from in vitro and in
vivo lesioning studies. The available mouse mutants that are
defective for either one of the three isoforms (Shull et al., 1992;
Kulkarni et al., 1993; Kaartinen et al., 1995; Proetzel et al., 1995;
Sanford et al., 1997) have failed to reveal overt deficits in neural
structure and function, possibly by virtue of compensatory effects of
the remaining isoforms. In contrast, mouse embryos deficient for the TGF- receptor type II (TRII; Oshima et al., 1996) are lethal at
E10.5, suggesting the general importance of TGF- signaling in
development. Functions of TGF- on neural cells in vitro
include regulation of extracellular matrix and proliferation of
astroglial cells (Flanders et al., 1993). TGF- can promote neuron
survival in vitro when administered to complex culture
systems (Krieglstein et al., 1995b), but shows little or no effect on
highly enriched or purified neurons (Krieglstein and Unsicker,
1996b; this study). All TGF- isoforms are known for the
strong contextuality of their actions (Nathan and Sporn, 1991).
Examples of contextual actions of TGF- on neural cells include
stimulation of proliferation of Schwann cells (Schubert, 1992) and
inhibition of astroglial cell proliferation in the absence and
presence, respectively, of FGF-2 (Flanders et al., 1993). The present
study adds a new dimension to the contextual potential of TGF- in
the nervous system by identifying TGF- as a molecule that crucially
determines the neurotrophic potential of GDNF.
Dependence of GDNF on the presence of a cofactor may not be surprising
in light of the reported neurotrophic potential of neurturin and
persephin, two recently discovered members of the GDNF subfamily
(Kotzbauer et al., 1996; Milbrandt et al., 1998). On dissociated rat
sympathetic neurons, neurturin is ineffective unless neurons are primed
with NGF (Creedon et al., 1997); persephin does not support peripheral
neurons at all (Milbrandt et al., 1998).
GDNF as a survival factor for peripheral and central neurons
GDNF is a distantly related member of the TGF- superfamily
classified by its structural characteristics (for review, see Lin et
al., 1993; Eigenbrot and Gerber, 1997; cf. Unsicker et al., 1998).
However, in contrast to TGF-, GDNF has been shown to employ for its
signaling a heteromeric tyrosine kinase receptor system that consists
of a GPI-linked -receptor (GFR, Jing et al., 1996; Treanor et
al., 1996) and the receptor tyrosine kinase Ret (Durbec et al., 1996;
Trupp et al., 1996). Striking similarities in the phenotypes of mice
deficient for GDNF (Moore et al., 1996; Pichel et al., 1996; Sanchez et
al., 1996) or its functional receptor Ret (Schuchardt et al.,
1994), respectively, suggest significant interdependence during
development. Kidney agenesis and the loss of the enteric nervous system
are the prominent features of the mice lacking either GDNF or its
receptor Ret. With regard to defects of the developing nervous system
deficits in the number of lumbar and trigeminal, but not facial
motoneurons, as well as trigeminal sensory, nodose-petrosal, dorsal
root ganglionic neurons, and sympathetic neurons have been reported
(Moore et al., 1996; Pichel et al., 1996). Surprisingly, the number of
TH-immunoreactive neurons in the substantia nigra and the density of
dopaminergic nerve terminals in the striatum seemed to be unaffected.
The present study underscores the developmental requirements of several
neuron populations for GDNF. With regard to dopaminergic neurons GDNF does not seem to be a major survival factor for prenatal neurons, but
may be more important in postnatal maturation of the nigrostriatal system. However, our analysis also shows that dopaminergic neurons, like the peripheral neurons tested, essentially require both GDNF and
its cofactor TGF-.
Molecular bases underlying the GDNF/TGF- cooperativity
Synergisms of TGF- and other cytokines, most notably FGF-2, in
regulating a variety of developmental processes, are well documented
in vitro. Thus, TGF- and FGF-2 synergistically promote early embryonic development in the fourth cell cycle (Larson et al.,
1992), induce chondrogenesis (Frenz et al., 1994) and angiogenesis (Gajdusek et al., 1993), and, most recently, have been shown to mediate
myogenic signals from the neural tube (Stern et al., 1997). However, in
none of the above scenarios the underlying molecular mechanisms have
been reported. Our study suggests that TGF-/GDNF cooperativity may
occur at two crucial steps of signal transduction. First, an important
component of GDNF/TGF- signaling has been identified as an
activation of PI-3 kinase. GDNF-induced Ret signaling has been shown to
activate at least two distinct pathways, the Ras-ERK2 and a PI-3
kinase-involving pathway (van Weering and Bos, 1997). Our data suggest
that the PI-3 kinase pathway, which previously has been shown to
mediate lamellipodia formation and probably neuritogenesis (van Weering
and Bos, 1997), is also important for mediating neuron survival. PI-3
kinase has also been implicated in neurotrophin-mediated neuron
survival (Kaplan and Miller, 1997). Second, our data point at another
important aspect of GDNF/TGF- cooperativity, the stabilization
and/or recruitment of the GPI-linked GDNF receptor GFR (Jing et
al., 1996; Treanor et al., 1996) by TGF- after PIPLC-mediated
hydrolysis of the GPI anchor. Finally, our results might suggest a role
for TGF- in recruiting GFR onto cells, which exclusively express
c-ret (Yu et al., 1998).
