Glial Cell Line-derived Neurotrophic Factor Binding Research Articles

Growth factor choice is critical for successful functionalization of nanoparticles.

Nanoparticles (NPs) show new characteristics compared to the corresponding bulk material. These nanoscale properties make them interesting for various applications in biomedicine and life sciences. One field of application is the use of magnetic NPs to support regeneration in the nervous system. Drug delivery requires a functionalization of NPs with bio-functional molecules. In our study, we functionalized self-made PEI-coated iron oxide NPs with nerve growth factor (NGF) and glial cell-line derived neurotrophic factor (GDNF). Next, we tested the bio-functionality of NGF in a rat pheochromocytoma cell line (PC12) and the bio-functionality of GDNF in an organotypic spinal cord culture. Covalent binding of NGF to PEI-NPs impaired bio-functionality of NGF, but non-covalent approach differentiated PC12 cells reliably. Non-covalent binding of GDNF showed a satisfying bio-functionality of GDNF:PEI-NPs, but turned out to be unstable in conjugation to the PEI-NPs. Taken together, our study showed the importance of assessing bio-functionality and binding stability of functionalized growth factors using proper biological models. It also shows that successful functionalization of magnetic NPs with growth factors is dependent on the used binding chemistry and that it is hardly predictable. For use as therapeutics, functionalization strategies have to be reproducible and future studies are needed.

Protein kinase C regulates self-renewal of mouse spermatogonial stem cells

Spermatogonial stem cells (SSCs) have unique abilities to self-renew and differentiate into committed cells that can finally produce sperm. Glial cell line-derived neurotrophic factor (GDNF) has been reported as a crucial factor for SSC self-renewal. However, the intracellular mechanisms of SSC self-renewal remain unclear. To investigate these mechanisms, we assessed the effect of GDNF-related signaling pathways on SSCs using an in vitro culture system combined with a transplantation technique to confirm stem cell activity and quantify stem cell numbers. Phosphoinositide-3 kinase (PI3K), Akt, Src family kinase (SFK), and protein kinase C (PKC) are downstream signaling pathways activated by GDNF binding to its receptors. Of these, little is known regarding the role of PKC signaling in SSC self-renewal. In this study, we compared the effects of PKC with other signaling pathways for SSC self-renewal. When SSCs were cultured with a chemical inhibitor of PKC for 8 days, self-renewal was rapidly prevented and the number of cultured cells decreased significantly. However, apoptosis analysis revealed that the number of apoptotic cells in the cells treated with PKC inhibitor was not significantly different from those in the control. In addition, no significant difference was observed in the differentiation of cells cultured in the presence of PKC inhibitor and untreated culture. Transplantation analysis showed colony formation in the recipient testis was similar for cells cultured with addition of each inhibitor compared with control, indicating that they are functionally normal. Collectively, these results demonstrate that PKC signaling is a crucial pathway for the selfrenewal division of SSCs.

FACS binding assay for analysing GDNF interactions

Glial cell-line derived neurotrophic factor (GDNF) is a secreted protein with great therapeutic potential. However, in order to analyse the interactions between GDNF and its receptors, researchers have been mostly dependent of radioactive binding assays. We developed a FACS-based binding assay for GDNF as an alternative to current methods. We demonstrated that the FACS-based assay using TGW cells allowed readily detection of GDNF binding and displacement to endogenous receptors. The dissociation constant and half maximal inhibitory concentration obtained were comparable to other studies using standard binding assays. Overall, this FACS-based, simple to perform and adaptable to high throughput setup, provides a safer and reliable alternative to radioactive methods.

The GTPase-activating protein Rap1GAP: A new player to modulate Ret signaling

Glial cell line-derived neurotrophic factor (GDNF) plays a critical role in orchestrating the development and maintenance of different populations of central and peripheral neurons. GDNF was initially discovered by its ability to promote the survival of ventral midbrain dopaminergic neurons 1. In addition, GDNF promotes the survival and control the differentiation of motor neurons and many peripheral neurons, including sensory and sympathetic neurons. Moreover, it is required to induce proliferation, migration and survival of enteric neurons. GDNF promotes these trophic effects through the activation of the receptor tyrosine kinase (RTK) Ret. A distinctive feature of the receptor complex for GDNF is the requirement of two types of receptors, one specialized in GDNF binding, represented by the glycosyl-phosphatidyl inositol (GPI)-linked co-receptor GFRα1, and another involved in transmembrane signaling, represented by the RTK Ret or the neural cell adhesion molecule NCAM 1, 2. Following homodimeric GDNF binding to GFRα1, Ret becomes dimerized and tyrosine phosphorylated, and triggers many different signaling pathways, including the Ras-Raf-MAPK (ERK1/2) cascade, the phosphatidylinositol-3-kinase (PI3K)-Akt, the PLC-γ and the Src signaling pathways 3.

