Glial Cell Line-derived Neurotrophic Factor Family Receptor Α1 Research Articles

Small-molecule agonists of the RET receptor tyrosine kinase activate biased trophic signals that are influenced by the presence of GFRa1 co-receptors

Glial cell line-derived neurotrophic factor (GDNF) is a growth factor that regulates the health and function of neurons and other cells. GDNF binds to GDNF family receptor α1 (GFRa1), and the resulting complex activates the RET receptor tyrosine kinase and subsequent downstream signals. This feature restricts GDNF activity to systems in which GFRa1 and RET are both present, a scenario that may constrain GDNF breadth of action. Furthermore, this co-dependence precludes the use of GDNF as a tool to study a putative functional cross-talk between GFRa1 and RET. Here, using biochemical techniques, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and immunohistochemistry in murine cells, tissues, or retinal organotypic cultures, we report that a naphthoquinone/quinolinedione family of small molecules (Q compounds) acts as RET agonists. We found that, like GDNF, signaling through the parental compound Q121 is GFRa1-dependent. Structural modifications of Q121 generated analogs that activated RET irrespective of GFRa1 expression. We used these analogs to examine RET-GFRa1 interactions and show that GFRa1 can influence RET-mediated signaling and enhance or diminish AKT Ser/Thr kinase or extracellular signal-regulated kinase signaling in a biased manner. In a genetic mutant model of retinitis pigmentosa, a lead compound, Q525, afforded sustained RET activation and prevented photoreceptor neuron loss in the retina. This work uncovers key components of the dynamic relationships between RET and its GFRa co-receptor and provides RET agonist scaffolds for drug development.

Expression of Glial Cell Line-derived Neurotrophic Factor in the Human Intervertebral Disc.

Biochemical and immunohistochemical analyses by the human intervertebral disc (IVD) cells and tissues. To examine the expression of glial cell line-derived neurotrophic factor (GDNF) and its receptors, GDNF family receptor (GFR) α1 and rearranged during transfection (RET) in the human IVD cells and the tissues with the early and advanced stages of degeneration. The neurotrophin family, including nerve growth factor, has been reported to be expressed in the IVDs and plays a role in hyperalgesia and neuronal sensitization. Despite having properties similar to the nerve growth factor, the expression of GDNF in the IVD remains unknown. Human IVD cells were cultured in monolayer. Immunohistochemical analyses and western blotting were performed to examine the protein levels of GDNF and its receptors. To examine the effect of proinflammatory cytokines, cells were cultured in the presence of interleukin-1β (IL-1β). The immunohistochemical expression of these proteins was also evaluated using human IVD tissues with different stages of degeneration. Immunofluorescent reactivity against anti-GDNF, GFRα1, and RET antibodies was identified in human IVD cells. In protein extracts from IVD cells, those protein expressions were also identified by Western blot. IL-1β significantly stimulated the mRNA expression of GDNF compared with that of the control group. There was no significant effect of IL-1β on the mRNA expression of GFRα1 and RET. The percentage of GDNF-immunopositive cells in advanced degenerated discs was significantly higher than that in early degenerated discs, whereas those of GFRα1 and RET showed no significant differences. GDNF and its receptors were constitutively expressed in the human IVD cells. GDNF expression was significantly enhanced by proinflammatory stimuli, and in the microenvironment with advanced tissue degeneration. N/A.

GDNF, A Neuron-Derived Factor Upregulated in Glial Cells during Disease.

