GDNF - a stranger in the TGF-beta superfamily?

Glial cell line-derived neurotrophic factor (GDNF) family, consisting of GDNF, neurturin, artemin and persephin are distant members of the transforming growth factor-beta (TGF-beta) superfamily. Unlike other members of the TGF-beta superfamily, which signal through the receptor serine-threonine kinases, GDNF family ligands activate intracellular signalling cascades via the receptor tyrosine kinase Ret. GDNF family ligands first bind to the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor alpha (GFRalpha) and then the GDNF family ligand-GFRalpha complex binds to and stimulates autophosphorylation of Ret. Alternatively, a preassociated complex between GFRalpha and Ret could form the binding site for the GDNF family ligand. GFRalpha1, GFRalpha2, GFRalpha3 and GFRalpha4 are the physiological coreceptors for GDNF, neurturin, artemin and persephin, respectively. Although all GDNF family ligands signal via activated Ret, GDNF can signal also via GFRalpha1 in the absence of Ret. GPI-anchored GFRalpha receptors are localized in plasma membrane to lipid rafts. GDNF binding to GFRalpha1 also recruits Ret to the lipid rafts and triggers association with Src, which is required for effective downstream signalling, leading to differentiation and neuronal survival. GDNF family ligands are potent survival factors for midbrain dopamine neurons, motoneurons, noradrenergic neurons, as well as for sympathetic, parasympathetic and sensory neurons. However, for most neuronal populations, except for motoneurons, TGF-beta is required as a cofactor for GDNF family ligand signalling. Because GDNF and neurturin can rescue dopamine neurons in the animal models of Parkinson disease, as well as motoneurons in vivo, hopes have been raised that GDNF family ligands may be new drugs for the treatment of neurodegenerative diseases. GDNF also has distinct functions outside the nervous system, promoting ureteric branching in kidney development and regulating spermatogenesis.     

GDNF and its receptors in the regulation of the ureteric branching.

Recent transgenic and organ culture experiments have inevitably shown that glial cell line-derived neurotrophic factor (GDNF) is a mesenchyme-derived signal for ureteric budding and branching. The signalling receptor complex for GDNF includes a dimer of Ret receptor tyrosine kinase and two molecules of GDNF family receptor alpha1. Alpha-receptors are not only needed for the ligand binding and Ret activation, but they might mediate signals without Ret. While GDNF is clearly required for ureteric branching, tissue recombination studies have shown that it is not sufficient for the completion of ureteric morphogenesis, and other signalling molecules are needed. Different experimental models have resulted in somewhat contradictory results on their molecular identity, but transforming growth factor-beta1, -beta2, fibroblast growth factor-7 and hepatocyte growth factor form, obviously among others, a redundant set of growth factors in ureteric differentiation. Three other members of the GDNF family, neurturin, artemin and persephin, are also expressed in the developing kidney, and at least neurturin and persephin promote ureteric branching in vitro, but their true in vivo roles are still unclear.     

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.     

Immunohistochemical expression of two members of the GDNF family of growth factors and their receptors in the olfactory system

The glial cell line-derived (GDNF) family of trophic factors, GDNF, neurturin, persephin and artemin, are known to support the survival and regulate differentiation of many neuronal populations, including peripheral autonomic, enteric and sensory neurons. Members of this family of related ligands bind to specific GDNF family receptor (GFR) proteins, which complex and signal through the Ret receptor tyrosine kinase. We showed previously that GDNF protein was detectable in olfactory sensory neurons (OSNs) in the olfactory neuroepithelium (ON). In this immunohistochemical study, we localized GDNF, neurturin, GFRα1, GFRα2 and Ret in the adult rat ON and olfactory bulb. We found that GDNF and Ret were widely expressed by immature and mature OSNs, while neurturin was selectively expressed in a subpopulation of OSNs zonally restricted in the ON. The GFRs had differential expression, with mature OSNs and their axons preferentially expressing GFRα1, whereas progenitors and immature neurons more avidly expressed GFRα2. In the bulb, GDNF was highly expressed by the mitral and tufted cells, and by periglomerular cells, and its distribution generally resembled that of Ret, with the exception that Ret was far more predominant on fibers than cell bodies. Neurturin, in contrast, was present at lower levels and was more restricted in its expression to the axonal compartment. GFRα2 appeared to be the dominant accessory protein in the bulb. These data are supportive of two members of this neurotrophic family, GDNF and neurturin, playing different physiological roles in the olfactory neuronal system.     