Medium-throughput computer aided micro-island method to assay embryonic dopaminergic neuron cultures in vitro

In Parkinson's disease (PD) midbrain dopaminergic (DA) neurons degenerate and die, causing loss of motor function. Currently no therapies exist to ameliorate neurodegeneration or to restore DA neurons, although neurotrophic factors (NTFs) are promising leads. Prior in vivo studies the NTFs are routinely assessed in vitro by quantifying the survival of DA neurons from embryonic rodent midbrain cultures. Current in vitro methods are limited in terms of assay reliability, arduous workflow, low throughput, low statistical power and may obscure detection of molecules with minor yet critically important therapeutic effects. We have developed a medium-throughput, micro-island culture method. It permits analysis of 10–12 data points from a single embryo – several fold more than any previously published method – and enables comparisons of DA neurons from a single gene knockout (KO) embryo. It is computer-aided, improves statistical power and decreases the number of animals and workload per experiment. This method enhances testing capabilities of NTFs and other factors, and enables small scale screening of chemical drug libraries. We have validated the method by confirming the known effects of glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN), and demonstrated additive effects via simultaneous addition of GDNF and heparin binding growth associated molecule (HB-GAM). We also show for the first time that DA neurons isolated from GDNF receptor RET-deficient mice are still GDNF responsive, suggesting the presence of an alternative non-RET receptor for GDNF in the DA system. Finally, the method can be adapted for analyses of other low abundance neuronal systems.

Silver Nanoparticles Disrupt GDNF/Fyn kinase Signaling in Spermatogonial Stem Cells

Silver nanoparticles (Ag-NPs) are being utilized in an increasing number of fields and are components of antibacterial coatings, antistatic materials, superconductors, and biosensors. A number of reports have now described the toxic effects of silver nanoparticles on somatic cells; however, no study has examined their effects on the germ line at the molecular level. Spermatogenesis is a complex biological process that is particularly sensitive to environmental insults. Many chemicals, including ultrafine particles, have a negative effect on the germ line, either by directly affecting the germ cells or by indirectly acting on the somatic cells of the testis. In the present study, we have assessed the impact of different doses of Ag-NPs, as well as their size and biocompatible coating, on the proliferation of mouse spermatogonial stem cells (SSCs), which are at the origin of the germ line in the adult testis. At concentrations >OR= 10 microg/ml, Ag-NPs induced a significant decline in SSCs proliferation, which was also dependent on their size and coating. At the concentration of 10 microg/ml, reactive oxygen species production and/or apoptosis did not seem to play a major role; therefore, we explored other mechanisms to explain the decrease in cell proliferation. Because glial cell line-derived neurotrophic factor (GDNF) is vital for SSC self-renewal in vitro and in vivo, we evaluated the effects of Ag-NPs on GDNF-mediated signaling in these cells. Although the nanoparticles did not reduce GDNF binding or Ret receptor activity, our data revealed that already at a concentration of 10 microg/ml, silver nanoparticles specifically interact with Fyn kinase downstream of Ret and impair SSC proliferation in vitro. In addition, we demonstrated that the particle coating was degraded upon interaction with the intracellular microenvironment, reducing biocompatibility.

