RET overactivation leads to concurrent Hirschsprung disease and intestinal ganglioneuromas.

Appropriately balanced RET signaling is of crucial importance during embryonic neural crest cell migration, proliferation and differentiation. RET deficiency, for example, leads to intestinal aganglionosis (Hirschsprung disease), whereas overactive RET can lead to multiple endocrine neoplasia (MEN) syndromes. Some RET mutations are associated with both intestinal aganglionosis and MEN-associated tumors. This seemingly paradoxical occurrence has led to speculation of a 'Janus mutation' in RET that causes overactivation or impairment of RET activity depending on the cellular context. Using an intestinal catenary culture system to test the effects of GDNF-mediated RET activation, we demonstrate the concurrent development of distal colonic aganglionosis and intestinal ganglioneuromas. Interestingly, the tumors induced by GDNF stimulation contain enteric neuronal progenitors capable of reconstituting an enteric nervous system when transplanted into a normal developmental environment. These results suggest that a Janus mutation may not be required to explain co-existing Hirschsprung disease and MEN-associated tumors, but rather that RET overstimulation alone is enough to cause both phenotypes. The results also suggest that reprogramming tumor cells toward non-pathological fates may represent a possible therapeutic avenue for MEN-associated neoplasms.

Levels of glial cell line-derived neurotrophic factor are decreased, but fibroblast growth factor 2 and cerebral dopamine neurotrophic factor are increased in the hippocampus in Parkinson's disease.

Growth factors can facilitate hippocampus-based learning and memory and are potential targets for treatment of cognitive dysfunction via their neuroprotective and neurorestorative effects. Dementia is common in Parkinson's disease (PD), but treatment options are limited. We aimed to determine if levels of growth factors are altered in the hippocampus of patients with PD, and if such alterations are associated with PD pathology. Enzyme-linked immunosorbent assays were used to quantify seven growth factors in fresh frozen hippocampus from 10 PD and nine age-matched control brains. Western blotting and immunohistochemistry were used to explore cellular and inflammatory changes that may be associated with growth factor alterations. In the PD hippocampus, protein levels of glial cell line-derived neurotrophic factor were significantly decreased, despite no evidence of neuronal loss. In contrast, protein levels of fibroblast growth factor 2 and cerebral dopamine neurotrophic factor were significantly increased in PD compared to controls. Levels of the growth factors epidermal growth factor, heparin-binding epidermal growth factor, brain-derived neurotrophic factor and mesencephalic astrocyte-derived neurotrophic factor did not differ between groups. Our data demonstrate changes in specific growth factors in the hippocampus of the PD brain, which potentially represent targets for modification to help attenuate cognitive decline in PD. These data also suggest that multiple growth factors and direction of change needs to be considered when approaching growth factors as a potential treatment for cognitive decline.

Enteric glial-mediated enhancement of intestinal barrier integrity is compromised by morphine.

BACKGROUND: The opioid epidemic is a growing concern, and emerging evidence suggests that morphine use may be associated with sepsis. Enteric glial cells (EGCs) are the most numerous cell type in the enteric nervous system and regulate gastrointestinal function through the production of trophic factors, including glial-derived neurotrophic factor (GDNF). We sought to determine the effect of morphine on enteric glia and hypothesized that morphine contributes to EGC dysfunction and increased gut permeability. MATERIALS AND METHODS: Rat intestinal epithelial cells (IECs) and EGC lines were purchased from ATCC. Immunocytochemistry was used to evaluate the impact of EGCs on IEC barrier proteins and detect the µ-opioid receptor. Co-culture assays were used to determine the effect of EGCs, GDNF, and morphine on barrier integrity. Quantitative polymerase chain reaction and western blotting were performed to determine the impact of morphine in GDNF production. Transepithelial resistance of IEC-6 cell monolayers was measured in the presence of EGC-conditioned media (EGC-CM) and morphine treated EGC-CM using electrical cell impedance sensing. RESULTS: EGC-CM enhanced tight junction organization in IECs. IEC barrier integrity was enhanced when co-cultured with unstimulated EGCs or with GDNF alone; this barrier protective effect was lost with morphine-treated EGCs. GDNF RNA and protein expression were decreased by morphine treatment. Transepithelial resistance was decreased in IEC confluent monolayers when exposed to morphine-treated EGC-CM compared with control. CONCLUSIONS: Morphine compromises intestinal epithelial cell barrier function through a mechanism which appears to involve GDNF. Further studies are warranted to delineate the role of enteric glial cell function in opioid signaling and sepsis.

