Growth/differentiation factor-15/macrophage inhibitory cytokine-1 is a novel trophic factor for midbrain dopaminergic neurons in vivo.

Transforming growth factor-betas (TGF-betas) constitute an expanding family of multifunctional cytokines with prominent roles in development, cell proliferation, differentiation, and repair. We have cloned, expressed, and raised antibodies against a distant member of the TGF-betas, growth/differentiation factor-15 (GDF-15). GDF-15 is identical to macrophage inhibitory cytokine-1 (MIC-1). GDF-15/MIC-1 mRNA and protein are widely distributed in the developing and adult CNS and peripheral nervous systems, including choroid plexus and CSF. GDF-15/MIC-1 is a potent survival promoting and protective factor for cultured and iron-intoxicated dopaminergic (DAergic) neurons cultured from the embryonic rat midbrain floor. The trophic effect of GDF-15/MIC-1 was not accompanied by an increase in cell proliferation and astroglial maturation, suggesting that GDF-15/MIC-1 probably acts directly on neurons. GDF-15/MIC-1 also protects 6-hydroxydopamine (6-OHDA)-lesioned nigrostriatal DAergic neurons in vivo. Unilateral injections of GDF-15/MIC-1 into the medial forebrain bundle just above the substantia nigra (SN) and into the left ventricle (20 microgram each) immediately before a 6-OHDA injection (8 microgram) prevented 6-OHDA-induced rotational behavior and significantly reduced losses of DAergic neurons in the SN. This protection was evident for at least 1 month. Administration of 5 microgram of GDF-15/MIC-1 in the same paradigm also provided significant neuroprotection. GDF-15/MIC-1 also promoted the serotonergic phenotype of cultured raphe neurons but did not support survival of rat motoneurons. Thus, GDF-15/MIC-1 is a novel neurotrophic factor with prominent effects on DAergic and serotonergic neurons. GDF-15/MIC-1 may therefore have a potential for the treatment of Parkinson's disease and disorders of the serotonergic system.

GDF-15/MIC-1 a novel member of the TGF-beta superfamily.

We have cloned, expressed, and raised antibodies against a novel member of the TGF-beta superfamily, growth/differentiation factor-15 (GDF-15). The predicted protein is identical to macrophage inhibitory cytokine-1 (MIC-1), which was discovered simultaneously. GDF-15 is a more distant member of the TGF-beta superfamily and does not belong to one of the known TGF-beta subfamilies. In the CNS, GDF-15/MIC-1 mRNA is abundantly expressed by the choroid plexus. In addition we have preliminary evidence that GDF-15/MIC-1 is a potent trophic factor for selected classes of neurons in vitro and in vivo. Thus, GDF-15 is a novel neurotrophic factor with prospects for the treatment of disorders of the CNS.

Characterization of bone morphogenetic protein family members as neurotrophic factors for cultured sensory neurons.

The bone morphogenetic proteins have been implicated in several inductive processes throughout vertebrate development including nervous system patterning. Recently, these proteins have also emerged as candidates for regulating survival of mesencephalic dopaminergic and sympathetic neurons. Interestingly, we have found that several bone morphogenetic proteins can be detected in developing embryonic day 14 rat dorsal root ganglia by means of reverse transcription-polymerase chain reaction and immunocytochemistry. To further elucidate their potential role during the period of ontogenetic neuron death, serum-free cultures of dorsal root sensory neurons from developing chick and rat embryos were treated with distinct bone morphogenetic proteins with or without simultaneous addition of other "established" neurotrophic factors. Our results show that bone morphogenetic proteins exert survival promoting effects on their own, and that they can positively modulate the effects of neurotrophins on sensory neurons. In particular, growth/differentiation factor-5, bone morphogenetic protein-2, -4, -7 and -12 significantly increased the survival promoting effects of neurotrophin-3 and nerve growth factor on cultured dorsal root ganglion neurons. These results fit well into the current concept that neurotrophic factors may act synergistically in ensuring neuronal survival. Moreover, these data suggest potential instructive interactions of bone morphogentic proteins and neurotrophins during sensory neuron development. Finally, the documented neurotrophic capacity of bone morphogenetic protein family members may have potential relevance for the treatment of peripheral neuropathies.