Biological significance of the GDNF/TGF- cooperativity
The classic neurotrophic factor concept implies that relevant
players are synthesized by the innervated target and retrogradely transported to the cell soma where they exert their functions (Levi-Montalcini, 1987; Thoenen et al., 1987). With regard to GDNF, its
synthesis in the target areas of responding neuron populations (Table
1) as well as retrograde transport (Yan et al., 1995; Tomac et al.,
1995b) have been well documented. TGF-, on the other hand,
represents a local factor acting in paracrine and/or autocrine manners
(Sporn and Roberts, 1992; Krieglstein and Unsicker, 1996b; Table 1).
TGF-2 and TGF-3 proteins are localized in close proximity of as
well as in neurons within developing ganglia of the peripheral nervous
system, motoneurons in the ventral spinal cord, and dopaminergic
neurons in the ventral mesencephalon (Table 1). Studies aiming at
revealing a retrograde axonal transport of TGF- have failed so far
(Blottner et al., 1996). Thus, a putative scenario for TGF-
and GDNF cooperativity could employ retrograde signaling by GDNF in
synergy with local actions of TGF-. In addition, both TGF- and
GDNF may be derived from identical cellular sources, e.g., cortical and
hippocampal neurons (Unsicker et al., 1991; Pochon et al., 1997). The
well documented colocalization of TGF- and GDNF in these neurons was
the rationale for using a model neuron, the neuroendocrine chromaffin
cell, for further characterization of the release mechanism. In
particular, chromaffin cells permitted us to use the endogenous stores
of GDNF and TGF- rather than overexpressing the growth factors.
Consistent with the notion that growth factors are acknowledged
modulators of synaptic plasticity (Korte and Bonhoeffer, 1997), our
data provide evidence that GDNF and TGF- both released from
chromaffin granules after cholinergic stimulation in a
calcium-dependent manner.
In summary, we have shown that neurotrophic effects that have been
attributed to GDNF result from a synergism of GDNF with TGF-. Our
findings have implications for a general understanding of mechanisms
underlying regulation of neuron survival in development and disease,
suggesting synergisms of growth factors as an important perspective in
future studies. Use of GDNF in clinical trials may benefit from
coadministration of TGF- or increasing levels of endogenous TGF-.
Along the same line, effects of heteromeric GDNF/TGF- complexes may
also be exploited for therapeutic interventions.
| |
FOOTNOTES |
Received March 10, 1998; revised Sept. 16, 1998; accepted Sept. 17, 1998.
This work was supported by grants from Deutsche Forschungsgemeinschaft
(SFB 317/D4, Un34/19-1-A2, and Un34/20-1) and BioMedII (BMH4-97-215).
We thank Drs. D. Rifkin (New York University), W. Huttner (University
of Heidelberg), V. Rosen (Genetics Institute, Cambridge, MA), and
M. B. Sporn (Dartmouth Medical College, Hanover, NH). We also
thank Ms. Jutta Fey, Ulla Hinz, Daniela Oguntke, and Ingrid Stenull for
excellent technical assistance.
Correspondence should be addressed to Dr. Kerstin Krieglstein,
Department of Neuroanatomy, University of Heidelberg, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany.
| |
REFERENCES |
-
Abe M,
Harpel JG,
Metz CN,
Nunes I,
Loskutoff DJ,
Rifkin DB
(1994)
An assay for transforming growth factor- using cells transfected with a plasminogen activator inhibitor-1 promotor-luciferase construct.
Anal Biochem
216:276-284[Web of Science][Medline].
-
Alexandrow MG,
Moses HL
(1995)
Transforming growth factor and cell cycle regulation.
Cancer Res
55:1452-1457[Free Full Text].
-
Arenas E,
Trupp M,
Akerud P,
Ibanez CF
(1995)
GDNF prevents degeneration and promotes the phenotype of brain noradrenergic neurons in vivo.
Neuron
15:1465-1473[Web of Science][Medline].
-
Avantaggiato V,
Dathan NA,
Grieco M,
Fabien N,
Lazzaro D,
Fusco A,
Simeone A,
Santoro M
(1994)
Developmental expression of the RET protooncogene.
Cell Growth Differ
5:305-311[Abstract].
-
Baloh RH,
Tansey MG,
Golden JP,
Creedon DJ,
Heuckeroth RO,
Keck CL,
Zimonjic DB,
Popescu NC,
Johnson Jr EM,
Milbrandt J
(1997)
TrnR2, a novel receptor that mediates neurturin and GDNF signaling through Ret.
Neuron
18:793-802[Web of Science][Medline].
-
Barbin G,
Manthorpe M,
Varon S
(1984)
Purification of the chick eye ciliary neurotrophic factor.
J Neurochem
43:1468-1478[Web of Science][Medline].
-
Beck KD,
Valverde J,
Alexi T,
Poulsen K,
Moffat B,
Vandlen RA,
Rosenthal A,
Hefti F
(1995)
Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain.
Nature
373:339-341[Medline].