In a healthy adult brain, glial cell line-derived neurotrophic factor (GDNF) is exclusively expressed by neurons, and, in some instances, it has also been shown to derive from a single neuronal subpopulation. Secreted GDNF acts in a paracrine fashion by forming a complex with the GDNF family receptor α1 (GFRα1), which is mainly expressed by neurons and can act in cis as a membrane-bound factor or in trans as a soluble factor. The GDNF/GFRα1 complex signals through interactions with the “rearranged during transfection” (RET) receptor or via the neural cell adhesion molecule (NCAM) with a lower affinity. GDNF can also signal independently from GFRα1 by interacting with syndecan-3. RET, which is expressed by neurons involved in several pathways (nigro–striatal dopaminergic neurons, motor neurons, enteric neurons, sensory neurons, etc.), could be the main determinant of the specificity of GDNF’s pro-survival effect. In an injured brain, de novo expression of GDNF occurs in glial cells. Neuroinflammation has been reported to induce GDNF expression in activated astrocytes and microglia, infiltrating macrophages, nestin-positive reactive astrocytes, and neuron/glia (NG2) positive microglia-like cells. This disease-related GDNF overexpression can be either beneficial or detrimental depending on the localization in the brain and the level and duration of glial cell activation. Some reports also describe the upregulation of RET and GFRα1 in glial cells, suggesting that GDNF could modulate neuroinflammation.

A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes in the mouse

Group III/IV muscle afferents transduce nociceptive signals and modulate exercise pressor reflexes (EPRs). However, the mechanisms governing afferent responsiveness to dually modulate these processes are not well characterized. We and others have shown that ischemic injury can induce both nociception-related behaviors and exacerbated EPRs in the same mice. This correlated with primary muscle afferent sensitization and increased expression of glial cell line-derived neurotrophic factor (GDNF) in injured muscle and increased expression of GDNF family receptor α1 (GFRα1) in dorsal root ganglia (DRG). Here, we report that increased GDNF/GFRα1 signaling to sensory neurons from ischemia/reperfusion-affected muscle directly modulated nociceptive-like behaviors and increased exercise-mediated reflexes and group III/IV muscle afferent sensitization. This appeared to have taken effect through increased cyclic adenosine monophosphate (cAMP) response element binding (CREB)/CREB binding protein-mediated expression of the purinergic receptor P2X5 in the DRGs. Muscle GDNF signaling to neurons may, therefore, play an important dual role in nociception and sympathetic reflexes and could provide a therapeutic target for treating complications from ischemic injuries.

Glycylglycine plays critical roles in theproliferation of spermatogonial stem cells.

Glial cell line-derived neurotrophic factor (GDNF) is critical for the proliferation of spermatogonial stem cells (SSCs), but the underlying mechanisms remain poorly understood. In this study, an unbiased metabolomic analysis was performed to examine the metabolic modifications in SSCs following GDNF deprivation, and 11 metabolites were observed to decrease while three increased. Of the 11 decreased metabolites identified, glycylglycine was observed to significantly rescue the proliferation of the impaired SSCs, while no such effect was observed by adding sorbitol. However, the expression of self-renewal genes, including B-cell CLL/lymphoma 6 member B, ETS variant 5, GDNF family receptor α1 and early growth response protein 4 remained unaltered following glycylglycine treatment. This finding suggests that although glycylglycine serves an important role in the proliferation of SSCs, it is not required for the self-renewal of SSCs.

Partial Reconstruction of the Nigrostriatal Circuit along a Preformed Molecular Guidance Pathway

The overall goal of our research is to establish a preformed molecular guidance pathway to direct the growth of dopaminergic axons from embryonic ventral mesencephalon (VM), tissue placed within the substantia nigra (SN), into the striatum to reconstruct the nigrostriatal pathway in a hemi-Parkinson’s disease rat model. Guidance pathways were prepared by injecting lentivirus encoding either GFP or a combination of glial-cell-line-derived neurotrophic factor (GDNF) with either GDNF family receptor α1 (GFRα1) or netrin1. In another cohort of animals, adeno-associated virus (AAV) encoding brain-derived neurotrophic factor (BDNF) was injected within the striatum after guidance pathway formation. GDNF combined with either GFRα1 or netrin significantly increased growth of dopaminergic axons out of transplants and along the pathway, resulting in a significant reduction in the number of amphetamine-induced rotations. Retrograde tract tracing showed that the dopaminergic axons innervating the striatum were from A9 neurons within the transplant. Increased dopaminergic innervation of the striatum and improved behavioral recovery were observed with the addition of BDNF. Preformed guidance pathways using a combination of GDNF and netrin1 can be used to reconstruct the nigrostriatal pathway and improve motor recovery.