ILC3s and the Willow Tree of Voices.

Type 3 innate lymphoid cells (ILC3s) and enteric glia, an essential structural component of gut innervation, are well-known regulators of intestinal homeostasis. Ibiza et al. (2016) uncover a new link between commensal bacteria, enteric glial cells, and ILC3s that is required for intestinal homeostasis and defense.

Lipid Rafts Are Physiologic Membrane Microdomains Necessary for the Morphogenic and Developmental Functions of Glial Cell Line-Derived Neurotrophic Factor In Vivo.

Glial cell line-derived neurotrophic factor (GDNF) promotes PNS development and kidney morphogenesis via a receptor complex consisting of the glycerophosphatidylinositol (GPI)-anchored, ligand binding receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Although Ret signal transduction in vitro is augmented by translocation into lipid rafts via GFRα1, the existence and importance of lipid rafts in GDNF-Ret signaling under physiologic conditions is unresolved. A knock-in mouse was produced that replaced GFRα1 with GFRα1-TM, which contains a transmembrane (TM) domain instead of the GPI anchor. GFRα1-TM still binds GDNF and promotes Ret activation but does not translocate into rafts. In Gfrα1(TM/TM) mice, GFRα1-TM is expressed, trafficked, and processed at levels identical to GFRα1. Although Gfrα1(+/TM) mice are viable, Gfrα1(TM/TM) mice display bilateral renal agenesis, lack enteric neurons in the intestines, and have motor axon guidance deficits, similar to Gfrα1(-/-) mice. Therefore, the recruitment of Ret into lipid rafts by GFRα1 is required for the physiologic functions of GDNF in vertebrates. Significance statement: Membrane microdomains known as lipid rafts have been proposed to be unique subdomains in the plasma membrane that are critical for the signaling functions of multiple receptor complexes. Their existence and physiologic relevance has been debated. Based on in vitro studies, lipid rafts have been reported to be necessary for the function of the Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors. The receptor for GDNF comprises the lipid raft-resident, glycerophosphatidylinositol-anchored receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Here we demonstrate, using a knock-in mouse model in which GFRα1 is no longer located in lipid rafts, that the developmental functions of GDNF in the periphery require the translocation of the GDNF receptor complex into lipid rafts.

Selective stimulatory action of olfactory ensheathing glia-conditioned medium on oligodendroglial differentiation, with additional reference to signaling mechanisms.

We examined the effects of conditioned medium from olfactory ensheathing glia (OEGCM) on the differentiation of oligodendrocytes in mixed cultures of early postnatal hippocampi. Differentiation was judged from the numerical density (ND) of cells immunoreactive to 2'3' cyclic nucleotide 3'phosphodiesterase (CNPase) and O4 antibodies. NDs increased according to inverted-U dose-response curves, particularly for CNPase+ cells (9-fold at optimal dilution) and these changes were blocked by inhibitors of ERK1, p38-MAPK, and PI3K. Our results raise the possibility that OEG secreted factor(s) may counteract demyelination induced by trauma, neurodegenerative diseases, and advanced age, and should stimulate novel methods to deliver these factors and/or potentiating chemicals.

Neurotrophic factors for retinal ganglion cell neuropathy - with a special reference to diabetic neuropathy in the retina.

Diabetes mellitus is a disease with a devastating impact on population. Recent data revealed that early retinal neuropathy in patients with diabetic retinopathy involved a reduced expression of brain-derived neurotrophic factor. Retinal ganglion cells (RGC) neuropathy is a progressive optic nerve neuropathy with RGC death and axonal degeneration, and it leads to blindness in the elderly population worldwide. Thus, neuroprotective therapies that rescue damaged RGCs and inhibit the progression of RGC loss and axonal degeneration are needed. This review introduces potential neuroprotective therapies using different neurotrophic factors for damaged RGC in eyes with RGC neuropathy associated diseases.

Biological basis of neuroprotection and neurotherapeutic effects of Whole Body Periodic Acceleration (pGz).