-
Bennett DL,
Michael GJ,
Ramachandran N,
Munson JB,
Averill S,
Yan Q,
McMahon SB,
Priestley JV
(1998)
A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurons after nerve injury.
J Neurosci
18:3059-3072[Abstract/Free Full Text].
-
Bieger SC,
Henkel AW,
Unsicker K
(1995)
Localization of basic fibroblast growth factor in bovine adrenal chromaffin cells.
J Neurochem
64:1521-1527[Web of Science][Medline].
-
Blottner D,
Wolf N,
Lachmund A,
Flanders KC,
Unsicker K
(1996)
TGF-beta rescues target-deprived preganglionic sympathetic neurons in the spinal cord.
Eur J Neurosci
8:202-210[Web of Science][Medline].
-
Bottenstein J,
Skaper S,
Varon S,
Sato G
(1980)
Selective survival of chick sensory ganglionic cultures utilizing serum-free supplement medium.
Exp Cell Res
125:183-190[Web of Science][Medline].
-
Buj-Bello A,
Buchman VL,
Horton A,
Rosenthal A,
Davies AM
(1995)
GDNF is an age-specific survival factor for sensory and autonomic neurons.
Neuron
15:821-828[Web of Science][Medline].
-
Burgoyne RD,
Morgan A,
O'Sullivan AJ
(1989)
The control of cytoskeletal actin and exocytosis in intact and permeabilized adrenal chromaffin cells: role of calcium and protein kinase C.
Cell Signal
1:323-334[Web of Science][Medline].
-
Camu W,
Henderson CE
(1992)
Purification of embryonic rat motoneurons by panning on a monoclonal antibody to the low-affinity NGF receptor.
J Neurosci Methods
44:59-70[Web of Science][Medline].
-
Chalazonitis A,
Kalberg J,
Twardzik DR,
Morrison RS,
Kessler JA
(1992)
Transforming growth factor beta has neurotrophic actions on sensory neurons in vitro and is synergistic with nerve growth factor.
Dev Biol
152:121-132[Web of Science][Medline].
-
Creedon DJ,
Tansey MG,
Baloh RH,
Osborne PA,
Lampe PA,
Fahrner TJ,
Heuckeroth RO,
Milbrandt J,
Johnson Jr EM
(1997)
Neurturin shares receptors and signal transduction pathways with glial cell line-derived neurotrophic factor in sympathetic neurons.
Proc Natl Acad Sci USA
94:7018-7023[Abstract/Free Full Text].
-
Durbec P,
Marcos-Gutierrez CV,
Kilkenny C,
Grigoriou M,
Wartiowaara K,
Suvanto P,
Smith D,
Ponder B,
Costantini F,
Saarma M,
Sariola H,
Pachnis V
(1996)
GDNF signalling through the ret receptor tyrosine kinase.
Nature
381:789-793[Medline].
-
Ebendal T,
Tomac A,
Hoffer BJ,
Olson L
(1995)
Glial cell line-derived neurotrophic factor stimulates fiber formation and survival in cultured neurons from peripheral autonomic ganglia.
J Neurosci Res
40:276-284[Web of Science][Medline].
-
Eckenstein FP,
Esch F,
Holbert T,
Blacher RW,
Nishi R
(1990)
Purification and characterization of a trophic factor for embryonic peripheral neurons: comparison with fibroblast growth factors.
Neuron
4:623-631[Web of Science][Medline].
-
Eigenbrot C,
Gerber N
(1997)
X-ray structure of glial cell-derived neurotrophic factor at 1.9A resolution and implications for receptor binding.
Nat Struct Biol
4:435-438[Web of Science][Medline].
-
Farkas L,
Suter-Crazzolara C,
Unsicker K
(1997)
GDNF induces the calretinin phenotype in cultures of embryonic striatal neurons.
J Neurosci Res
50:361-372[Web of Science][Medline].
-
Ferro-Novick S,
Jahn R
(1994)
Vesicle fusion from yeast to man.
Nature
370:191-193[Medline].
-
Flanders KC,
Lüdecke G,
Engels S,
Cissel DS,
Roberts AB,
Kondaiah P,
Lafyatis R,
Sporn MB,
Unsicker K
(1991)
Immunohistochemical localization of transforming growth factor-s in the nervous system of the mouse embryo.
Development
113:183-191[Abstract].
-
Flanders KC,
Lüdecke G,
Renzig J,
Hamm C,
Cissel DS,
Unsicker K
(1993)
Effect of TGF-s and bFGF on astroglial cell growth and gene expression in vitro.
Mol Cell Neurosci
4:406-417.
-
Frenz DA,
Liu W,
Williams JD,
Hatcher V,
Galinovic-Schwartz V,
Flanders KC,
Van de Water TR
(1994)
Induction of chondrogenesis: requirement for synergistic interaction of basic fibroblast growth factor and transforming growth factor-beta.
Development
120:415-424[Abstract].
-
Gajdusek CM,
Luo Z,
Mayberg MR
(1993)
Basic fibroblast growth factor and transforming growth factor beta-1: synergistic mediators of angiogenesis in vitro.
J Cell Physiol
157:133-144[Web of Science][Medline].