AKT3 Is a Pivotal Molecule of Cadherin-22 and GDNF Family Receptor-α1 Signal Pathways Regulating Self-Renewal in Female Germline Stem Cells.

Female germline stem cells (FGSCs) are rare population residing in cortex of ovary, with the potential to rescue female infertility caused by ovary failure. Recently, we reported that cadherin-22 (CDH22), a member of cadherin family, regulates self-renewal of mouse FGSCs via interaction with JAK-STAT signal pathway and β-catenin. In this study, the expression profiles of FGSCs and spermatogonial stem cells (SSCs) were analyzed to further reveal their similarity and difference, and AKT3 was predicted as a pivotal molecule for FGSCs self-renewal. Then, we demonstrated that CDH22 interacted with PI3K to phosphorylate AKT3 and subsequently enhanced the expression levels of N-myc and cyclin family in FGSCs to promote self-renewal. Moreover, glial cell line-derived neurotrophic factor (GDNF) was identified as an essential factor for FGSCs self-renewal with a more complicated mechanism: GDNF-GFRA1 activates AKT3 via PI3K or Src family kinase (SFK), and SFK upregulates its target genes, Bcl6b, Etv5, and Lhx1, to promote self-renewal of FGSCs. However, Src, the key intermediate factor for SSCs, was not the functional molecule of SFK family in the GDNF signal network of FGSCs. Based on the observations of bioinformatics analysis and molecular evidence, we demonstrate the underlying links of potential factors which are critical to the self-renewal in FGSC and imply the therapeutic potentials of FGSCs in cure of female infertility. Stem Cells 2019;37:1095-1107.

GDNF-Induced Downregulation of miR-145-5p Enhances Human Oocyte Maturation and Cumulus Cell Viability.

Although glial cell line-derived neurotrophic factor (GDNF) and microRNAs (miRNAs) have been shown to regulate mammalian oocyte maturation, little is known about their effects on human oocyte maturation and the underlying molecular mechanisms. To examine the effects of GDNF on both nuclear and cytoplasmic maturation in cultured immature human oocytes and to investigate the involvement of miRNAs in GDNF-induced oocyte maturation. A total of 200 human immature oocytes were used to evaluate the effects of GDNF on oocyte maturation. The involvement of miRNAs in GDNF-induced oocyte maturation was identified by comparing the miRNA expression profiles of cumulus cells (CCs) either with or without GDNF stimulation. An in vitro fertilization center at the Women's Hospital, Zhejiang University School of Medicine. Agilent human miRNA (8*60K) arrays were used to examine the miRNA expression patterns of human CCs either with or without GDNF stimulation. miR-145-5p inhibitor and mimic transfections were performed to study downstream gene expression in human CCs. During the in vitro maturation process, GDNF significantly increased the percentage of metaphase II-stage oocytes and downregulated the expression of miR-145-5p in cultured human CCs. Expression of miR-145-5p in CCs is negatively correlated with oocyte maturation. miR-145-5p mimic significantly decreased the expression of GDNF family receptor-α1, ret proto-oncogene, and epidermal growth factor receptor, whereas miR-145-5p inhibitor reversed these effects. GDNF treatment inhibited cell apoptosis in cultured CCs, and this suppressive effect was reversed by transfection with the miR-145-5p mimic. Downregulation of miR-145-5p may contribute to GDNF-induced enhancement of oocyte maturation and of cell viability against cell apoptosis.