Heparin-binding determinants of GDNF reduce its tissue distribution but are beneficial for the protection of nigral dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) protects and repairs dopamine neurons. It binds to GDNF family receptor α1 (GFRα1) and activates receptor tyrosine kinase. Heparan sulphate proteoglycans (HSPGs) also participate in the signalling of GDNF, though binding to HS may hinder the diffusion of infused GDNF. We assessed the importance of heparin-binding determinants in the neuroprotective effects of GDNF in the 6-OHDA rat model of Parkinson's disease. We utilized a truncated, non-heparin-binding Δ38N-GDNF or combined wtGDNF with heparin-binding growth-associated molecule (HB-GAM, pleiotrophin). Tissue diffusion of wtGDNF ± HB-GAM and Δ38N-GDNF was also compared. A protective effect against ipsilateral d-amphetamine-induced turning was seen with 10 μg wtGDNF, 17 μg HB-GAM + 10 μg wtGDNF or 10 μg Δ38N-GDNF at 8 weeks post lesion. This effect was most pronounced with wtGDNF alone. HB-GAM (17 or 50 μg) also reduced rotational behaviour, but did not protect dopaminergic cells. Otherwise, the survival of TH-positive cells in the substantia nigra correlated with the behavioural data. Although Δ38N-GDNF was more widely distributed than wtGDNF (irrespective of its origin), stable in a brain extract, and potent in mitogen-activated kinase assay, it was inferior in vivo. The results imply that GDNF binding to HSs is needed for the optimum neuroprotective effect.

Insights into GFRα1 Regulation of Neural Cell Adhesion Molecule (NCAM) Function from Structure-Function Analysis of the NCAM/GFRα1 Receptor Complex

The neural cell adhesion molecule NCAM binds glial cell line-derived neurotrophic factor (GDNF) through specific determinants located in its third immunoglobulin (Ig) domain. However, high affinity GDNF binding and downstream signaling depend upon NCAM co-expression with the GDNF co-receptor GFRalpha1. GFRalpha1 promotes high affinity GDNF binding to NCAM and down-regulates NCAM-mediated homophilic cell adhesion, but the mechanisms underlying these effects are unknown. NCAM and GFRalpha1 interact at the plasma membrane, but the molecular determinants involved have not been characterized nor is it clear whether their interaction is required for GFRalpha1 regulation of NCAM function. We have investigated the structure-function relationships underlying GFRalpha1 binding to NCAM in intact cells. The fourth Ig domain of NCAM was both necessary and sufficient for the interaction of NCAM with GFRalpha1. Moreover, although the N-terminal domain of GFRalpha1 had previously been shown to be dispensable for GDNF binding, we found that it was both necessary and sufficient for the efficient interaction of this receptor with NCAM. GFRalpha1 lacking its N-terminal domain was still able to potentiate GDNF binding to NCAM and assemble into a tripartite receptor complex but showed a reduced capacity to attenuate NCAM-mediated cell adhesion. On its own, the GFRalpha1 N-terminal domain was sufficient to decrease NCAM-mediated cell adhesion. These results indicate that direct receptor-receptor interactions are not required for high affinity GDNF binding to NCAM but play an important role in the regulation of NCAM-mediated cell adhesion by GFRalpha1.

Lrig1 Is an Endogenous Inhibitor of Ret Receptor Tyrosine Kinase Activation, Downstream Signaling, and Biological Responses to GDNF

Glial cell line-derived neurotrophic factor (GDNF)/Ret signaling has potent trophic effects on ventral midbrain dopaminergic, motor, sensory, and sympathetic neurons. The molecular mechanisms that restrict Ret receptor tyrosine kinase activation are not well understood. Here, we show that Lrig1, a transmembrane protein containing leucine-rich repeats and Ig-like domains in its extracellular region, acts in a negative feedback loop to regulate the activity of Ret receptor tyrosine kinase. In particular, we demonstrate that Lrig1 is capable of physically interacting with Ret and that Lrig1/Ret association inhibits GDNF binding, recruitment of Ret to lipid rafts, receptor autophosphorylation, and mitogen-activated protein kinase (MAPK) activation in response to GDNF. In neuronal cells, Lrig1 overexpression also inhibits GDNF/Ret-induced neurite outgrowth in a cell-autonomous manner. Downregulation of Lrig1 using small interference RNA knock-down experiments potentiates both neuronal differentiation and MAPK activation in response to GDNF. Together, these results provide an insight into Lrig1 function and establish a new physiological mechanism to restrict signaling and biological responses induced by GDNF and Ret in neuronal cells.