Chat in the trophic web: NGF activates Ret by inter-RTK signaling.

In this issue of Neuron, show that in rodent SCG neurons NGF activates Ret, the signaling component of the multisubunit GDNF receptor, in vitro and in vivo by a mechanism, which is independent of GFL ligands and GFRalpha coreceptors. NGF-dependent Ret phosphorylation regulates soma size and metabolism but not survival of maturing postnatal sympathetic neurons.     

Tyrosine 981, a novel ret autophosphorylation site, binds c-Src to mediate neuronal survival

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are neurotrophic factors that influence several aspects of the developing and injured nervous system. GFLs signal through a common receptor tyrosine kinase (Ret) and one of the four ligand-binding co-receptors (GFRalpha1 to 4). Ligand-induced translocation of Ret to lipid rafts, where it interacts with the nonreceptor tyrosine kinase Src, is a prerequisite for full biological activity of these neurotrophic factors. This interaction and subsequent activation of Src are required for GFL-mediated neuronal survival, neurite outgrowth, or cell proliferation. Here we show by multiple approaches that Ret tyrosine 981 constitutes the major binding site of the Src homology 2 domain of Src and therefore the primary residue responsible for Src activation upon Ret engagement. Other tyrosines such as 1015 and 1029 may contribute to the overall interaction between Ret and Src, as judged by overexpression experiments. By generating a phosphospecific antibody, we demonstrate that tyrosine 981 is a novel autophosphorylation site in Ret. Importantly, we also show that this tyrosine becomes phosphorylated in dissociated sympathetic neurons after ligand stimulation. Mutation of tyrosine 981 to phenylalanine reduces GDNF-mediated survival in a transfected cerebellar granule neuron paradigm.     

The neuron-specific Rai (ShcC) adaptor protein inhibits apoptosis by coupling Ret to the phosphatidylinositol 3-kinase/Akt signaling pathway

Rai is a recently identified member of the family of Shc-like proteins, which are cytoplasmic signal transducers characterized by the unique PTB-CH1-SH2 modular organization. Rai expression is restricted to neuronal cells and regulates in vivo the number of postmitotic sympathetic neurons. We report here that Rai is not a common substrate of receptor tyrosine kinases under physiological conditions and that among the analyzed receptors (Ret, epidermal growth factor receptor, and TrkA) it is activated specifically by Ret. Overexpression of Rai in neuronal cell lines promoted survival by reducing apoptosis both under conditions of limited availability of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) and in the absence of Ret activation. Overexpressed Rai resulted in the potentiation of the Ret-dependent activation of phosphatidylinositol 3-kinase (PI3K) and Akt. Notably, increased Akt phosphorylation and PI3K activity were also found under basal conditions, e.g., in serum-starved neuronal cells. Phosphorylated and hypophosphorylated Rai proteins form a constitutive complex with the p85 subunit of PI3K: upon Ret triggering, the Rai-PI3K complex is recruited to the tyrosine-phosphorylated Ret receptor through the binding of the Rai PTB domain to tyrosine 1062 of Ret. In neurons treated with low concentrations of GDNF, the prosurvival effect of Rai depends on Rai phosphorylation and Ret activation. In the absence of Ret activation, the prosurvival effect of Rai is, instead, phosphorylation independent. Finally, we showed that overexpression of Rai, at variance with Shc, had no effects on the early peak of mitogen-activated protein kinase (MAPK) activation, whereas it increased its activation at later time points. Phosphorylated Rai, however, was not found in complexes with Grb2. We propose that Rai potentiates the MAPK and PI3K signaling pathways and regulates Ret-dependent and -independent survival signals.     