Exercise is a well known neuroprotective and neurotherapeutic strategy in animal models and humans with brain injury and cognitive dysfunction. In part, exercise induced beneficial effects relate to endothelial derived nitric oxide (eNO) production and induction of the neurotrophins; Brain Derived Neurotrophic Factor (BDNF) and Glial Derived Neurotrophic Factor (GDNF). Whole Body Periodic Acceleration (WBPA (pGz), is the motion of the supine body headward to footward in a sinusoidal fashion, at frequencies of 100-160 cycles/min, inducing pulsatile shear stress to the vascular endothelium. WBPA (pGz) increases eNO in the cardiovascular system in animal models and humans. We hypothesized that WBPA (pGz) has neuroprotective and neurotherapeutic effects due to enhancement of biological pathways that include eNOS, BDNF and GDNF. We discuss protein expression analysis of these in brain of rodents. Animal and observational human data affirm a neuroprotective and neurotherapeutic role for WBPA (pGz). These findings suggest that WBPA (pGz) in addition to its well known beneficial cardiovascular effects can be a simple non-invasive neuroprotective and neurotherapeutic strategy with far reaching health benefits.

Glial cell line-derived neurotrophic factor induces cell migration in human oral squamous cell carcinoma.

OBJECTIVES: Perineural invasion is a prominent clinical feature of various cancers, which causes difficulty in curative resection. Glial cell-derived neurotrophic factor (GDNF), a potent neurotrophic factor, plays an important role in the invasive and metastatic behavior of various cancers. The aim of this study was to examine the role of GDNF on oral squamous cell carcinoma. MATERIALS AND METHODS: GDNF expression in tissue samples was analyzed by immunohistochemistry. Transwell assay, zymography, Western blot, reverse transcription-PCR, and electrophoretic mobility shift assay (EMSA) were carried out to assess the effects of GDNF on oral cancer cells. RESULTS: Human oral cancer tissues showed higher GDNF expression than that in normal tissues. We also found that application of human GDNF enhanced the cell migration ability of human oral cancers. Moreover, treatment with GDNF increased matrix metalloproteinase (MMP)-9 and MMP-13 expression in oral cancer. Inhibition of MMP-9 and MMP-13 in oral cancer cells by pharmacological inhibitors or neutralizing antibodies reduced GDNF-enhanced cell migration. Moreover, transfection with siRNA against MMP-13 inhibited GDNF-enhanced cell migration. Treatment with GDNF also increased ERK, p38 and JNK phosphorylation, and AP-1 DNA binding activity in human oral cancer cells. Inhibition of MAP kinase or AP-1 also reduced GDNF-induced oral cancer cell migration. In migration-prone sublines, oral cancer cells showed a higher migration ability than that of the original oral cancer cells. Surprisingly, the enhancement of cell migratory activity in migration-prone sublines was reduced by a GDNF-neutralizing antibody. Importantly, migration-prone sublines of oral cancer revealed higher GDNF expression. CONCLUSION: These results indicate a regulatory effect on cell migration by GDNF in oral squamous cancer.

Therapeutic potential of mesenchymal stromal cells and MSC conditioned medium in Amyotrophic Lateral Sclerosis (ALS)--in vitro evidence from primary motor neuron cultures, NSC-34 cells, astrocytes and microglia.

Administration of mesenchymal stromal cells (MSC) improves functional outcome in the SOD1G93A mouse model of the degenerative motor neuron disorder amyotrophic lateral sclerosis (ALS) as well as in models of other neurological disorders. We have now investigated the effect of the interaction between MSC and motor neurons (derived from both non-transgenic and mutant SOD1G93A transgenic mice), NSC-34 cells and glial cells (astrocytes, microglia) (derived again from both non-transgenic and mutant SOD1G93A ALS transgenic mice) in vitro. In primary motor neurons, NSC-34 cells and astrocytes, MSC conditioned medium (MSC CM) attenuated staurosporine (STS) - induced apoptosis in a concentration-dependent manner. Studying MSC CM-induced expression of neurotrophic factors in astrocytes and NSC-34 cells, we found that glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) gene expression in astrocytes were significantly enhanced by MSC CM, with differential responses of non-transgenic and mutant astrocytes. Expression of Vascular Endothelial Growth Factor (VEGF) in NSC-34 cells was significantly upregulated upon MSC CM-treatment. MSC CM significantly reduced the expression of the cytokines TNFα and IL-6 and iNOS both in transgenic and non-transgenic astrocytes. Gene expression of the neuroprotective chemokine Fractalkine (CX3CL1) was also upregulated in mutant SOD1G93A transgenic astrocytes by MSC CM treatment. Correspondingly, MSC CM increased the respective receptor, CX3CR1, in mutant SOD1G93A transgenic microglia. Our data demonstrate that MSC modulate motor neuronal and glial response to apoptosis and inflammation. MSC therefore represent an interesting candidate for further preclinical and clinical evaluation in ALS.