Disruption of the GDNF Binding Site in NCAM Dissociates Ligand Binding and Homophilic Cell Adhesion

Most plasma membrane proteins are capable of sensing multiple cell-cell and cell-ligand interactions, but the extent to which this functional versatility is founded on their modular design is less clear. We have identified the third immunoglobulin domain of the Neural Cell Adhesion Molecule (NCAM) as the necessary and sufficient determinant for its interaction with Glial Cell Line-derived Neurotrophic Factor (GDNF). Four charged contacts were identified by molecular modeling as the main contributors to binding energy. Their mutation abolished GDNF binding to NCAM but left intact the ability of NCAM to mediate cell adhesion, indicating that the two functions are genetically separable. The GDNF-NCAM interface allows complex formation with the GDNF family receptor alpha1, shedding light on the molecular architecture of a multicomponent GDNF receptor.

Heparin/heparan sulphate binding in the TGF-β cytokine superfamily

The TGF-beta (transforming growth factor-beta) cytokine superfamily in mammals contains some 30 members. These dimeric proteins are characterized by a strongly conserved cystine knot-based structure. They regulate the proliferation, differentiation and migration of many cell types, and therefore have important roles in morphogenesis, organogenesis, tissue maintenance and wound healing. Thus far, around one-quarter of these cytokines have been shown to bind to heparin and heparan sulphate. Well-established examples are the TGF-beta isoforms 1 and 2, and the BMPs (bone morphogenetic proteins) -2 and -4. In studies in my laboratory, we have shown that GDNF (glial-cell-line-derived neurotrophic factor) and its close relatives neurturin and artemin bind to heparin and heparan sulphate with high affinity. We have reported previously that binding of GDNF is highly dependent on the presence of 2-O-sulphate groups. More recently, we and others have been investigating the heparin/heparan sulphate-binding properties of BMP-7, which is a representative of a distinct BMP subgroup from that of BMPs -2 and -4. Interestingly, several of the various specific BMP antagonist proteins also bind to heparin and heparan sulphate. Much remains to be learnt about the nature and role of glycosaminoglycan interactions in the TGF-beta superfamily, but current work suggests that these cytokines do not share a single highly conserved heparin/heparan sulphate-binding site.

The first cysteine-rich domain of the receptor GFRα1 stabilizes the binding of GDNF

The GDNF (glial cell line-derived neurotrophic factor)-binding receptor GFRalpha1 (GDNF family receptor alpha1) is attached to the membrane by a GPI (glycosylphosphatidylinositol) anchor and consists of three cysteine-rich domains. The region corresponding to the second and third domains has been shown previously to participate in ligand binding, and to interact with the transmembrane tyrosine kinase receptor RET. No function has so far been found for the N-terminal, first domain (D1). Here we show that the GPI-anchored full-length receptor binds 125I-GDNF two times more tightly than does a GPI-anchored truncated receptor lacking D1. Scintillation proximity assays with purified receptor proteins also show that the GDNF-binding capacity of the soluble full-length GFRalpha1 is two times higher than the GDNF-binding capacity of the soluble D1-truncated GFRalpha1. As RET stabilizes the binding of GDNF equally well to the full-length and truncated receptors, D1 seems not to be involved in the interaction between GFRalpha1 and RET. Moreover, soluble full-length GFRalpha1 mediates GDNF-promoted neurite outgrowth in PC6-3 cells more efficiently than the soluble truncated GFRalpha1 protein. At low concentrations, the soluble fulllength receptor mediates the phosphorylation of RET more efficiently than the soluble truncated receptor. However, when the receptors are overexpressed on the cell surface as GPI-anchored proteins, or added to the growth medium at high concentrations as soluble proteins, full-length and truncated GFRalpha1 are indistinguishable in GDNF-dependent RET-phosphorylation assays. High levels of the receptors can thus mask a slightly impaired function in the phosphorylation assay. Based on assays with both GPI-anchored and soluble receptors, we therefore conclude that D1 contributes to the optimal function of GFRalpha1 by stabilizing the interaction between GFRalpha1 and GDNF.