Identification of the key amino acids of glial cell line-derived neurotrophic factor family receptor alpha1 involved in its biological function

Glial cell line-derived neurotrophic factor (GDNF) plays a critical role in neurodevelopment and survival of midbrain dopaminergic and spinal motor neurons in vitro and in vivo. The biological actions of GDNF are mediated by a two-receptor complex consisting of a glycosylphosphatidylinositol-linked cell surface molecule, the GDNF family receptor alpha1 (GFRalpha1), and receptor protein tyrosine kinase Ret. Although structural analysis of GDNF has been extensively examined, less is known about the structural basis of GFRalpha1 function. In this study, based on evolutionary trace method and relative solvent accessibility prediction of residues, a set of trace residues that are solvent-accessible was selected for site-directed mutagenesis. A series of GFRalpha1 mutations was made, and PC12 cell lines stably expressing different GFRalpha1 mutants were generated. According to the survival and differentiation responses of these stable PC12 cells upon GDNF stimulation and the GDNF-GFRalpha1-Ret interaction assay, residues 152NN153, Arg259, and 316SNS318 in the GFRalpha1 central region were found to be critical for GFRalpha1 binding to GDNF and eliciting downstream signal transduction. The single mutation R259A in the GFRalpha1 molecule simultaneously lost its binding ability to GDNF and Ret. However N152A/N153A or S316A/N317A/S318A mutation in the GFRalpha1 molecule still retained the ability to bind with Ret. These findings suggest that distinct structural elements in GFRalpha1 may be involved in binding to GDNF and Ret.     

胶质源性神经营养因子对不同组织细胞的影响

GDNF来自于小胶质神经元,首先作为中脑多巴胺能神经元的复活因子被发现,可促进细胞存活,并有增加多巴胺神经元细胞大小及轴突长度的作用。GDNF通过与锚定蛋白细胞表面受体糖基磷脂酰肌醇的相互作用来调节细胞活性。GDNF家族a-1受体,通过跨膜酪氨酸受体或者神经元细胞黏附分子,来促进细胞存活,神经突生长,以及突触发育。后续的研究提示,无论未成年还是成体大脑,GDNF对多种神经细胞都有复活的作用,并与一些周围神经复活、迁移、分化相关。不同的脑缺血实验模型均证实了外源性GDNF对于病灶部位及全脑的神经保护作用,包括局部应用营养因子,利用病毒载体运载GDNF基因以及移植表达GDNF的细胞。近来研究还证实,GDNF不仅对多巴胺能神经元,中枢和周围神经系统的运动、感觉神经元,以及自主神经元有营养和保护作用,对于非神经系统也有不同调节作用。本文将重点讨论这些GDNF作用的不同策略以及机制。     

Rapid down-regulation of Ret following exposure of dopaminergic neurons to neurotoxins

The survival and functional maintenance of vertebrate neurons depends on the availability of specific neurotrophic factors. We studied the influence of neurotrophic support on responses of dopaminergic neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin known to damage the nigrostriatal dopaminergic pathway in humans and other mammals. Treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine caused decreases in levels of Ret, a tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF) in the striatum, under the condition in which tyrosine hydroxylase was moderately decreased and the GDNF family receptor alpha1, another receptor of GDNF that is the ligand-binding subunit, were unaffected. Down-regulation of Ret was also observed in dopamine-producing PC12 cells undergoing apoptosis induced by rotenone, another toxic substance for dopaminergic neurons, while other cellular components were not affected. Ret was also extremely vulnerable to other apoptotic inducing conditions. Taken together, these results indicate that Ret, an important signal molecule in dopaminergic neurons, may be down-regulated in the early stages of neuronal degeneration caused by various neurotoxic substances, and may lead to reduced neurotrophic influences.     

GDNF Selectively Induces Microglial Activation and Neuronal Survival in CA1/CA3 Hippocampal Regions Exposed to NMDA Insult through Ret/ERK Signalling

The glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for several neuronal populations in different brain regions, including the hippocampus. However, no information is available on the: (1) hippocampal subregions involved in the GDNF-neuroprotective actions upon excitotoxicity, (2) identity of GDNF-responsive hippocampal cells, (3) transduction pathways involved in the GDNF-mediated neuroprotection in the hippocampus. We addressed these questions in organotypic hippocampal slices exposed to GDNF in presence of N-methyl-D-aspartate (NMDA) by immunoblotting, immunohistochemistry, and confocal analysis. In hippocampal slices GDNF acts through the activation of the tyrosine kinase receptor, Ret, without involving the NCAM-mediated pathway. Both Ret and ERK phosphorylation mainly occurred in the CA3 region where the two activated proteins co-localized. GDNF protected in a greater extent CA3 rather than CA1 following NMDA exposure. This neuroprotective effect targeted preferentially neurons, as assessed by NeuN staining. GDNF neuroprotection was associated with a significant increase of Ret phosphorylation in both CA3 and CA1. Interestingly, confocal images revealed that upon NMDA exposure, Ret activation occurred in microglial cells in the CA3 and CA1 following GDNF exposure. Collectively, this study shows that CA3 and CA1 hippocampal regions are highly responsive to GDNF-induced Ret activation and neuroprotection, and suggest that, upon excitotoxicity, such neuroprotection involves a GDNF modulation of microglial cell activity.     