Structural studies of GDNF family ligands with their receptors-Insights into ligand recognition and activation of receptor tyrosine kinase RET.

RET is the receptor for glial cell line-derived neurotrophic factor family of ligands (GFLs). It is different from most other members in the receptor tyrosine kinase (RTK) family with the requirement of a co-receptor, GFRα, for ligand recognition and activation. Through the common signal transducer RET, GFLs are crucial for the development and maintenance of distinct sets of central and peripheral neurons, which has led to a series of studies towards understanding the structure, function and signaling mechanisms of GFLs with GFRα and RET receptors. Here I summarize our current understanding of the molecular basis underlying ligand recognition and activation of RET, focusing on the interactions of GFLs with their respective GFRα receptors, the recently determined crystal structure of RET extracellular region and a proposed GFL-GFRα-RET ternary complex model based on extensive structural, biochemical and functional data. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.

Neuroprotective cyclopentenone prostaglandins up-regulate neurotrophic factors in C6 glioma cells.

In a previous study, we developed newly synthesized arylthio derivatives of cyclopentenone prostaglandins (GIF-0642, GIF-0643, GIF-0644, GIF-0745 and GIF-0747), which are neuroprotective against both manganese toxicity in PC12 cells and glutamate toxicity in HT22 cells. In the present study, we showed that these compounds and their lead compound, NEPP11, are potent inducers of glial cell line-derived neurotrophic factor (GDNF) expression in C6 glioma cells and primary astrocytes. These neuroprotective cyclopentenone prostaglandins also induced the gene expression of nerve growth factor and, to a lesser extent, brain-derived neurotrophic factor. The induction of GDNF mRNA was transcription-dependent, and the overexpression of dominant-negative Nrf2 attenuated the ability of the (arylthio)cyclopentenone prostaglandins to stimulate GDNF gene expression. These results suggest that (arylthio)cyclopentenone prostaglandins increase GDNF gene expression partly via the Keap1/Nrf2 pathway. A growing number of reports demonstrate the importance of increasing the amounts of neurotrophic factors, especially GDNF, in neuropathological states. Although the precise mechanisms by which the GIF compounds inhibit cell death are under investigation, an increase in neurotrophic factors may contribute to the diverse pharmacological properties of (arylthio)cyclopentenone prostaglandins in vivo and will make them potentially valuable in the treatment of neurodegenerative disorders.

Role of glial cell line-derived neurotrophic factor in perineural invasion of pancreatic cancer.

Perineural invasion (PNI) is the initial infiltration of tumor cells into the retroperitoneal nerve plexus and along the nerves. It precludes curative resection, is thought to be the major cause of local recurrence following resection, and is a special metastatic route in pancreatic cancer. Glial cell line-derived neurotrophic factor (GDNF) was recently recognized as a key player in the PNI process. This review covers the most recently published studies on the role of GDNF in pancreatic cancer. We introduce the players in PNI, summarize the distribution of GDNF and its receptors in pancreatic cancer, and discuss the effects and underlying mechanism of GDNF in the PNI process. Finally, we also review some potential inhibitors for GDNF-targeted therapy.

GDNF family ligands: a potential future for Parkinson's disease therapy.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by motor dysfunction that occurs secondary to loss of dopaminergic neurons in the nigrostriatal pathway. Current pharmacotherapies focus on the replacement of lost dopamine to alleviate disease symptoms. However, over time this method of therapy loses effectiveness due to the continued death of dopaminergic neurons. Alternative strategies for the treatment of PD are aimed at modifying the disease state through the preservation of remaining dopamine neurons or even the regeneration of dopamine innervation through the use of neurotrophic factors. Neurotrophic factors are specialized proteins that can promote neuronal development, maintain neuronal health and modulate neuronal function in the ventral midbrain, making them candidates for the treatment of PD. Preclinial studies indicate that members of the glial cell line-derived neurotrophic factor family of ligands are capable of preserving the degenerating dopamine neurons. These promising results moved neurotrophic factor therapy to clinical trials in PD patients. To date, neurotrophic factor therapy is proven to be safe and well-tolerated in humans, but conclusive evidence of efficacy in the clinic remains to be determined. This review will discuss the preclinical and clinical experiments of glial cell line-derived neurotrophic factor family ligands for the treatment of PD.