Glial Cell Line-Derived Neurotrophic Factor (GDNF) Receptor α-1 (GFRα1) Is Highly Selective for GDNF versus Artemin

To clarify whether glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFRalpha1), the glycosylphosphatidylinositol (GPI)-linked coreceptor for GDNF, is also a functional coreceptor for artemin (ART), we have studied receptor binding, signaling, and neuronal survival. In cell-free binding studies, GFRalpha1-Ig displayed strong preferential binding to GDNF, though in the presence of soluble RET, weak binding to ART could also be detected. However, using GFRalpha1-transfected NB41A3 cells, ART showed no detectable competition against the binding of (125)I-labeled GDNF. Moreover, ART failed to induce phosphorylation of extracellular signal-related kinase (ERK) and Akt in these cells and was >10(4)-fold less potent than GDNF in stimulating RET phosphorylation. When rat primary dorsal root ganglion (DRG) neurons were used, only the survival promoting activity of GDNF and not that of ART was blocked by an anti-GFRalpha1 antibody. These results indicate that although ART can interact weakly with soluble GFRalpha1 constructs under certain circumstances in vitro, in cell-based functional assays GFRalpha1 is at least 10 000-fold selective for GDNF over ART. The extremely high selectivity of GFRalpha1 for GDNF over ART and the low reactivity of ART for this receptor suggest that GFRalpha1 is not likely to be a functional coreceptor for ART in vivo.

The structure of GFRalpha1 domain 3 reveals new insights into GDNF binding and RET activation.

Glial cell line-derived neurotrophic factor (GDNF) binds to the GDNF family co-receptor alpha1 (GFRalpha1) and activates RET receptor tyrosine kinase. GFRalpha1 has a putative domain structure of three homologous cysteine-rich domains, where domains 2 and 3 make up a central domain responsible for GDNF binding. We report here the 1.8 A crystal structure of GFRalpha1 domain 3 showing a new protein fold. It is an all-alpha five-helix bundle with five disulfide bridges. The structure was used to model the homologous domain 2, the other half of the GDNF-binding fragment, and to construct the first structural model of the GDNF-GFRalpha1 interaction. Using site-directed mutagenesis, we identified closely spaced residues, Phe213, Arg224, Arg225 and Ile229, comprising a putative GDNF-binding surface. Mutating each one of them had slightly different effects on GDNF binding and RET phosphorylation. In addition, the R217E mutant bound GDNF equally well in the presence and absence of RET. Arg217 may thus be involved in the allosteric properties of GFRalpha1 or in binding RET.

The binding of human glial cell line-derived neurotrophic factor to heparin and heparan sulfate: importance of 2-O-sulfate groups and effect on its interaction with its receptor, GFRalpha1.

We report ELISA studies of the glycosaminoglycan binding properties of recombinant human glial cell line-derived neurotrophic factor (GDNF). We demonstrate relatively high affinity binding as soluble heparin competes with an IC50 of 0.1 micro g/ml. The binding of GDNF to heparin is particularly dependent on the presence of 2-O-sulfate groups. Highly sulfated heparan sulfate is also an effective competitor for GDNF binding. We also show that heparin at low concentrations protects GDNF from proteolytic modification by an endoprotease and also promotes the binding of GDNF to its receptor polypeptide, GFRalpha1. In both of these actions, 2-O-desulfated heparin is less effective. Considered overall, these findings provide strong support for a hypothesis that the bioactivity of GDNF during prenatal development is essentially dependent on the binding of this growth factor to 2-O-sulfate-rich heparin-related glycosaminoglycan.

XIB4035, a novel nonpeptidyl small molecule agonist for GFRα-1

We investigated the agonistic activities of N4-{7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl}-N1,N1-diethyl-pentane-1,4-diamine (XIB4035), at the glial cell line-derived neurotrophic factor (GDNF) family receptorα-1(GFRα-1) in Neuro-2A cells, a mouse neuroblastoma cell line which is a suitable model for investigating functions mediated through GFRα-1. XIB4035 concentration-dependently inhibited [125I]GDNF binding in Neuro-2A cells with an IC50 of 10.4μM. GDNF induced autophosphorylation of Ret protein, and promoted neurite outgrowth in Neuro-2A cells. XIB4035, like GDNF, induced Ret autophosphorylation in the Neuro-2A cells. Moreover, XIB4035 promoted neurite outgrowth in a concentration-dependent manner. These results show that XIB4035 may act as an agonist at GFRα-1 receptor complex, and mimic neurotrophic effects of GDNF in Neuro-2A cells. This is an interesting finding showing that a nonpeptidyl small molecule is capable of inducing activation of a receptor that normally bind a relatively large protein ligand such as GDNF.

