Glial Cell Line-derived Neurotrophic Factor Receptor Research Articles

Regulation of Glial Cell Line-Derived Neurotrophic Factor Responsiveness in Developing Rat Sympathetic Neurons by Retinoic Acid and Bone Morphogenetic Protein-2

There are several lines of evidence suggesting that, in addition to neurotrophins, member(s) of glial cell line-derived neurotrophic factor (GDNF) family play important roles in the development of sympathetic neurons. However, the mechanism regulating the responsiveness of the neurons to GDNF family members is not known. Previously, we reported on the cooperative roles of bone morphogenetic protein-2 (BMP2) and retinoic acid (RA) in the enhancement of neurotrophin-3 (NT3) responsiveness in cultured sympathetic neurons dissociated from perinatal rat superior cervical ganglia (SCG). In the present study, we further examined the effects of BMP2 and RA on the regulation of the responsiveness of SCG neurons to GDNF family members. Consequently, we found that RA alone induced the responsiveness of SCG neurons specifically to GDNF by upregulating the ligand-specifying receptor for GDNF (GFRalpha-1) at both the mRNA and protein levels. The expression levels of mRNAs for other ligand-specifying receptors for GDNF family (GFRalpha-2 and GFRalpha-3) were unaffected by RA. Although the upregulation of signal-transducing receptor Ret by the RA treatment was rather small, this treatment significantly increased the efficacy of tyrosine phosphorylation of Ret by GDNF. Experiments using synthetic retinoids suggested that RA acts through alpha-type of nuclear retinoic acid receptor to exert the induction of GDNF responsiveness. On the other hand, BMP2, which had no significant effect by itself on the GDNF responsiveness, promoted the action of RA to upregulate GFRalpha-1 and enhance the GDNF responsiveness. These results indicate that RA and BMP2 play important roles in the induction of GDNF responsiveness, as well as NT3 responsiveness, of developing SCG neurons.

Glial cell line-derived neutrotrophic factor and neurturin can act as paracrine growth factors stimulating DNA synthesis of Ret-expressing spermatogonia.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are related growth factors which exert trophic effects on several neuronal populations and developing kidney. GDNF-family ligands interact with membrane receptors designated GFRalphas which, in turn, mediate stimulation of the Ret receptor tyrosine kinase. Here we show that Ret, GFRalpha-1 (the GDNF receptor), and GFRalpha-2 (the NTN receptor) are expressed by testicular germ cells, while GDNF and NTN are expressed by Sertoli cells. Both GDNF and NTN stimulate DNA synthesis in spermatogonia. Furthermore, Ret, ligands and co-receptors are expressed in germ cell tumors. Thus, GDNF-family ligands may act as paracrine factors in spermatogenesis and this circuit may be active in germ cell tumors.

GDNF AND CISPLATIN NEURONOPATHY

GDNF is a growth factor produced in the central nervous system by glial cells and has a trophic action on dopaminergic neurons. However, GDNF mRNA is expressed also in the dorsal root ganglia (DRG), suggesting that it may act as a neurotrophic factor on different neuronal populations. The three different GDNF receptor components are highly expressed in a group of small primary sensory neurons and these neurons do not express any of the specific Trk receptors for the neurotrophins. Furthermore, GDNF receptor expression has been also found in DRG neurons of all sizes, indicating that these cells too may be responsive to GDNF. We have, therefore, evaluated the possible neuroprotective effect of GDNF (1 mg/kg, s.c. three times a week, donated by Amgen Inc) against cisplatin (CDDP, 2 mg/Kg i.p. twice a week for 9 times) DRG neuronopathy.Using our experimental paradigm, GDNF had no positive effect with both electrophysiological and morphometric determinations. The lack of GDNF activity in preventing the signs of peripheral nerve involvement in CDDP neuronopathy with respect to NGF and NT3 may be explained by a different distribution of their specific receptors in DRG neurons. In fact, the specific NT‐3 TrkC receptor is mainly expressed by the large DRG neurons, which are prominently involved in cisplatin neuronopathy, while GDNF receptors are mainly, although not exclusively, expressed by small‐ and medium‐sized DRG neurons. Small‐ and medium‐sized DRG neurons are also involved in the pathological changes of cisplatin neuronopathy, but to a lesser degree than the large ones. Furthermore, GDNF, in other models of peripheral neuropathies, may be active in an indirect way, mediated by its capacity to activate macrophages and inflammatory responses.