Neuroprotective effects and mechanisms of exercise in a chronic mouse model of Parkinson's disease with moderate neurodegeneration.

The protective impact of exercise on neurodegenerative processes has not been confirmed, and the mechanisms underlying the benefit of exercise have not been determined in human Parkinson's disease or in chronic animal disease models. This research examined the long-term neurological, behavioral, and mechanistic consequences of endurance exercise in experimental chronic parkinsonism. We used a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease with moderate neurodegeneration and examined the effects of treadmill exercise on movement and balance coordination, changes in dopamine neuron biomarkers, mitochondrial functions, and neurotrophic factor activities in the nigrostriatal system. The exercise results were compared with those of the control and sedentary chronic parkinsonian animals. After 18 weeks of exercise training in the chronic parkinsonian mice, we observed a significant deterrence in the loss of neuronal dopamine-producing cells and other functional indicators. The impaired movement and balance incoordination in the chronic parkinsonian mice were also markedly reduced following exercise. Mechanistic investigations revealed that the neuronal and behavioral recovery produced by exercise in the chronic parkinsonian mice was associated with an improved mitochondrial function and an increase in the brain region-specific levels of brain-derived and glial cell line-derived neurotrophic factors. Our findings indicate that exercise not only produces neuronal and mitochondrial protection, it also boosts nigrostriatal neurotrophic factor levels in the chronic parkinsonian mice with moderate neurodegeneration. Therefore, modifying lifestyle with increased exercise activity would be a non-pharmacological neuroprotective approach for averting neurodegenerative processes, as demonstrated in experimental chronic parkinsonism.

Exercise exerts neuroprotective effects on Parkinson's disease model of rats.

Recent studies demonstrate that rehabilitation ameliorates physical and cognitive impairments of patients with stroke, spinal cord injury, and other neurological diseases and that rehabilitation also has potencies to modulate brain plasticity. Here we examined the effects of compulsive exercise on Parkinson's disease model of rats. Before 6-hydroxydopamine (6-OHDA, 20 microg) lesion into the right striatum of female SD rats, bromodeoxyuridine (BrdU) was injected to label the proliferating cells. Subsequently, at 24 h after the lesion, the rats were forced to run on the treadmill (5 days/week, 30 min/day, 11 m/min). As behavioral evaluations, cylinder test was performed at 1, 2, 3, and 4 weeks and amphetamine-induced rotational test was performed at 2 and 4 weeks with consequent euthanasia for immunohistochemical investigations. The exercise group showed better behavioral recovery in cylinder test and significant decrease in the number of amphetamine-induced rotations, compared to the non-exercise group. Correspondingly, significant preservation of tyrosine hydroxylase (TH)-positive fibers in the striatum and TH-positive neurons in the substantia nigra pars compacta (SNc) was demonstrated, compared to the non-exercise group. Additionally, the number of migrated BrdU- and Doublecortin-positive cells toward the lesioned striatum was increased in the exercise group. Furthermore, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor increased in the striatum by exercise. The results suggest that exercise exerts neuroprotective effects or enhances the neuronal differentiation in Parkinson's disease model of rats with subsequent improvement in deteriorated motor function.

Neurotrophic factor-expressing mesenchymal stem cells survive transplantation into the contused spinal cord without differentiating into neural cells.