-
Gash DM,
Zhang Z,
Ovadia A,
Cass WA,
Yi A,
Simmerman L,
Russell D,
Martin D,
Lapchak PA,
Collins F,
Hoffer BJ,
Gerhardt GA
(1996)
Functional recovery in parkinsonian monkeys treated with GDNF.
Nature
380:252-255[Medline].
-
Heller S,
Finn TP,
Huber J,
Nishi R,
Geissen M,
Puschel AW,
Rohrer H
(1995)
Analysis of function and expression of the chick GPA receptor (GPAR alpha) suggests multiple roles in neuronal development.
Development
121:2681-2693[Abstract].
-
Henderson CE,
Phillips HS,
Pollock RA,
Davies AM,
Lemeulle C,
Armanini MP,
Simpson LC,
Moffet B,
Vandlen RA,
Koliatsos VE,
Rosenthal A
(1994)
GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle.
Science
266:1062-1064[Abstract/Free Full Text].
-
Hoffer BJ,
Hoffman A,
Bowenkamp K,
Huettl P,
Hudson J,
Martin D,
Lin LFH,
Gerhardt GA
(1994)
Glial cell line-derived neurotrophic factor reverses toxin-induced injury to midbrain dopaminergic neurons in vivo.
Neurosci Lett
182:107-111[Web of Science][Medline].
-
Hogan BL
(1996)
Bone morphogenetic proteins: multifunctional regulators of vertebrate development.
Genes Dev
10:1580-1594[Free Full Text].
-
Huttner WB,
Gerdes H-H,
Rosa P
(1991)
Chromogranins/secretogranins: widespread constituents of the secretory granule matrix in endocrine cells and neurons.
In: Markers from neural and endocrine cells: molecular and cell biology, diagnostic applications (Gratzl M,
ed), pp 93-131. Weinheim: VCH.
-
Jahn R,
Südhoff TC
(1994)
Synaptic vesicles and exocytosis.
Annu Rev Neurosci
17:219-246[Web of Science][Medline].
-
Jing S,
Wen D,
Yu Y,
Holst PL,
Luo Y,
Fang M,
Tamir R,
Antonio L,
Hu Z,
Cupples R,
Louis JC,
Hu S,
Altrock BW,
Fox GM
(1996)
GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF.
Cell
85:1113-1124[Web of Science][Medline].
-
Kaartinen V,
Voncken JW,
Shuler C,
Warburton D,
Bu D,
Heisterkamp N,
Groffen J
(1995)
Abnormal lung development and cleft palate in mice lacking TGF- 3 indicates defects of epithelial-mesenchymal interaction.
Nat Genet
11:415-421[Web of Science][Medline].
-
Kaplan DR,
Miller FD
(1997)
Signal transduction by the neurotrophin receptors.
Curr Opin Cell Biol
9:213-221[Web of Science][Medline].
-
Kingsley DM
(1994)
The TGF- superfamily: new members, new receptors, and new genetic tests of function in different organisms.
Genes Dev
8:133-146[Free Full Text].
-
Korte M,
Bonhoeffer T
(1997)
Activity-dependent synaptic plasticity: a new face of action for neurotrophins.
Mol Psychiatry
2:197-199[Web of Science][Medline].
-
Kotzbauer PT,
Lampe PA,
Heuckeroth RO,
Golden JP,
Creedon DJ,
Johnson Jr EM,
Milbrandt J
(1996)
Neurturin, a relative of glial-cell-line-derived neurotrophic factor.
Nature
384:467-470[Medline].
-
Krieglstein K,
Unsicker K
(1994)
Transforming growth factor- promotes survival of midbrain dopaminergic neurons and protects them against N-methyl-4-phenylpyridinium ion toxicity.
Neuroscience
63:1189-1196[Web of Science][Medline].
-
Krieglstein K,
Unsicker K
(1995)
Bovine chromaffin granules contain and release a transforming growth factor--like activity.
J Neurochem
65:1423-1426[Web of Science][Medline].
-
Krieglstein K,
Unsicker K
(1996a)
Proteins from chromaffin granules promote survival of dorsal root ganglionic neurons: comparison with neurotrophins.
Dev Brain Res
93:10-17[Medline].
-
Krieglstein K,
Unsicker K
(1996b)
Distinct modulatory actions of TGF- and LIF on neurotrophin-mediated survival of developing sensory neurons.
Neurochem Res
21:849-856.
-
Krieglstein K,
Unsicker K
(1997)
Proteins from chromaffin granules promote survival of mesencephalic dopaminergic neurons by an EGF-receptor ligand-mediated pathway.
J Neurosci Res
48:18-30[Web of Science][Medline].
-
Krieglstein K,
Rufer M,
Suter-Crazzolara C,
Unsicker K
(1995a)
Neural functions of the transforming growth factor .
Int J Dev Neurosci
13:301-315[Web of Science][Medline].
-
Krieglstein K,
Suter-Crazzolara C,
Fischer WH,
Unsicker K
(1995b)
TGF- superfamily members promote survival of midbrain dopaminergic neurons and protect them against MPP+ toxicity.
EMBO J
14:736-742[Web of Science][Medline].