A dynamic regulation of GDNF-family receptors correlates with a specific trophic dependency of cranial motor neuron subpopulations during development.

Glial cell line-derived neurotrophic factor (GDNF) family ligands promote the survival of developing motor neurons in vivo and in vitro. However, not all neurons survive with any single ligand in culture and GDNF null mutant mice display only a partial motor neuron loss. An interesting possibility is that subpopulations of motor neurons based on their function and/or their myotopic organization require distinct members of GDNF family ligands. Because responsiveness to the different ligands depends on the expression of their cognate ligand-binding receptor we have herein addressed this issue by examining the expression of GDNF-family receptors (gfr) during development and in the adult in cranial motor nuclei subpopulations. We have furthermore examined the in vivo role of GDNF for cranial motor neuron subpopulations. The shared ret receptor was expressed in all somatic, branchial and visceral cranial embryonic motor nuclei examined, showing that they are all competent to respond to GDNF family ligands during development. At early stages of development both the GDNF receptor, gfralpha1, and the neurturin (NTN) receptor, gfralpha2, were expressed in the oculomotor, facial and spinal accessory, and only gfralpha1 in the trochlear, superior salivatory, trigeminal, hypoglossal and weakly in the dorsal motor nucleus of the vagus and the ambiguous nucleus. The abducens nucleus was negative for both gfralpha1 and gfralpha2. The artemin (ART) receptor, gfralpha3, was expressed only in the superior salivatory nucleus. A motor neuron subnuclei-specific expression of gfralpha1 and gfralpha2 was seen in the facial and trigeminal nuclei which corresponded to their dependence on GDNF in null mutant mice. We found that the expression was dynamic in these nuclei, which may reflect developmental changes in their trophic factor dependency. Analysis of GDNF null mutant mice revealed that the dynamic receptor expression is regulated by the ligand in vivo, indicating that the attainment of changes in dependency could be ligand induced. Our results indicate that specific GDNF family ligands support selective muscle-motor neuron circuits during development.

Neurturin, RET, GFRα-1 and GFRα-2, but not GFRα-3, mRNA are expressed in mice gonads

The gonads are known to produce numerous hormones and also neurotrophins and their receptors. Here we demonstrate expression of glial-cell-line-derived neurotrophic factor (GDNF) family ligands and related receptors in adult mice gonads by in situ hybridization. GDNF mRNA was expressed in the ovary, but was not detectable in testis. Neurturin (NTN), another ligand in this family, gave rise to strong mRNA hybridization signals in a mosaic pattern in the seminiferous tubules of the testis at stages IX-XII and I-II of the cycle. NTN mRNA signals were also found in uterus and the oviduct. In testis, the transducing receptor RET as well as GDNF receptor alpha-1 (GFR)alpha-1 and GFRalpha-2 were distributed in complementary and overlapping patterns, the former at stages XI-XII-I and the latter at stages VII and VIII. GFRalpha-3 could not be detected. Expression of these trophic molecules suggests involvement of GDNF family ligands and related receptor components in reproduction.

Differential regulation of trophic factor receptor mRNAs in spinal motoneurons after sciatic nerve transection and ventral root avulsion in the rat.