The aim of this study was to assess the feasibility of transplanting mesenchymal stem cells (MSCs), genetically modified to express glial-derived neurotrophic factor (GDNF), to the contused rat spinal cord, and to subsequently assess their neural differentiation potential. MSCs expressing green fluorescent protein were transduced with a retroviral vector to express the neurotrophin GDNF. The transduction protocol was optimized by using green fluorescent protein-expressing retroviral constructs; approximately 90% of MSCs were transduced successfully after G418 selection. GDNF-transduced MSCs expressed the transgene and secreted growth factor into the media (approximately 12 ng/500,000 cells secreted into the supernatant 2 weeks after transduction). Injuries were established using an impactor device, which applied a given, reproducible force to the exposed spinal cord. GDNF-expressing MSCs were transplanted rostral and caudal to the site of injury. Spinal cord sections were analyzed 2 and 6 weeks after transplantation. We demonstrate that GDNF-transduced MSCs engraft, survive, and express the therapeutic gene up to 6 weeks posttransplantation, while maintaining an undifferentiated phenotype. In conclusion, transplanted MSCs have limited capacity for the replacement of neural cells lost as a result of a spinal cord trauma. However, they provide excellent opportunities for local delivery of neurotrophic factors into the injured tissue. This study underlines the therapeutic benefits associated with cell transplantation and provides a good example of the use of MSCs for gene delivery.

GDNF and GFRalpha: a versatile molecular complex for developing neurons.

The GDNF family ligands (GFLs) signal through the canonical signaling receptor Ret and a glycosyl-phosphatidylinositol-anchored co-receptor, GFRalpha. In recent years, signaling by GFLs has been shown to be more complex than originally assumed. The discrepant expression between GFRalphas and Ret has suggested the existence of additional signal-transducing GDNF receptors, such as NCAM. Here we summarize novel functions and Ret-independent signaling mechanisms for GDNF and GFRalpha, focusing on developing neurons. Emerging evidence indicates a prominent role of GDNF and GFRalpha in the control of neuroblast migration and chemoattraction and in the formation of neuronal synapses by a new mechanism of ligand-induced cell adhesion. Therefore, these data highlight the importance of this versatile molecular complex for nervous system development, function and regeneration.

Specificity in the crosstalk of TGFbeta/GDNF family members is determined by distinct GFR alpha receptors.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) are neurotrophic factors for parasympathetic neurons including ciliary ganglion (CG) neurons. Recently, we have shown that survival and signaling mediated by GDNF in CG neurons essentially requires transforming growth factor beta (TGFbeta). We have provided evidence that TGFbeta regulates the availability of the glycosyl phosphatidylinositol (GPI)-anchored GDNF receptor alpha 1 (GFRalpha1) by promoting the recruitment of the receptor to the plasma membrane. We report now that in addition to GDNF, NRTN, but not persephin (PSPN) or artemin (ARTN), is able to promote survival of CG neurons. Interestingly, in contrast to GDNF, NRTN is not dependent on cooperation with TGFbeta, but efficiently promotes neuronal survival and intracellular signaling in the absence of TGFbeta. Additional treatment with TGFbeta does not further increase the NRTN response. Both NRTN and GDNF exclusively bind to and activate their cognate receptors, GFRalpha2 and GFRalpha1, respectively, as shown by the use of receptor-specific neutralizing antibodies. Immunocytochemical staining for the two receptors on the surface of CG neurons reveals that, in contrast to the effect on GFRalpha1, TGFbeta is not required for recruitment of GFRalpha2 to the plasma membrane. Moreover, binding of radioactively labeled GDNF but not NRTN is increased upon treatment of CG neurons with TGFbeta. Disruption of TGFbeta signaling does interfere with GDNF-, but not NRTN-mediated signaling and survival. We propose a model taking into account data from GFRalpha1 crystallization and ontogenetic development of the CG that may explain the differences in TGFbeta-dependence of GDNF and NRTN.

Loss of neurturin in frog--comparative genomics study of GDNF family ligand-receptor pairs.

Four different GDNF family ligand (GFL)-receptor (GFRalpha) binding pairs exist in mammals, and they all signal via the RET receptor tyrosine kinase. However, the evolution of these molecules is poorly understood. We identified orthologs of all four GFRalpha receptors and GRAL (GDNF Receptor Alpha-Like) in all vertebrate classes, and a predicted GFR-like protein in several invertebrates. In addition, Gas1 (growth arrest-specific 1), a distant member of the GFR-superfamily, is present in both vertebrates and invertebrates. Analysis of exon structures suggests a common origin of GFR-superfamily proteins and early divergence of Gas1 from the common ancestor. Bony fishes have orthologs of all four mammalian GFLs, consistent with genome duplications in early vertebrates. Surprisingly, the clawed frog and chicken have only three GFLs: synteny analysis indicates loss of neurturin in frog and of persephin in chicken. Evolutionary trace analysis and protein structure homology modeling points at GDNF as the endogenous ligand of frog GFRalpha2.