-
Krieglstein K,
Deimling F,
Suter-Crazzolara C,
Unsicker K
(1996)
Expression and localization of GDNF in developing and adult adrenal chromaffin cells.
Cell Tissue Res
286:263-268[Web of Science][Medline].
-
Krieglstein K, Farkas L, Unsicker K (1998) TGF- regulates
the survival of ciliary ganglionic neurons synergistically with ciliary
neurotrophic factor and neurotrophins. J Neurobiol, in
press.
-
Kulkarni AB,
Huh C-G,
Becker D,
Geiser A,
Lyght M,
Flanders KC,
Roberts AB,
Sporn MB,
Ward JM,
Karlsson S
(1993)
Transforming growth factor 1 null mutation in mice causes excessive inflammatory response and early death.
Proc Natl Acad Sci USA
90:770-774[Abstract/Free Full Text].
-
Lachmund A,
Gehrke D,
Krieglstein K,
Unsicker K
(1994)
Trophic factors from chromaffin granules promote survival of peripheral and central nervous system neurons.
Neuroscience
62:361-370[Web of Science][Medline].
-
Larson RC,
Ignotz GG,
Currie WB
(1992)
Transforming growth factor and basic fibroblast growth factor synergistically promote early bovine embryo development during the fourth cell cycle.
Mol Reprod Dev
33:432-435[Web of Science][Medline].
-
Levi-Montalcini R
(1987)
The nerve growth factor 35 years later.
Science
237:1154-1162[Free Full Text].
-
Lin LFH,
Doherty DH,
Lile JD,
Bektesh S,
Collins F
(1993)
GDNF - a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons.
Science
260:1130-1132[Abstract/Free Full Text].
-
Martinou J-C,
le van Thai A,
Valette A,
Weber MJ
(1990)
Transforming growth factor b1 is a potent survival factor for rat embryo motoneurons in culture.
Dev Brain Res
52:175-181[Medline].
-
Maysinger D,
Krieglstein K,
Filipovic-Grcic J,
Sendtner M,
Unsicker K,
Richardson P
(1996)
Microencapsulated ciliary neurotrophic factor: physical properties and biological activities.
Exp Neurol
138:177-188[Web of Science][Medline].
-
Milbrandt J,
de Sauvage FJ,
Fahrner TJ,
Baloh RH,
Leitner ML,
Tansey MG,
Lampe PA,
Heuckeroth RO,
Kotzbauer PT,
Simburger KS,
Golden JP,
Davies JA,
Vejsada R,
Kato AC,
Hynes M,
Sherman D,
Nishimura M,
Wang L-C,
Vandlen R,
Moffat B,
Klein RD,
Poulsen K,
Gray C,
Garces A,
Henderson CE,
Philips HS,
Johnson Jr EM
(1998)
Persephin, a novel neurotrophic factor related to GDNF and neurturin.
Neuron
20:245-253[Web of Science][Medline].
-
Moore MW,
Klein RD,
Farinas I,
Sauer H,
Armanini M,
Phillips H,
Reichardt LF,
Ryan AM,
Carver-Moore K,
Rosenthal A
(1996)
Renal and neuronal abnormalities in mice lacking GDNF.
Nature
382:76-79[Medline].
-
Nakamura M,
Ohta K,
Hirokawa K,
Fukushima M,
Uchino M,
Ando M,
Tanaka H
(1996)
Developmental and denervation changes in c-ret proto-oncogene expression in chick motoneurons.
Brain Res Mol Brain Res
39:1-11[Medline].
-
Nathan C,
Sporn M
(1991)
Cytokines in context.
J Cell Biol
113:981-986[Free Full Text].
-
Neher E,
Marty A
(1982)
Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells.
Proc Natl Acad Sci USA
79:6712-6716[Abstract/Free Full Text].
-
Nosrat CA,
Tomac A,
Lindqvist E,
Lindskog S,
Humpel C,
Stromberg I,
Ebendal T,
Hoffer BJ,
Olson L
(1996)
Cellular expression of GDNF mRNA suggests multiple functions inside and outside the nervous system.
Cell Tissue Res
286:191-207[Web of Science][Medline].
-
Oshima M,
Oshima H,
Taketo MM
(1996)
TGF- receptor type II deficiency results in defects of yolk sac hematopoiesis and vasculogenesis.
Dev Biol
179:297-302[Web of Science][Medline].
-
Pachnis V,
Mankoo B,
Costantini F
(1993)
Expression of the c-ret proto-oncogene during mouse embryogenesis.
Development
119:1005-1017[Abstract].
-
Pichel JP,
Shen L,
Sheng HZ,
Granholm A-C,
Drago J,
Grinberg A,
Lee EJ,
Huang SP,
Saarma M,
Hoffer BJ,
Sariola H,
Westphal H
(1996)
Defects in enteric innervation and kidney development in mice lacking GDNF.
Nature
382:73-76[Medline].
-
Pochon NA-M,
Menoud A,
Tseng JL,
Zurn AD,
Aebischer P
(1997)
Neuronal GDNF expression in the adult rat nervous system identified by in situ hybridization.
Eur J Neurosci
9:463-471[Web of Science][Medline].