After sciatic nerve lesion in the adult rat, motoneurons survive and regenerate, whereas the same lesion in the neonatal animal or an avulsion of ventral roots from the spinal cord in adults induces extensive cell death among lesioned motoneurons with limited or no axon regeneration. A number of substances with neurotrophic effects have been shown to increase survival of motoneurons in vivo and in vitro. Here we have used semiquantitative in situ hybridization histochemistry to detect the regulation in motoneurons of mRNAs for receptors to ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) 1-42 days after the described three types of axon injury. After all types of injury, the mRNAs for GDNF receptors (GFRalpha-1 and c-RET) and the LIF receptor LIFR were distinctly (up to 300%) up-regulated in motoneurons. The CNTF receptor CNTFRalpha mRNA displayed only small changes, whereas the mRNA for membrane glycoprotein 130 (gp130), which is a critical receptor component for LIF and CNTF transduction, was profoundly down-regulated in motoneurons after ventral root avulsion. The BDNF full-length receptor trkB mRNA was up-regulated acutely after adult sciatic nerve lesion, whereas after ventral root avulsion trkB was down-regulated. The NT-3 receptor trkC mRNA was strongly down-regulated after ventral root avulsion. The results demonstrate that removal of peripheral nerve tissue from proximally lesioned motor axons induces profound down-regulations of mRNAs for critical components of receptors for CNTF, LIF, and NT-3 in affected motoneurons, but GDNF receptor mRNAs are up-regulated in the same situation. These results should be considered in relation to the extensive cell death among motoneurons after ventral root avulsion and should also be important for the design of therapeutical approaches in cases of motoneuron death.

Regulation of GFR?-1 and GFR?-2 mRNAs in rat brain by electroconvulsive seizure

The influence of both acute and chronic electroconvulsive seizure (ECS) or antidepressant drug treatments on expression of mRNAs encoding glial cell line-derived neurotrophic factor (GDNF) and its receptors, GFRalpha-1, GFRalpha-2, and c-Ret proto-oncogene (RET) in the rat hippocampus was examined by in situ hybridization. Two hours after acute ECS, levels of GFRalpha-1 mRNA in the dentate gyrus were significantly increased. This increase peaked to nearly 3-fold at 6 h after acute ECS and returned to basal levels 24 h after treatment. Chronic (once daily for 10 days) ECS significantly increased the expression of GFRalpha-1 mRNA nearly 5-fold after the last treatment. Levels of GFRalpha-2 mRNA in the dentate gyrus were also significantly increased by acute and chronic ECS, although this effect was less than that observed for GFRalpha-1. Maximum induction of GFRalpha-2 was 30% and 70% compared to sham in response to acute or chronic ECS, respectively. Levels of GDNF and RET mRNAs were not significantly changed following either acute or chronic ECS treatment at the time points examined. Chronic (14 days) administration of different classes of antidepressant drugs, including tranylcypromine, desipramine, or fluoxetine, did not significantly affect the GDNF, GFRalpha-1, GFRalpha-2, or RET mRNA levels in CA1, CA3, and dentate gyrus areas of hippocampus. The results demonstrate that acute ECS increases the expression of GFRalpha-1 and GFRalpha-2 and that these effects are enhanced by chronic ECS. The results also imply that regulation of the binding components of GDNF receptor complex may mediate the adaptive responses of the GDNF system to acute and chronic stimulation.

Expression Pattern of GDNF, c-ret, and GFRαs Suggests Novel Roles for GDNF Ligands during Early Organogenesis in the Chick Embryo

We have cloned a partial cDNA of chicken glial cell line-derived neurotrophic factor (GDNF) and systematically examined its expression pattern as well as that of GDNF-binding components (GDNF family receptor alpha-1 and 2: GFRα-1 and 2) and a common signal transduction receptor (c-ret protooncogene: RET) during very early developmental stages. In addition, we also examined the expression pattern of an apparent avian-specific binding component, GFRα-4. The cloned chicken cDNA for GDNF had approximately 80% homology to mammalian counterparts. The expression of GDNF mRNA occurred in many spatially and temporally discrete regions such as the intermediate mesoderm, the floor plate of the spinal cord, pharyngeal endoderm contacting the epibranchial placodes, distal ganglia of cranial nerves, subpopulations of mesenchyme cells in the craniofacial region, and in the mesodermal wall of the digestive tract. Both a GDNF receptor signal transduction component (RET) and a binding component (GFRα-1 or GFRα-2) were independently expressed in nearby interacting tissues such as the somites, peripheral and central nervous system, and mesenchyme cells in the craniofacial region. These observations suggest that possible combinations of novel unidentified receptors acting with RET or with GFRαs may mediate GDNF-derived signals and indicate that GDNF or other family members may have previously unidentified actions in early organogenesis in the chick embryo.