(144) - Ischemia and reperfusion injury induces acute and chronic muscle pain-related behaviors through increased expression of the GFRα1 receptor

Muscle pain is a common complication of conditions that produce ischemia/reperfusion injuries (I/R), like peripheral artery disease (PAD) or sickle cell anemia. Previously, we have found that I/R injury in mouse induces increased pain-related behaviors that correlate with group III/IV muscle afferent sensitization and increased gene expression in the affected muscles and the dorsal root ganglia (DRG). In particular, glial cell line-derived neurotrophic factor (GDNF) expression was increased in the injured muscle tissue, while its receptor, GDNF family receptor α1 (GFRα1) was upregulated in DRGs. Both GDNF and GFRα1 have been linked to behavioral changes as well as afferent sensitization in other models of muscle pain. We hypothesized that enhanced expression of GFRα1 in sensory neurons regulates the development of pain-related behaviors after I/R. To test this, we selectively knocked down GFRα1 in mice by median and ulnar nerve injection of Penetratin1-linked GFRα1-targeting siRNAs in vivo prior to unilateral I/R injury of the forelimb. We then performed pain-related behavioral tests and single unit recordings of group III/IV muscle afferents along with real-time PCR on the I/R affected DRGs. Selective knockdown of GFRα1 expression in the DRGs effectively decreased the I/R-induced pain related behaviors and the observed reduction in mechanical thresholds in group III/IV afferents. GFRα1 inhibition also specifically prevented I/R-induced upregulation of the purinergic receptor, P2X5 and acid sensing ion channel 3 (ASIC3). Finally, repetitive I/R injury induced prolonged spontaneous pain-like behaviors (paw guarding) compared to animals with a single injury. This also correlated with increased GFRα1 expression in DRGs. These results suggest that GFRα1 upregulation may play an important role in the development of pain-related behaviors via group III/IV muscle afferent sensitization. GFRα1 and the receptors downstream of its signaling, may provide potential targets for therapies aimed at patients experiencing acute or chronic pain from ischemia/reperfusion injuries.

RET-mediated glial cell line-derived neurotrophic factor signaling inhibits mouse prostate development.

In humans and rodents, the prostate gland develops from the embryonic urogenital sinus (UGS). The androgen receptor (AR) is thought to control the expression of morphogenetic genes in inductive UGS mesenchyme, which promotes proliferation and cytodifferentiation of the prostatic epithelium. However, the nature of the AR-regulated morphogenetic genes and the mechanisms whereby AR controls prostate development are not understood. Glial cell line-derived neurotrophic factor (GDNF) binds GDNF family receptor α1 (GFRα1) and signals through activation of RET tyrosine kinase. Gene disruption studies in mice have revealed essential roles for GDNF signaling in development; however, its role in prostate development is unexplored. Here, we establish novel roles of GDNF signaling in mouse prostate development. Using an organ culture system for prostate development and Ret mutant mice, we demonstrate that RET-mediated GDNF signaling in UGS increases proliferation of mesenchyme cells and suppresses androgen-induced proliferation and differentiation of prostate epithelial cells, inhibiting prostate development. We also identify Ar as a GDNF-repressed gene and Gdnf and Gfrα1 as androgen-repressed genes in UGS, thus establishing reciprocal regulatory crosstalk between AR and GDNF signaling in prostate development.

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.

Expression patterns of gdnf and gfrα1 in rainbow trout testis

In mice, glial cell line-derived neurotrophic factor (GDNF) is essential for normal spermatogenesis and in vitro culture of spermatogonial stem cells. In murine testes, GDNF acts as paracrine factor; Sertoli cells secrete it to a subset of spermatogonial cells expressing its receptor, GDNF family receptor α1 (GFRα1). However, in fish, it is unclear what types of cells express gdnf and gfrα1. In this study, we isolated the rainbow trout orthologues of these genes and analyzed their expression patterns during spermatogenesis. In rainbow trout testes, gdnf and gfrα1 were expressed in almost all type A spermatogonia (ASG). Noticeably, unlike in mice, the expression of gdnf was not observed in Sertoli cells in rainbow trout. During spermatogenesis, the expression levels of these genes changed synchronously; gdnf and gfrα1 showed high expression in ASG and decreased dramatically in subsequent developmental stages. These results suggested that GDNF most likely acts as an autocrine factor in rainbow trout testes.