-
Poulsen KT,
Armanini MP,
Klein RD,
Hynes MA,
Phillips HS,
Rosenthal A
(1994)
TGF2 and TGF3 are potent survival factors for midbrain dopaminergic neurons.
Neuron
13:1245-1252[Web of Science][Medline].
-
Proetzel G,
Pawlowski SA,
Wiles MV,
Yin M,
Boivin GP,
Howles PN,
Ding J,
Ferguson MW,
Doetschman T
(1995)
Transforming growth factor-beta 3 is required for secondary palate fusion.
Nat Genet
11:409-414[Web of Science][Medline].
-
Roberts AB,
Sporn MB
(1990)
The transforming growth factors-s.
In: Handbook of experimental pharmacology, Vol 95 (Sporn MB,
Roberts AB,
eds), pp 419-472. Heidelberg: Springer.
-
Sanchez MP,
Silos-Santiago I,
Frisen J,
He B,
Lira SA,
Barbacid M
(1996)
Renal agenesis and the absence of enteric neurons in mice lacking GDNF.
Nature
382:70-73[Medline].
-
Sanford LP,
Ormsby I,
Gittenberger de Groot AC,
Sariola H,
Friedman R,
Boivin GP,
Cardell EL,
Doetschman T
(1997)
TGF-2 knockout mice have multiple developmental defects that are non-overlapping with other TGF knockout phenotypes.
Development
124:2659-2670[Abstract].
-
Sauer H,
Rosenblad C,
Björklund A
(1995)
GDNF but not TGF-3 prevents delayed degeneration of nigral dopaminergic neurons following striatal 6-hydroxydopamine lesion.
Proc Natl Acad Sci USA
92:8935-8939[Abstract/Free Full Text].
-
Schubert D
(1992)
Synergistic interactions between transforming growth factor beta and fibroblast growth factor regulate Schwann cell mitosis.
J Neurobiol
23:143-148[Web of Science][Medline].
-
Schuchardt A,
D'Agati V,
Larsson-Blomberg L,
Costantini F,
Pachnis V
(1994)
Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret.
Nature
367:380-383[Medline].
-
Shull MM,
Ormsby I,
Kier AB,
Pawlowski S,
Diebold RJ,
Yin M,
Allen R,
Sidman C,
Proetzel G,
Calvin D,
Annunziata N,
Doetschman T
(1992)
Targeted disruption of the mouse transforming growth factor-1 gene results in multifocal inflammatory disease.
Nature
359:693-699[Medline].
-
Sporn MB,
Roberts AB
(1992)
Autocrine Secretion: 10 years later.
Ann Intern Med
117:408-414.
-
Stachowiak MK,
Moffett J,
Joy A,
Puchacz E,
Florkiewicz R,
Stachowiak EK
(1994)
Regulation of bFGF gene expression and subcellular distribution of bFGF protein in adrenal medullary cells.
J Cell Biol
127:203-223[Abstract/Free Full Text].
-
Stern HM,
Lin-Jones J,
Hauschka SD
(1997)
Synergistic interactions between bFGF and a TGF- family member may mediate myogenic signals from neural tube.
Development
124:3511-3523[Abstract].
-
Thoenen H,
Bandtlow C,
Heumann R
(1987)
The physiological function of nerve growth factor in the central nervous system: comparison with the periphery.
Rev Physiol Biochem Pharmacol
109:145-178[Web of Science][Medline].
-
Tomac A,
Lindqvist E,
Lin LFH,
Ögren SO,
Young D,
Hoffer BJ,
Olson L
(1995a)
Protection and repair of the nigrostriatal dopaminergic system by GDNF in vivo.
Nature
373:335-339[Medline].
-
Tomac A,
Widenfalk J,
Lin LF,
Kohno T,
Ebendal T,
Hoffer BJ,
Olson L
(1995b)
Retrograde axonal transport of glial cell line-derived neurotrophic factor in the adult nigrostriatal system suggests a trophic role in the adult.
Proc Natl Acad Sci USA
92:8274-8278[Abstract/Free Full Text].
-
Treanor JJS,
Goodman L,
de Sauvage F,
Stone DM,
Poulsen KT,
Beck CD,
Gray C,
Armanini MP,
Pollock RA,
Hefti F,
Phillips HS,
Goddard A,
Moore MW,
Buj-Bello A,
Davies AM,
Asai N,
Takahashi M,
Vandlen R,
Henderson CE,
Rosenthal A
(1996)
Characterization of a multicomponent receptor for GDNF.
Nature
382:80-83[Medline].
-
Trupp M,
Ryden M,
Jornvall H,
Funakoshi H,
Timmusk T,
Arenas E,
Ibanez CF
(1995)
Peripheral expression and biological activities of GDNF, a new neurotrophic factor for avian and mammalian peripheral neurons.
J Cell Biol
130:137-148[Abstract/Free Full Text].
-
Trupp M,
Arenas E,
Fainzilber M,
Nilsson AS,
Sieber BA,
Grigoriou M,
Kilkenny C,
Salazar-Grueso E,
Pachnis V,
Arumäe U,
Sariola H,
Saarma M,
Ibanez CF
(1996)
Functional receptor for GDNF encoded by the c-ret proto-oncogene.