Molecular Modeling of the Extracellular Domain of the RET Receptor Tyrosine Kinase Reveals Multiple Cadherin-like Domains and a Calcium-binding Site

Using bioinformatic tools, mutagenesis, and binding studies, we have investigated the structural organization of the extracellular region of the RET receptor tyrosine kinase, a functional receptor for glial cell line-derived neurotrophic factor (GDNF). Multiple sequence alignments of seven vertebrate sequences and one invertebrate RET sequence delineated four distinct N-terminal domains, each of about 110 residues, containing many of the consensus motifs of the cadherin fold. Based on these alignments and the crystal structures of epithelial and neural cadherins, we have generated molecular models of each of the four cadherin-like domains in the extracellular region of human RET. The modeled structures represent realistic models from both energetic and geometrical points of view and are consistent with previous observations gathered from biochemical analyses of the effects of Hirschsprung's disease mutations affecting the folding and stability of the RET molecule, as well as our own site-directed mutagenesis studies of RET cadherin-like domain 1. We have also investigated the role of Ca(2+) in ligand binding by RET and found that Ca(2+) ions are required for RET binding to GDNF but not for GDNF binding to the GFRalpha1 co-receptor. In agreement with these results, RET, but not GFRalpha1, was found to bind Ca(2+) directly. Our results indicate that the overall architecture of the extracellular region of RET is more closely related to cadherins than previously thought. The models of the cadherin-like domains of human RET represent valuable tools with which to guide future site-directed mutagenesis studies aimed at identifying residues involved in ligand binding and receptor activation.

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.

Binding of GDNF and Neurturin to Human GDNF Family Receptor α 1 and 2: INFLUENCE OF cRET AND COOPERATIVE INTERACTIONS

The members of the glial cell line-derived neurotrophic factor (GDNF) family signal via binding to the glycosyl phosphatidylinositol-anchored membrane proteins, the GDNF family receptors alpha (GFRalpha), and activation of cRET. We performed a detailed analysis of the binding of GDNF and neurturin to their receptors and investigated the influence of cRET on the binding affinities. We show that the rate of dissociation of (125)I-GDNF from GFRalpha1 is increased in the presence of 50 nm GDNF, an effect that can be explained by the occurrence of negative cooperativity. Scatchard plots of the ligand concentration binding isotherms reveal a pronounced downward curvature at low (125)I-GDNF concentrations suggesting the presence of positive cooperativity. This effect is observed in the range of GDNF concentrations responsible for biological activity (1-20 pm) and may have an important role in cRET-independent signaling. A high affinity site with a K(D) of 11 pm for (125)I-GDNF is detected only when GFRalpha1 is co-expressed with cRET at a DNA ratio of 1:3. These results suggest an interaction of GFRalpha1 and cRET in the absence of GDNF and demonstrate that the high affinity binding can be measured only when cRET is present.

Expression of GFRalpha-1, receptor for GDNF, in rat brain capillary during postnatal development of the BBB.

It is well known that the blood-brain barrier (BBB) matures at approximately 2 wk after birth in the rat. Recently, we showed that glial cell line-derived neurotrophic factor (GDNF) enhances the barrier function of porcine endothelial cells forming the BBB in culture. In the present study, we examined the relation between permeability of the BBB, using Evans blue as a tracer, and expression of the GDNF family receptor (GFRalpha-1) during postnatal development of the BBB. Morphometric analysis showed that exudation of Evans blue from capillaries of the cerebral cortex progressively decreased until postnatal day 21. Inversely, immunohistochemical examinations showed expression of GFRalpha-1 in the capillaries at postnatal day 3 and expression that reached the same levels as observed in adult rats by postnatal day 10. However, c-ret, which is thought to mediate a signal evoked by binding of GDNF to GFRalpha-1, was not expressed in the capillaries of the brain cortex in 3-mo-old rats. On the other hand, the tight junction proteins occludin and ZO-1 appeared to be fully expressed at birth. The reciprocal relation between GFRalpha-1 expression and the permeability of the BBB strongly suggests active participation of GDNF in postnatal development of the BBB, although the mechanism(s) involved is still veiled.