An Improved Protocol for Isolation and Culturing of Mouse Spermatogonial Stem Cells

Spermatogonial stem cells (SSCs) represent a unique testicular cell type that has the capacity for proliferating, differentiating, and transmitting genetic information. This particular cell type is a strong focus of stem cell research, with isolation and maintenance of SSCs as an important issue. Therefore, we attempted to optimize SSCs handling and to analyze different media and feeder layers, such as adult and embryonic Sertoli cells. The expression patterns of SSC-specific proteins (α6 and β1 integrins, Stra8, and DAZL) and restoration of spermatogenesis were chosen as parameters to demonstrate the efficacy of the protocol. SSCs were isolated from testes of 3- to 6-day-old mice using a magnetic activated cell-sorting system and Thy-1 antibody. After enrichment, SSCs were cultured for 7 days with different media and feeder layers. Then, SSCs were transplanted to recipient mice. Culturing on adult and embryonic Sertoli cells and in the presence of different growth factors [glial cell line-derived neurotrophic factor (GDNF), GDNF family receptor α1 (GFR-α1), and basic fibroblast growth factor (bFGF) resulted in an undifferentiated SSC phenotype with typical stem cell characteristics observed in vivo. The established co-culture model could help to improve the recovery and quality of stem cell preparation of mammalian testis.

Stage specific requirement of Gfrα1 in the ureteric epithelium during kidney development

Glial cell line-derived neurotrophic factor (GDNF) binds a coreceptor GDNF family receptor α1 (GFRα1) and forms a signaling complex with the receptor tyrosine kinase RET. GDNF-GFRα1-RET signaling activates cellular pathways that are required for normal induction of the ureteric bud (UB) from the Wolffian duct (WD). Failure of UB formation results in bilateral renal agenesis and perinatal lethality. Gfrα1 is expressed in both the epithelial and mesenchymal compartments of the developing kidney while Ret expression is specific to the epithelium. The biological importance of Gfrα1’s wider tissue expression and its role in later kidney development are unclear. We discovered that conditional loss of Gfrα1 in the WD epithelium prior to UB branching is sufficient to cause renal agenesis. This finding indicates that Gfrα1 expressed in the nonepithelial structures cannot compensate for this loss. To determine Gfrα1’s role in branching morphogenesis after UB induction we used an inducible Gfrα1-specific Cre-deletor strain and deleted Gfrα1 from the majority of UB tip cells post UB induction in vivo and in explant kidney cultures. We report that Gfrα1 excision from the epithelia compartment after UB induction caused a modest reduction in branching morphogenesis. The loss of Gfrα1 from UB-tip cells resulted in reduced cell proliferation and decreased activated ERK (pERK). Further, cells without Gfrα1 expression are able to populate the branching UB tips. These findings delineate previously unclear biological roles of Gfrα1 in the urinary tract and demonstrate its cell-type and stage-specific requirements in kidney development.

Glial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis in the cochlear spiral ganglion via neural cell adhesion molecule (NCAM)