GDNF promotes tubulogenesis of GFRalpha1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase

Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor alpha1 (GFRalpha1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRalpha1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRalpha1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRalpha1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRalpha1-positive and GFRalpha1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.     

Gas1 is related to the glial cell-derived neurotrophic factor family receptors alpha and regulates Ret signaling

The growth arrest-specific gene 1 (Gas1) protein has been proposed to function during development as an inhibitor of growth and a mediator of cell death and is also re-expressed in adult neurons during excitotoxic insult. Here we have demonstrated that the Gas1 protein shows high structural similarity to the glial cell-derived neurotrophic factor (GDNF) family receptors alpha, which mediate GDNF responses through the receptor tyrosine kinase Ret. We found that Gas1 binds Ret in a ligand-independent manner and sequesters Ret in lipid rafts. Signaling downstream of Ret is thus modified through a mechanism that involves the adaptor protein Shc as well as ERK, eventually blocking Akt activation. Consequently, when Gas1 is induced, Ret-mediated GDNF-dependent survival effects are compromised.     

Coordinated activation of autophosphorylation sites in the RET receptor tyrosine kinase: importance of tyrosine 1062 for GDNF mediated neuronal differentiation and survival.

The catalytic and signaling activities of RET, a tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF), are controlled by the autophosphorylation of several tyrosine residues in the RET cytoplasmic domain. To analyze the phosphorylation state of individual tyrosines, we generated antibodies recognizing specific phosphotyrosine sites involved in the catalytic (Tyr(905)) and downstream signaling (Tyr(1015), Tyr(1062), and Tyr(1096)) activities of this receptor. Stimulation with GDNF induced coordinated phosphorylation of the 4 tyrosine residues in neuronal cell lines and in primary cultures of sympathetic neurons isolated from rat superior cervical ganglia. Neurturin and artemin, two other members of the GDNF ligand family, also induced synchronized phosphorylation of RET tyrosines with kinetics comparable to those observed with GDNF. Tyrosine phosphorylation was maximal 15 min after ligand stimulation, decaying thereafter with similar kinetics in all 4 residues. Co-stimulation with a soluble form of the GFRalpha1 co-receptor potentiated ligand-dependent phosphorylation of different intracellular tyrosines to a similar extent and increased the survival of superior cervical ganglion neurons compared with treatment with GDNF alone. In vivo, high levels of phosphorylated Tyr(905), Tyr(1015), and Tyr(1062) were detected in embryonic mouse dorsal root ganglia, with a sharp decline at early postnatal stages. Protein transduction of anti-Tyr(P)(1062) antibodies into cultured cells reduced activation of MAPKs ERK1 and ERK2 and the AKT kinase in response to GDNF and diminished GDNF-dependent neuronal differentiation and survival of embryonic sensory neurons from the nodose ganglion. These results demonstrate synchronized utilization of individual RET tyrosine residues in neurons in vivo and reveal an important role for RET Tyr(1062) in mediating neuronal survival by GDNF.     

Neurturin is a neurotrophic factor for penile parasympathetic neurons in adult rat

Neurturin (NRTN), a member of the GDNF family of neurotrophic factors, promotes the survival and function of several neuronal populations in the peripheral and central nervous system. Recent gene ablation studies have shown that NRTN is a neurotrophic factor for many cranial parasympathetic and enteric neurons, whereas its significance for the sacral parasympathetic neurons has not been studied. NRTN signals via a receptor complex composed of the high-affinity binding receptor component GFRalpha2 and the transmembrane tyrosine kinase Ret. The aim of this study was to determine whether NRTN could be an endogenous trophic factor for penis-projecting parasympathetic neurons. NRTN mRNA was expressed in smooth muscle of penile blood vessels and corpus cavernosum in adult rat as well as in several intrapelvic organs, whereas GFRalpha2 and Ret mRNAs were expressed in virtually all cell bodies of the penile neurons, originating in the major pelvic ganglia. (125)I-NRTN injected into the shaft of the penis was retrogradely transported into the major pelvic and dorsal root ganglia. Mice lacking the GFRalpha2 receptor component had significantly less nitric oxide synthase-containing nerve fibers in the dorsal penile and cavernous nerves. In conclusion, these data suggest that NRTN acts as a target-derived survival and/or neuritogenic factor for penile erection-inducing postganglionic neurons.     