Glial cell line-derived neurotrophic factor rescues target-deprived sympathetic spinal cord neurons but requires transforming growth factor-beta as cofactor in vivo.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for several populations of CNS and peripheral neurons. Synthesis and storage of GDNF by the neuron-like adrenal medullary cells suggest roles in adrenal functions and/or in the maintenance of spinal cord neurons that innervate the adrenal medulla. We show that unilateral adrenomedullectomy causes degeneration of all sympathetic preganglionic neurons within the intermediolateral column (IML) of spinal cord segments T7-T10 that project to the adrenal medulla. In situ hybridization revealed that IML neurons express the glycosylphosphatidylinositol-linked alpha receptor 1 and c-Ret receptors, which are essential for GDNF signaling. IML neurons also display immunoreactivity for transforming growth factor-beta (TGF-beta) receptor II. Administration of GDNF (recombinant human, 1 microg) in Gelfoam implanted into the medullectomized adrenal gland rescued all Fluoro-Gold-labeled preganglionic neurons projecting to the adrenal medulla after four weeks. Cytochrome c applied as a control protein was not effective. The protective effect of GDNF was prevented by co-administration to the Gelfoam of neutralizing antibodies recognizing all three TGF-beta isoforms but not GDNF. This suggests that the presence of endogenous TGF-beta was essential for permitting a neurotrophic effect of GDNF. Our data indicate that GDNF has a capacity to protect a population of autonomic spinal cord neurons from target-deprived cell death. Furthermore, our results demonstrate for the first time that the previously reported requirement of TGF-beta for permitting trophic actions of GDNF in vitro (Kreiglstein et al., 1998) also applies to the in vivo situation.

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

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.

GDNF-related factor persephin is widely distributed throughout the nervous system.

Persephin (PSP) is the most recently discovered member of the GDNF family of neurotrophic factors. We have used an RT-PCR approach to start addressing the putative functional significance of PSP by determining sites of its synthesis in the neonatal rat brain. Generally, two transcripts were found. Sequence analysis of the transcripts identifies an 88 bp intronic sequence. Neural tissues analysed included cortex, hippocampus, striatum, diencephalon, mesencephalon, cerebellum, hindbrain and spinal cord as well as superior cervical, dorsal root ganglia, adrenal gland, and PC12 pheochromocytoma cells. As non-neuronal tissues, sciatic nerve, optic nerve, primary astroglial, oligodendroglial, O2A progenitor, and glioma cells (C6, B49) were also included. All tissues/cells except oligodendrocytes and O2A progenitor cells were strongly positive for PSP mRNA. To test the hypothesis of whether PSP might act as a target-derived factor, as suggested for GDNF, the motoneuron-muscle axis has been analysed. PSP is synthesized in skeletal muscle and, to a higher extent, in the spinal cord. Moreover, PSP is synthesized in purified embryonic motoneurons. Together, these data do not support a role for PSP as a typical target-derived neurotrophic factor for motoneurons. We conclude that PSP is synthesized throughout the nervous system and that it is presumably of both astroglial and neuronal origin, in contrast to GDNF and neurturin, which seem to be predominantly of neuronal origin.

Effects of glutamate-induced excitotoxicity on calretinin-expressing neuron populations in the area postrema of the rat.

We mapped the distribution of calretinin-immunoreactive neuron populations in a circumventricular organ of the rat, the area postrema, and investigated their sensitivity to excitotoxic stimuli mediated by subcutaneously administered monosodium glutamate. We were specifically interested to ascertain whether the presence of calretinin can, per se, confer an in vivo intrinsic resistance for area postrema neurons to glutamate excitotoxicity. We found that dense populations of calretinin-positive neurons displayed a subregional compartmentation in coronal sections of the area postrema along its rostrocaudal axis. We demonstrated that calretinin-positive neurons differ in their sensitivities to monosodium glutamate depending on their position within the area postrema. Neurons in the caudal area postrema were the most sensitive ones, while those in the rostral area postrema were spared of degeneration. We conclude that calretinin-positive neurons in the area postrema are not uniformly protected against glutamate excitotoxicity. It is possible that differences in the local concentrations of monosodium glutamate due to regional heterogeneities in density and permeability of the capillary bed rather than neuronal expression of calretinin account for the observed effects.