Glial cell line-derived neurotrophic factor (GDNF) increases survival and neurite extension of spiral ganglion neurons (SGNs), the primary neurons of the auditory system, via yet unknown signaling mechanisms. In other cell types, signaling is achieved by the GPI-linked GDNF family receptor α1 (GFRα1) via recruitment of transmembrane receptors: Ret (re-arranged during transformation) and/or NCAM (neural cell adhesion molecule). Here we show that GDNF enhances neuritogenesis in organotypic cultures of spiral ganglia from 5-day-old rats and mice. Addition of GFRα1-Fc increases this effect. GDNF/GFRα1-Fc stimulation activates intracellular PI3K/Akt and MEK/Erk signaling cascades as detected by Western blot analysis of cultures prepared from rats at postnatal days 5 (P5, before the onset of hearing) and 20 (P20, after the onset of hearing). Both cascades mediate GDNF stimulation of neuritogenesis, since application of the Akt inhibitor Wortmannin or the Erk inhibitor U0126 abolished GDNF/GFRα1-Fc stimulated neuritogenesis in P5 rats. Since cultures of P5 NCAM-deficient mice failed to respond by neuritogenesis to GDNF/GFRα1-Fc, we conclude that NCAM serves as a receptor for GDNF signaling responsible for neuritogenesis in early postnatal spiral ganglion.

Neuroprotection by GDNF in the ischemic brain

The glial cell line-derived neurotrophic factor (GDNF) was first identified as a survival factor for midbrain dopaminergic neurons, but additional studies provided evidences for a role as a trophic factor for other neurons of the central and peripheral nervous systems. GDNF regulates cellular activity through interaction with glycosyl-phosphatidylinositol-anchored cell surface receptors, GDNF family receptor-α1, which might signal through the transmembrane Ret tyrosine receptors or the neural cell adhesion molecule, to promote cell survival, neurite outgrowth, and synaptogenesis. The neuroprotective effect of exogenous GDNF has been shown in different experimental models of focal and global brain ischemia, by local administration of the trophic factor, using viral vectors carrying the GDNF gene and by transplantation of GDNF-expressing cells. These different strategies and the mechanisms contributing to neuroprotection by GDNF are discussed in this review. Importantly, neuroprotection by GDNF was observed even when administered after the ischemic injury.

Cyclical and Patch-Like GDNF Distribution along the Basal Surface of Sertoli Cells in Mouse and Hamster Testes

Background and AimsIn mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs) through GDNF family receptor α1 (GFRα1). It remains unclear as to when, where and how GDNF molecules are produced and exposed to the GFRα1-positive spermatogonia in vivo.Methodology and Principal FindingsHere we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFRα1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFRα1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFRα1-positive spermatogonia. In both species, GFRα1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules.Conclusion/SignificanceOur data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFRα1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules.

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.

The Neurotrophic Effects of Glial Cell Line-Derived Neurotrophic Factor on Spinal Motoneurons Are Restricted to Fusimotor Subtypes

Glial cell line-derived neurotrophic factor (GDNF) regulates multiple aspects of spinal motoneuron (MN) development, including gene expression, target selection, survival, and synapse elimination, and mice lacking either GDNF or its receptors GDNF family receptor alpha1 (GFRalpha1) and Ret exhibit a 25% reduction of lumbar MNs at postnatal day 0 (P0). Whether this loss reflects a generic trophic role for GDNF and thus a reduction of all MN subpopulations, or a more restricted role affecting only specific MN subpopulations, such as those innervating individual muscles, remains unclear. We therefore examined MN number and innervation in mice in which Ret, GFRalpha1, or GDNF was deleted and replaced by reporter alleles. Whereas nearly all hindlimb muscles exhibited normal gross innervation, intrafusal muscle spindles displayed a significant loss of innervation in most but not all muscles at P0. Furthermore, we observed a dramatic and restricted loss of small myelinated axons in the lumbar ventral roots of adult mice in which the function of either Ret or GFRalpha1 was inactivated in MNs early in development. Finally, we demonstrated that the period during which spindle-innervating MNs require GDNF for survival is restricted to early neonatal development, because mice in which the function of Ret or GFRalpha1 was inactivated after P5 failed to exhibit denervation of muscle spindles or MN loss. Therefore, although GDNF influences several aspects of MN development, the survival-promoting effects of GDNF during programmed cell death are mostly confined to spindle-innervating MNs.

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.