Novel role of neuronal Ca2+ sensor-1 as a survival factor up-regulated in injured neurons

A molecular basis of survival from neuronal injury is essential for the development of therapeutic strategy to remedy neurodegenerative disorders. In this study, we demonstrate that an EF-hand Ca2+-binding protein neuronal Ca2+ sensor-1 (NCS-1), one of the key proteins for various neuronal functions, also acts as an important survival factor. Overexpression of NCS-1 rendered cultured neurons more tolerant to cell death caused by several kinds of stressors, whereas the dominant-negative mutant (E120Q) accelerated it. In addition, NCS-1 proteins increased upon treatment with glial cell line-derived neurotrophic factor (GDNF) and mediated GDNF survival signal in an Akt (but not MAPK)-dependent manner. Furthermore, NCS-1 is significantly up-regulated in response to axotomy-induced injury in the dorsal motor nucleus of the vagus neurons of adult rats in vivo, and adenoviral overexpression of E120Q resulted in a significant loss of surviving neurons, suggesting that NCS-1 is involved in an antiapoptotic mechanism in adult motor neurons. We propose that NCS-1 is a novel survival-promoting factor up-regulated in injured neurons that mediates the GDNF survival signal via the phosphatidylinositol 3-kinase-Akt pathway.     

The isolectin IB4 binds RET receptor tyrosine kinase in microglia

Ret receptor tyrosine kinase is the signaling component of the receptor complex for the family ligands of the glial cell line‐derived neurotrophic factor (GDNF). Ret is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons, and it is necessary for the post‐natal maintenance of dopaminergic neurons. Ret expression has been as well demonstrated on microglia and several evidence indicate that GDNF regulates not only neuronal survival and maturation but also certain functions of microglia in the brain. Here, we demonstrated that the plant lectin Griffonia (Bandeiraea) simplicifolia lectin I, isolectin B4 (IB4), commonly used as a microglial marker in the brain, binds to the glycosylated extracellular domain of Ret on the surface of living NIH3T3 fibroblasts cells stably transfected with Ret as well as in adult rat brain as revealed by immunoblotting. Furthermore, confocal immunofluorescence analysis demonstrated a clear overlap in staining between pRet and IB4 in primary microglia cultures as well as in adult rat sections obtained from control or post‐ischemic brain after permanent middle artery occlusion (pMCAO). Interestingly, IB4 staining identified activated or ameboid Ret‐expressing microglia under ischemic conditions. Collectively, our data indicate Ret receptor as one of the IB4‐reactive glycoconjugate accounting for the IB4 stain in microglia under physiological and ischemic conditions.     

Dok-6, a Novel p62 Dok family member, promotes Ret-mediated neurite outgrowth

Activation of Ret, the receptor-tyrosine kinase for the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), results in the recruitment and assembly of adaptor protein complexes that function to transduce signals downstream of the receptor. Here we identify Dok-6, a novel member of the Dok-4/5 subclass of the p62 Dok family of intracellular adaptor molecules, and characterize its interaction with Ret. Expression analysis reveals that Dok-6 is highly expressed in the developing central nervous system and is co-expressed with Ret in several locations, including sympathetic, sensory, and parasympathetic ganglia, as well as in the ureteric buds of the developing kidneys. Pull-down assays using the Dok-6 phosphotyrosine binding (PTB) domain and GDNF-activated Ret indicate that Dok-6 binds to the phosphorylated Ret Tyr(1062) residue. Moreover, ligand activation of Ret resulted in phosphorylation of tyrosine residue(s) located within the unique C terminus of Dok-6 predominantly through a Src-dependent mechanism, indicating that Dok-6 is a substrate of the Ret-Src signaling pathway. Interestingly, expression of Dok-6 potentiated GDNF-induced neurite outgrowth in GDNF family receptor alpha1 (GFRalpha1)-expressing Neuro2A cells that was dependent upon the C-terminal residues of Dok-6. Taken together, these data identify Dok-6 as a novel Dok-4/5-related adaptor molecule that may function in vivo to transduce signals that regulate Ret-mediated processes such as axonal projection.