GDNF triggers a novel Ret-independent Src kinase family-coupled signaling via a GPI-linked GDNF receptor α1

Glial cell line-derived neurotrophic factor (GDNF) has potentially great clinical importance in the treatment of Parkinson’s disease and several other neurodegenerative diseases, however its intracellular signaling mechanisms are poorly understood. Here we show that upon GDNF binding glycosyl-phosphatidylinositol (GPI)-linked GDNF receptor α1 (GFRα1) activates cytoplasmic Src family tyrosine kinase(s) in Ret tyrosine kinase-deficient cultured mouse dorsal root ganglion neurons and in two Ret-negative cell lines. GFRα1-mediated Src-type kinase activation subsequently triggers phosphorylation of mitogen-activated protein kinase, cAMP response element binding protein and phospholipase Cγ. We therefore conclude that GDNF can activate intracellular signaling pathways Ret-independently via GPI-linked GFRα1.

Expression of the GDNF receptors Ret and GFRα1 in the developing avian enteric nervous system

Expression of the GDNF receptors Ret and GFRα1 in the developing avian enteric nervous system

Rat primary cultured microglia express glial cell line-derived neurotrophic factor receptors

The neurotrophic effect of glial cell line-derived neurotrophic factor (GDNF) has been well documented in both the central and peripheral nervous systems. From the histological findings, target cells of GDNF have been considered to be neurons. In the present study, the expression of GDNF receptors, ret and GFRα-1, was demonstrated in rat primary cultured microglia by reverse transcription-polymerase chain reaction and the protein-level expression of Ret was also confirmed by Western-blotting analyses. Moreover, GDNF stimulated the phosphorylation of MAP kinase (ERK1/2) in the cells. These results suggest that GDNF regulates not only neuronal survival and maturation but also certain functions of microglia in the brain.

Glial cell line-derived neurotrophic factor. Potential for otoprotection.

Sensorineural hearing loss results from the degeneration of hair cells and/or auditory neurons in the cochlea of the inner ear. BDNF and NT-3 were shown to support survival of auditory neurons both in vitro and in vivo. Cochlea from P3-P4 rats were cultured as floating explants and hair cells in the organ of Corti were identified by phalloidin-FITC immunostaining. Treatment with cisplatin (35 micrograms/mL) or neomycin (0.6 mM) resulted in 21.2 +/- 6.0% and 7.4 +/- 4.7% surviving hair cells, respectively, after 3 days in culture. GDNF, added together with the ototoxins, increased their number to 46.7% and 37.4%, respectively. In cultures of dissociated cochlea from 4-week-old rat, cisplatin (5 mg/mL) added 24 h after seeding resulted in only 6.1 +/- 1.2% surviving neurons. However, when cisplatin was added together with GDNF (10 ng/mL), 32.8 +/- 1.0% of the neurons survived. The efficacy of GDNF in animal models of ototoxicity was tested next. Guinea pigs were pretreated with GDNF in one ear, delivered either by infusion into the inner ear (scala tympani) with Alzet minipumps (50 ng/mL at a 0.5 microL/h), or injected into the middle ear (120 microL at 1 mg/mL) through the tympanic membrane. The ear that did not receive GDNF always served as control. Ototoxicity was induced systemically either by intraperitoneal cisplatin injections (1 mg/kg/day for 15 days or two injections of 7.5 mg/kg at a 5-day interval or by a combination of kanamycin (200-300 mg/kg, administered subcutaneously) and ethacrinic acid (40 mg/kg, intravenous). It was found that the number of surviving hair cells in GDNF-treated ears was about twice that of control ears in animals exposed to the ototoxins. The transducing GDNF receptor (ret) is expressed in the inner ear.

