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. 1998 Dec 1;18(23):9822-34.
doi: 10.1523/JNEUROSCI.18-23-09822.1998.

Glial cell line-derived neurotrophic factor requires transforming growth factor-beta for exerting its full neurotrophic potential on peripheral and CNS neurons

K Krieglstein  1 , P Henheik, L Farkas, J Jaszai, D Galter, K Krohn, K Unsicker
Affiliations

Glial cell line-derived neurotrophic factor requires transforming growth factor-beta for exerting its full neurotrophic potential on peripheral and CNS neurons

K Krieglstein et al. J Neurosci. .

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-beta. Immunoneutralization of endogenous TGF-beta provided by serum or TGF-beta-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-beta identifies 60 pg/ml of either factor combined with 2 ng/ml of the other factor as the EC50. GDNF/TGF-beta 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-beta 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 alpha. The biological significance of the trophic synergism of GDNF and TGF-beta is underscored by colocalization of the receptors for TGF-beta and GDNF on all investigated GDNF-responsive neuron populations in vivo. Moreover, the in vivo relevance of the TGF-beta/GDNF synergism is highlighted by the co-storage of TGF-beta 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.

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Figures

Fig. 1.
Fig. 1.
Survival of peripheral autonomic and sensory neurons by the synergistic action of GDNF and TGF-β.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-β1, TGF-β2, and TGF-β3 (a-TGF-β, 10 μg/ml) reduced neuron survival to levels seen with the addition of serum alone, suggesting that GDNF required TGF-β in the serum to achieve its survival-promoting effect. In serum-free conditions, GDNF and TGF-β-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-β antibody recognizes TGF-β1 and TGF-β3, but not any other TGF-β superfamily or neurotrophin family member tested. C, Dose–response curve for the combined action of GDNF and TGF-β 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-β1. Circlesrepresent neuron survival achieved in the presence of a constant amount of TGF-β1 (2 ng/ml) plus the indicated amounts of GDNF.
Fig. 2.
Fig. 2.
Assay as performed in Figure1A using neurons from the respective ganglia of chick E10 (A) and E12 (B) embryos. Data indicate that the GDNF/TGF-β 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.
Fig. 3.
Fig. 3.
Survival-promoting effect of GDNF and TGF-β 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-β antibodies (10 μg/ml), or NGF or GDNF plus anti-TGF-β 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-β3 (2 ng/ml), GDNF plus TGF-β3, or GDNF plus anti-TGF-β 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-β1 or anti-TGF-β antibodies (10 μg/ml). Data are given as mean ± SEM (n = 4) and expressed in percent of cells present at day 1.
Fig. 4.
Fig. 4.
Mechanisms underlying the synergistic actions of GDNF and TGF-β. A, Chick ciliary ganglionic neurons (E8) were treated with GDNF (2 ng/ml), TGF-β1 (2 ng/ml), GDNF plus TGF-β1 (2 ng/ml each), and GDNF plus TGF-β1 plus wortmannin (0.25 μm), a specific inhibitor of PI-3 kinase. Wortmannin abolishes GDNF/TGF-β-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-β. Data are given as mean ± SEM (n = 6), pvalues 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-β1 (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-β consistent with the essential role of a GPI-linked GFRα in GDNF signal transduction. Addition of both PIPLC and TGF-β to isolated ciliary neurons protects the GPI-linked α-receptors, suggesting that the synergistic neurotrophic action of GDNF and TGF-β may be caused by a protective action of TGF-β on the GFRα. 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-β1.
Fig. 5.
Fig. 5.
RT-PCR (30 cycles) of P0 rat tissues and E14 purified motoneurons with primers specific for TGF-β2 (355 bp), TGF-β3 (290 bp), TβR-II (296 bp), GFRα-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.
Fig. 6.
Fig. 6.
Immunohistochemistry showing localization of TGF-β2 (A, B), TGF-β3 (C, D), and TβR-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.
Fig. 7.
Fig. 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-βs TGF-β1, TGF-β2, and TGF-β3 (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-β are the long-sought ciliary neurotrophic proteins contained in VP. Data are given as mean ± SEM (n = 6), pvalues derived from Student’s t test are ***p < 0.001 for decreased survival after antibody treatment.
Fig. 8.
Fig. 8.
RT-PCR analysis of bovine chromaffin cells (lanes 3 and 4) using primers specific for ribosomal S6 (293 bp), GDNF (415 bp), TGF-β1 (279 bp), TGF-β2 (359 bp), and TGF-β3 (291 bp). Bovine chromaffin cells were analyzed after 18 hr of culture with lanes 3 and4, representing two different RNA preparations. Inlane 2 RNA from cultured cortical astrocytes is amplified. Lane 1 contains a negative control with water instead of cDNA.
Fig. 9.
Fig. 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.
Fig. 10.
Fig. 10.
GDNF as well as TGF-β1 are released from bovine chromaffin cells after cholinergic stimulation. A, Western blot showing immunoprecipitated TGF-β1 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-β1 and protein extracts from isolated bovine chromaffin granules (VP) were used. Theasterisks 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.
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References

    1. Abe M, Harpel JG, Metz CN, Nunes I, Loskutoff DJ, Rifkin DB. An assay for transforming growth factor-β using cells transfected with a plasminogen activator inhibitor-1 promotor-luciferase construct. Anal Biochem. 1994;216:276–284. - PubMed
    1. Alexandrow MG, Moses HL. Transforming growth factor β and cell cycle regulation. Cancer Res. 1995;55:1452–1457. - PubMed
    1. Arenas E, Trupp M, Akerud P, Ibanez CF. GDNF prevents degeneration and promotes the phenotype of brain noradrenergic neurons in vivo. Neuron. 1995;15:1465–1473. - PubMed
    1. Avantaggiato V, Dathan NA, Grieco M, Fabien N, Lazzaro D, Fusco A, Simeone A, Santoro M. Developmental expression of the RET protooncogene. Cell Growth Differ. 1994;5:305–311. - PubMed
    1. Baloh RH, Tansey MG, Golden JP, Creedon DJ, Heuckeroth RO, Keck CL, Zimonjic DB, Popescu NC, Johnson EM, Jr, Milbrandt J. TrnR2, a novel receptor that mediates neurturin and GDNF signaling through Ret. Neuron. 1997;18:793–802. - PubMed

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