Nature
381:785-789[Medline].
-
Trupp M,
Belluardo N,
Funakoshi H,
Ibanez CF
(1997)
Complementary and overlapping expression of glial cell line-derived neurotrophic factor (GDNF), c-ret proto-oncogene, and GDNF receptor-alpha indicates multiple mechanisms of trophic actions in the adult rat CNS.
J Neurosci
17:3554-3567[Abstract/Free Full Text].
-
Tsuzuki T,
Takahashi M,
Asai N,
Iwashita T,
Matsuyama M,
Asai J
(1995)
Spatial and temporal expression of the ret proto-oncogene product in embryonic, infant and adult rat tissues.
Oncogene
10:191-198[Web of Science][Medline].
-
Unsicker K
(1993)
The chromaffin cell: paradigm in cell development and growth factor biology.
J Anat
183:207-221.
-
Unsicker K,
Krieglstein K
(1996)
Growth factors in chromaffin cells.
Prog Neurobiol
48:307-324[Web of Science][Medline].
-
Unsicker K,
Lietzke R
(1987)
Chromaffin cells: modified neurons that are both targets and storage sites of neuronotrophic and neurite promoting factors.
In: Glial-neuronal communication in development and regeneration (Althaus H,
Seifert W,
eds), pp 365-384. Heidelberg: Springer.
-
Unsicker K,
Griesser GH,
Lindmar R,
Löffelholz U,
Wolf U
(1980)
Establishment, characterization and fibre outgrowth of isolated bovine adrenal medullary cells in long-term cultures.
Neuroscience
5:1445-1460[Web of Science][Medline].
-
Unsicker K,
Reichert-Preibsch H,
Schmidt R,
Pettmann B,
Labourdette G,
Sensenbrenner M
(1987)
Astroblast and fibroblast growth factors have neurotrophic functions for peripheral and central nervous system neurons.
Proc Natl Acad Sci USA
84:5459-5463[Abstract/Free Full Text].
-
Unsicker K,
Flanders KC,
Cissel DS,
Lafyatis R,
Sporn MB
(1991)
Transforming growth factor beta isoforms in the adult rat central and peripheral nervous system.
Neuroscience
44:613-625[Web of Science][Medline].
-
Unsicker K,
Meier C,
Krieglstein K,
Sartor BM,
Flanders KC
(1996)
Expression, localization, and function of transforming growth factor-s in embryonic chick spinal cord, hindbrain, and dorsal root ganglia.
J Neurobiol
29:262-276[Web of Science][Medline].
-
Unsicker K,
Suter-Crazzolara C,
Krieglstein K
(1998)
Neurotrophic roles of GDNF and related factors.
In: Neurotrophic factors, handbook of experimental pharmacology, Vol 134 (Hefti F,
ed), pp 189-224. Heidelberg: Springer.
-
van Weering DHJ,
Bos JL
(1997)
Glial cell line-derived neurotrophic factor induces ret-mediated lamellipodia formation.
J Biol Chem
272:249-254[Abstract/Free Full Text].
-
Widenfalk J,
Nosrat C,
Tomac A,
Westphal H,
Hoffer B,
Olson L
(1997)
Neurturin and glial cell line-derived neurotrophic factor receptor- (GDNFR-), novel proteins related to GDNF and GDNFR- with specific cellular patterns of expression suggesting roles in the developing and adult nervous system and in peripheral organs.
J Neurosci
17:8506-8519[Abstract/Free Full Text].
-
Winkler H,
Fischer-Colbrie R
(1992)
The chromogranins A and B: the first 25 years and future perspectives.
Neuroscience
49:497-528[Web of Science][Medline].
-
Wright DE,
Snider WD
(1996)
Focal expression of glial cell line-derived neurotrophic factor in developing mouse limb bud.
Cell Tissue Res
286:209-217[Web of Science][Medline].
-
Yan Q,
Matheson C,
Lopez OT
(1995)
In vivo neurotrophic effects of GDNF on neonatal and adult facial motor neurons.
Nature
373:341-344[Medline].
-
Yu T,
Scully S,
Yu Y,
Fox GM,
Jing S,
Zhou R
(1998)
Expression of GDNF family receptor components during development: implications in the mechanisms of interaction.
J Neurosci
18:4684-4696[Abstract/Free Full Text].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18239822-13$05.00/0
This article has been cited by other articles:
|
|
|
|
 
J. Strelau, A. Strzelczyk, P. Rusu, G. Bendner, S. Wiese, F. Diella, A. L. Altick, C. S. von Bartheld, R. Klein, M. Sendtner, et al.
Progressive Postnatal Motoneuron Loss in Mice Lacking GDF-15
J. Neurosci.,
October 28, 2009;
29(43):
13640 - 13648.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Song, G. Chen, Y. Wang, G. Mao, Y. Wang, and H. Bai
Chemically defined sequential culture media for TH+ cell derivation from human embryonic stem cells
Mol. Hum. Reprod.,
November 1, 2008;
14(11):
619 - 625.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Feng and C.-P. Ko
Schwann Cells Promote Synaptogenesis at the Neuromuscular Junction via Transforming Growth Factor-{beta}1
J. Neurosci.,
September 24, 2008;
28(39):
9599 - 9609.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Murase and R. D. McKay
A specific survival response in dopamine neurons at most risk in Parkinson's disease.