Expression of the GDNF receptors Ret and GFR?1 in the developing avian enteric nervous system

The formation of the enteric nervous system (ENS) from neural crest-derived cell precursors requires the growth factor glial cell line-derived neurotrophic factor (GDNF) and the receptors Ret and GDNF family receptor alpha 1 (GFRalpha1). We investigated the location(s), the timing, and the extent to which these GDNF receptors appear in the population of crest-derived precursors that form the avian ENS using immunohistochemistry and in situ hybridization. Sections and whole mounts of embryonic chick gastrointestinal tract were costained with antibodies to the receptors and to HNK-1, a marker for crest-derived cells. Neural crest-derived precursors migrate through the primitive esophagus to colonize the gizzard where an extensive cellular network forms. Ret-immunoreactivity (ir) was found in a network of cells in the gizzard at embryonic day (E)3.5. As development proceeded, Ret-immunoreactive cells appeared at progressively more caudal positions and were present in the colon at E7.5. Costaining with Ret and HNK-1 was performed to determine the number of Ret-immunoreactive cells in the crest-derived population. Ret appeared in some HNK-1 cells in the esophagus and gizzard at embryonic day (E)3.5. During development, the number of crest cells with Ret increased in the ganglia of the gizzard and small intestine. GFRalpha1-ir was also found in HNK-1 cells in the esophagus at E3.5 but did not appear in the gizzard until E4.5. Surprisingly, the colonizing vanguard of crest-derived cells lacked both Ret- and GFRalpha-ir. Between E4.5 and E6.5, the fraction of HNK-1-positive cells expressing GFRalpha1 increased considerably in the foregut. Ret and GFRalpha1 were coexpressed in many cells at E6.5, and the number of such cells increased as development progressed. In the adult, GFRalpha1 and Ret were found in the neuropil of enteric ganglia. We conclude that the population of cells expressing the receptors increases during development and persists in the adult, findings that support a neurotrophic role for GDNF in the formation and maintenance of the avian ENS.

Expression of Neurturin, GDNF, and GDNF Family-Receptor mRNA in the Developing and Mature Mouse

The GDNF family of neurotrophic factors currently has four members: neurturin (NRTN), glial cell line-derived neurotrophic factor (GDNF), persephin, and artemin. These proteins are potent survival factors for several populations of central and peripheral neurons. The receptors for these factors are complexes that include the Ret tyrosine kinase receptor and a GPI-linked, ligand-binding component called GDNF family receptor α 1–4 (GFRα1–4). We have used in situ hybridization to study the mRNA expression of NRTN, GDNF, Ret, GFRα1, and GFRα2 during embryonic development and in the adult mouse. GDNF receptors were prominently expressed during embryonic development in the nervous system, the urogenital system, the digestive system, the respiratory system, and in developing skin, bone, muscle, and endocrine glands. In some regions, incomplete receptor complexes were expressed suggesting that other, as yet unidentified, receptor components exist or that receptor complexes are formed in trans. NRTN and GDNF were expressed in many trigeminal targets during embryonic development including the nasal epithelium, the teeth, and the whisker follicles. NRTN and GDNF were also expressed in the developing limbs and urogenital system. In the embryo, GDNF factors and receptors were expressed at several sites of mesenchyme/epithelial induction, including the kidney, tooth, and submandibular gland. This expression pattern is consistent with the possibility that the GDNF factors function in inductive processes during embryonic development and with the recently discovered role of NRTN as a necessary trophic factor for the development of some parasympathetic neurons. In the mature animal, receptor expression was more limited than in the embryo. In the adult mouse, NRTN was most prominently expressed in the gut, prostate testicle, and oviduct; GDNF was most prominently expressed in the ovary.

Induction of glial cell line-derived neurotrophic factor receptor proteins in cerebral cortex and striatum after permanent middle cerebral artery occlusion in rats

In an attempt to elucidate whether glial cell line-derived neurotrophic factor (GDNF) receptors are induced after ischemic brain injury, possible expression of immunoreactive GDNF receptor-α1 (GFRα-1) and c-ret (RET) was examined at 3, 8, or 24 h after permanent middle cerebral artery occlusion (MCAO) in rats. Immunohistochemical study showed that both GFRα-1 and RET staining cells which were not detected in sham control brain, were present in the ipsilateral cortex and caudate at 3 to 8 h after permanent MCAO, and then decreased but remained to some extent at 24 h. Positive cells for both GDNF receptors were predominantly in cortical neurons of ischemic penumbral area. Western blot analysis confirmed the induction of those receptors after permanent MCAO. This rapid induction of GFRα-1 and RET, which correlates with the similar induction of GDNF under these conditions, may play a role in the early response to ischemic brain injury.

Ret-dependent and -independent Mechanisms of Glial Cell Line-derived Neurotrophic Factor Signaling in Neuronal Cells

Glial cell line-derived neurotrophic factor (GDNF) has been shown to signal through a multicomponent receptor complex consisting of the Ret receptor tyrosine kinase and a member of the GFRalpha family of glycosylphosphatidylinositol-anchored receptors. In the current model of GDNF signaling, Ret delivers the intracellular signal but cannot bind ligand on its own, while GFRalphas bind ligand but are thought not to signal in the absence of Ret. We have compared signaling pathways activated by GDNF in two neuronal cell lines expressing different complements of GDNF receptors. In a motorneuron-derived cell line expressing Ret and GFRalphas, GDNF stimulated sustained activation of the Ras/ERK and phosphatidylinositol 3-kinase/Akt pathways, cAMP response element-binding protein phosphorylation, and increased c-fos expression. Unexpectedly, GDNF also promoted biochemical and biological responses in a line of conditionally immortalized neuronal precursors that express high levels of GFRalphas but not Ret. GDNF treatment did not activate the Ras/ERK pathway in these cells, but stimulated a GFRalpha1-associated Src-like kinase activity in detergent-insoluble membrane compartments, rapid phosphorylation of cAMP response element-binding protein, up-regulation of c-fos mRNA, and cell survival. Together, these results offer new insights into the dynamics of GDNF signaling in neuronal cells, and indicate the existence of novel signaling mechanisms directly or indirectly mediated by GFRalpha receptors acting in a cell-autonomous manner independently of Ret.

POU domain factor Brn-3a controls the differentiation and survival of trigeminal neurons by regulating Trk receptor expression.

Mice lacking the POU domain-containing transcription factor Brn-3a have several neuronal deficits. In the present paper, we show that Brn-3a plays two distinct roles during development of the trigeminal ganglion. In this ganglion, neurons expressing the neurotrophin receptors, TrkB and TrkC, are born between E9.5 and E11.5. In the absence of Brn-3a, very few neurons ever express TrkC, but TrkB-expressing neurons are present at E12.5 in elevated numbers, suggesting that Brn-3a may be a constituent of a regulatory circuit determining which Trk receptor is expressed by these early-born neurons. Most neurons expressing the neurotrophin receptor TrkA are generated between E11.5 and E13.5 in this ganglion and their initial generation is not prevented by absence of Brn-3a. However, after E12. 5, absence of Brn-3a results in a progressive loss in neuronal TrkA and TrkB expression, which leads to a massive wave of apoptosis that peaks at E15.5. Despite complete absence of the Trk receptors at E17. 5 and P0, approximately 30% of the normal complement of neurons survive to birth in Brn-3a mutants. Approximately 70% of these express the GDNF receptor subunit, c-ret; many can be sustained by GDNF, but not by NGF in culture. Thus, the vast majority of surviving neurons are probably sustained in vivo by trophic factor(s) whose receptors are not regulated by Brn-3a. In conclusion, our data indicate the specific functions of Brn-3a in controlling the survival and differentiation of trigeminal neurons by regulating expression of each of the three Trk receptors.

Expression of human GFRα-1 (GDNF receptor) at the neuromuscular junction and myelinated nerves

Motor neurons have been known to require a wide variety of neurotrophic factors for their survival. As one of the target-derived trophic factors, glial cell line-derived neurotrophic factor (GDNF) has been shown to exert its effects on motor neurons via a receptor complex including GDNF receptor alpha 1 (GFRα-1). Immunoreactivity of GFRα-1 was observed at myelinated peripheral nerves and neuromuscular junction (NMJ) of human skeletal muscles. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that mRNA of GFRα-1 existed in the ventral horn of human spinal cord, but not in the skeletal muscles. The results suggested that GFRα-1 might play a key role for uptake and internalization of GDNF at the human NMJ.

Differences and Developmental Changes in the Responsiveness of PNS Neurons to GDNF and Neurturin

We have studied the ability of GDNF and neurturin to promote the in vitro survival of populations of embryonic chicken parasympathetic, sympathetic, and sensory neurons. We show that these neurons are more responsive to one or other of these factors at particular stages of development. Whereas the parasympathetic neurons are more sensitive to neurturin at late embryonic stages, sympathetic neurons are more sensitive to neurturin at early stages. In contrast, sensory neurons of the nodose ganglion are more sensitive to GDNF throughout embryonic development. Using competitive RT/PCR, we measured the levels of mRNAs encoding GDNF and neurturin receptors in purified neurons. All neurons expressed Ret mRNA, which encodes the common receptor tyrosine kinase for GDNF and neurturin. Neurons that were more sensitive to GDNF expressed higher levels of GFRα-1 mRNA than GFRα-2 mRNA and neurons that were more sensitive to neurturin expressed higher levels of GFRα-2 mRNA than GFRα-1 mRNA. These results show that populations of PNS neurons differ markedly in their responsiveness to GDNF and neurturin at certain stages of the development and suggest that these differences are governed in part by the relative levels of expression of members of the GFRα family of GPI-linked receptors.

GDNF family members and their receptors: expression and functions in two oligodendroglial cell lines representing distinct stages of oligodendroglial development.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), and persephin (PSP) constitute a subfamily of transforming growth factor-betas (TGF-betas) with prominent roles in the regulation of neuron survival and differentiation. Although numerous members of the TGF-beta superfamily are important regulators of glial cell functions in health and disease, it is unknown whether any member of the GDNF subfamily may have functions in normal or pathological glial cell performances. To begin to address this issue, we have studied expression and putative functions of GDNF, NTN, PSP, and their receptors in two cell lines representing models for oligodendrocyte progenitor cells (OLI-neu) and immature oligodendrocytes (OLN-93), respectively. RT-PCR analysis revealed expression of all three growth factor mRNAs in OLI-neu and OLN-93 cells. Expression was weak in OLI-neu cells, while both NTN and PSP mRNAs were strongly expressed in OLN-93 cells. Furthermore, OLI-neu and OLN-93 cells expressed transcripts encoding the GDNF receptors Ret and GFRalpha-1. The two splice variants for GFRalpha-2 were exclusively synthesized in OLI-neu cells. Similarly, primary O-2A progenitor cells and enriched mature oligodendrocytes expressed Ret, GFRalpha-1 and GFRalpha-2 mRNAs. Both GDNF and NTN stimulated DNA synthesis monitored by BrdU incorporation of OLI-neu cells in a dose-dependent fashion. Co-administration of TGF-beta significantly reduced this effect. Similarly, PDGF co-applied with GDNF or NTN down-regulated proliferation in OLI-neu cells. In contrast, OLN-93 cells did not respond to GDNF or NTN with increased incorporation of BrdU. Expression of GDNF, NTN, and their receptors and distinct effects in two model cell lines of oligodendrocyte development suggest that functions of members of the GDNF family and their receptors may not be restricted to neurons and may be implicated in oligodendrocyte development.