J. Neurosci.,
September 20, 2006;
26(38):
9750 - 9760.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Vargas-Leal, R. Bruno, T. Derfuss, M. Krumbholz, R. Hohlfeld, and E. Meinl
Expression and Function of Glial Cell Line-Derived Neurotrophic Factor Family Ligands and Their Receptors on Human Immune Cells
J. Immunol.,
August 15, 2005;
175(4):
2301 - 2308.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. M. Farkas, N. Dunker, E. Roussa, K. Unsicker, and K. Krieglstein
Transforming Growth Factor-{beta}s Are Essential for the Development of Midbrain Dopaminergic Neurons In Vitro and In Vivo
J. Neurosci.,
June 15, 2003;
23(12):
5178 - 5186.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Subramaniam, J. Strelau, and K. Unsicker
Growth Differentiation Factor-15 Prevents Low Potassium-induced Cell Death of Cerebellar Granule Neurons by Differential Regulation of Akt and ERK Pathways
J. Biol. Chem.,
March 7, 2003;
278(11):
8904 - 8912.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. M. Dhandapani, V. B. Mahesh, and D. W. Brann
Astrocytes and Brain Function: Implications for Reproduction
Experimental Biology and Medicine,
March 1, 2003;
228(3):
253 - 260.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Peterziel, K. Unsicker, and K. Krieglstein
TGF{beta} induces GDNF responsiveness in neurons by recruitment of GFR{alpha}1 to the plasma membrane
J. Cell Biol.,
October 14, 2002;
159(1):
157 - 167.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Grammas and R. Ovase
Cerebrovascular Transforming Growth Factor-{beta} Contributes to Inflammation in the Alzheimer's Disease Brain
Am. J. Pathol.,
May 1, 2002;
160(5):
1583 - 1587.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. F. Espejo, M. C. Gonzalez-Albo, J.-P. Moraes, F. El Banoua, J. A. Flores, and I. Caraballo
Functional Regeneration in a Rat Parkinson's Model after Intrastriatal Grafts of Glial Cell Line-Derived Neurotrophic Factor and Transforming Growth Factor beta 1-Expressing Extra-Adrenal Chromaffin Cells of the Zuckerkandl's Organ
J. Neurosci.,
December 15, 2001;
21(24):
9888 - 9895.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Boillee, J. Cadusseau, M. Coulpier, G. Grannec, and M.-P. Junier
Transforming Growth Factor {alpha}: A Promoter of Motoneuron Survival of Potential Biological Relevance
J. Neurosci.,
September 15, 2001;
21(18):
7079 - 7088.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Dunker, N. Schuster, and K. Krieglstein
TGF-{beta} modulates programmed cell death in the retina of the developing chick embryo
Development,
June 1, 2001;
128(11):
1933 - 1942.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. Nicole, C. Ali, F. Docagne, L. Plawinski, E. T. MacKenzie, D. Vivien, and A. Buisson
Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway
J. Neurosci.,
May 1, 2001;
21(9):
3024 - 3033.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. T. Erickson, T. A. Brosenitsch, and D. M. Katz
Brain-Derived Neurotrophic Factor and Glial Cell Line-Derived Neurotrophic Factor Are Required Simultaneously for Survival of Dopaminergic Primary Sensory Neurons In Vivo
J. Neurosci.,
January 15, 2001;
21(2):
581 - 589.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Strelau, A. Sullivan, M. Bottner, P. Lingor, E. Falkenstein, C. Suter-Crazzolara, D. Galter, J. Jaszai, K. Krieglstein, and K. Unsicker
Growth/Differentiation Factor-15/Macrophage Inhibitory Cytokine-1 Is a Novel Trophic Factor for Midbrain Dopaminergic Neurons In Vivo
J. Neurosci.,
December 1, 2000;
20(23):
8597 - 8603.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. J. Symes, R. L. Pitts, J. Conover, K. Kos, and J. Coulombe
Synergy of Activin and Ciliary Neurotrophic Factor Signaling Pathways in the Induction of Vasoactive Intestinal Peptide Gene Expression
Mol. Endocrinol.,
March 1, 2000;
14(3):
429 - 439.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
D. Worley, J. Pisano, E. Choi, L Walus, C. Hession, R. Cate, M Sanicola, and S. Birren
Developmental regulation of GDNF response and receptor expression in the enteric nervous system
Development,
January 10, 2000;
127(20):
4383 - 4393.
[Abstract]
[PDF]
|
|
|
|
|
|
|
A. Schober, R. Hertel, U. Arumae, L. Farkas, J. Jaszai, K. Krieglstein, M. Saarma, and K. Unsicker
Glial Cell Line-Derived Neurotrophic Factor Rescues Target-Deprived Sympathetic Spinal Cord Neurons But Requires Transforming Growth Factor-beta as Cofactor In Vivo
J. Neurosci.,
March 15, 1999;
19(6):
2008 - 2015.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction
