Glial cell line-derived neurotrophic factor-induced signaling in Schwann cells

Glial cell line-derived neurotrophic factor (GDNF), a known survival factor for neurons, has recently been shown to stimulate the migration of Schwann cells (SCs) and to enhance myelination. GDNF exerts its biological effects by activating the Ret tyrosine kinase in the presence of glycosylphosphatidylinositol-linked receptor, GDNF family receptor (GFR) alpha1. In Ret-negative cells, the alternative transmembrane coreceptor is the 140-kDa isoform of neural cell adhesion molecule (NCAM) associated with a non-receptor tyrosine kinase Fyn. We confirmed that GDNF, GFRalpha1 and NCAM are expressed in neonatal rat SCs. We found that GDNF induces an increase in the partitioning of NCAM and heparan sulfate proteoglycan agrin into lipid rafts and that heparinase inhibits GDNF-signaling in SCs. In addition to activation of extracellular signal-regulated kinases, and phosphorylation of cAMP response element binding protein, we found that cAMP-dependent protein kinase A and protein kinase C are involved in GDNF-mediated signaling in SCs. Although GDNF did not promote the differentiation of purified SCs into the myelinating phenotype, it enhanced myelination in neuron-SC cocultures. We conclude that GDNF utilizes NCAM signaling pathways to regulate SC function prior to myelination and at early stages of myelin formation.     

Glial cell line-derived neurotrophic factor family ligands and their therapeutic potential

Four glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) have been characterized: GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN). These proteins support and restore multiple neuronal populations such as dopaminergic, sensory, motor, hippocampal, basal forebrain, enteric, sympathetic and parasympathetic neurons. Therefore, GFLs attracted significant attention as a potential cure for the diseases caused by neuronal injury and degeneration. Results of multiple experiments indicate that GFLs can alleviate behavioral symptoms and restore affected neurons in animal models of several neurological disorders including, among others, Parkinson’s disease (PD). During the last decade, GDNF protein and NRTN gene therapy have been tested in several clinical trials in patients with PD. Although the results of phase I clinical trials were positive, phase II clinical trials failed to reach primary end-points. Poor pharmacokinetic properties of GFLs (inability to penetrate tissues barriers, high affinity for extracellular matrix, etc.) could contribute to the absence of clear clinical benefits of these proteins for the patients. The purpose of this paper was to review therapeutic potential of GFLs and discuss possibilities to overcome difficulties associated with pharmacokinetic properties and delivery of GFLs to target neurons.     

Glial cell line-derived neurotrophic factor promotes sleep in rats and rabbits

Various growth factors (e.g., growth hormone-releasing hormone, acidic fibroblast growth factor, nerve growth factor, brain-derived neurotrophic factor, and interleukin-1) are implicated in sleep regulation. It is hypothesized that neuronal activity enhances the production of such growth factors, and they in turn form part of the sleep regulatory mechanism. Glial cell line-derived neurotrophic factor (GDNF) promotes development, differentiation, maintenance, and regeneration of neurons, and its production is induced by well-characterized sleep regulatory substances such as interleukin-1 and tumor necrosis factor. Therefore, we investigated whether GDNF would promote sleep. Twenty-six male Sprague-Dawley rats and 30 male New Zealand White rabbits were surgically implanted with electroencephalogram (EEG) and electromyogram (EMG; rats only) electrodes, a brain thermistor, and a lateral intracerebroventricular cannula. The animals were injected intracerebroventricularly with pyrogen-free saline and on a separate day with one of the following doses of GDNF: 5, 50, and 500 ng in rabbits and 50 and 500 ng in rats. The EEG, brain temperature, EMG (in rats), and motor activity (in rabbits) were recorded for 23 h after the intracerebroventricular injection. GDNF (500-ng dose) increased the time spent in nonrapid eye movement sleep in both rats and rabbits. Rapid eye movement sleep was not affected by the lower doses of GDNF but was inhibited in rabbits after the high dose. EEG slow-wave activity was not affected by GDNF. The current results provide further evidence that various growth factors are involved in sleep regulation.     

Glial cell line-derived neurotrophic factor (GDNF) is a proliferation factor for rat C6 glioma cells: evidence from antisense experiments

Growth factors play an important role in proliferation and differentiation of malignant brain gliomas in humans. Glial cell line-derived neurotrophic factor (GDNF) has been shown recently to be highly expressed in human glioblastomas and in rat glial cell lines B49 and C6. The aim of the present study was to knockdown GDNF, its receptor GFR-alpha1, and the related family member persephin by using antisense oligonucleotides and to observe the effects on cell proliferation. To enhance cellular uptake into C6 glioma cells, 15-mer phosphorothioate oligonucleotides were complexed with the cationic lipid Lipofectamine. The complex was applied for 3 x 12 hours to C6 glioma cells, and cells were allowed to recover for 24 hours after each transfection and then analyzed. This protocol markedly reduced GDNF and GFR-alpha1 protein levels in C6 glioma cells compared with control oligonucleotides. Knockdown of C6 cells with GDNF and GFR-alpha1 but not with persephin antisense oligonucleotides significantly decreased the number of C6 glioma cells and also inhibited the incorporation of bromodeoxyuridine as a sign of reduced DNA synthesis. In conclusion, it is shown that GDNF but not persephin is a potent proliferation factor for rat glioma cells. Knockdown of GDNF using antisense oligonucleotides complexed with lipids as carriers may be useful in gene therapeutic approaches in vitro and possibly also in vivo.     

Glial cell line-derived neurotrophic factor alters the growth characteristics and genomic imprinting of mouse multipotent adult germline stem cells

This study evaluated the essentiality of glial cell line-derived neurotrophic factor (GDNF) for in vitro culture of established mouse multipotent adult germline stem (maGS) cell lines by culturing them in the presence of GDNF, leukemia inhibitory factor (LIF) or both. We show that, in the absence of LIF, GDNF slows the proliferation of maGS cells and result in smaller sized colonies without any change in distribution of cells to different cell-cycle stages, expression of pluripotency genes and in vitro differentiation potential. Furthermore, in the absence of LIF, GDNF increased the expression of male germ-line genes and repopulated the empty seminiferous tubule of W/W^v mutant mouse without the formation of teratoma. GDNF also altered the genomic imprinting of Igf2, Peg1, and H19 genes but had no effect on DNA methylation of Oct4, Nanog and Stra8 genes. However, these effects of GDNF were masked in the presence of LIF. GDNF also did not interfere with the multipotency of maGS cells if they are cultured in the presence of LIF. In conclusion, our results suggest that, in the absence of LIF, GDNF alters the growth characteristics of maGS cells and partially impart them some of the germline stem (GS) cell-like characteristics.     

Glial cell line-derived neurotrophic factor alters lipid composition and protein distribution in MPP+-injured differentiated SH-SY5Y cells

Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive loss of dopaminergic neurons in the substantia nigra and striatum. Glial cell line-derived neurotrophic factor (GDNF) can effectively promote the differentiation and survival of many types of neurons, especially dopaminergic neurons, suggesting it could be a treatment for PD. Lipid rafts are highly dynamic cell membrane domains that contain numerous signal protein receptors, providing an important platform for signal transduction. Compelling evidence indicates that alterations in lipid rafts are associated with PD, and some studies have reported that GDNF can regulate the expression of caveolin-1, a lipid raft-marker protein. However, the precise effects of GDNF on lipid rafts remain unknown. We developed a cellular PD model, purified detergent-resistant membranes (membrane rafts), and performed proteomic and lipid metabolomics analyses to examine changes in lipid rafts after GDNF treatment. The results showed considerable protein and lipid alterations in response to GDNF, especially altered levels of dopamine-β-hydroxylase, heat shock 70 kDa protein, neural cell adhesion molecule, cytoskeletal proteins, and long-chain polysaturated/unsaturated fatty acids. These findings reveal a new avenue to explore the relationships between GDNF, lipid rafts, and PD and support the hypothesis that GDNF may be a useful treatment for PD.     

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for sensory neurons: Comparison with the effects of the neurotrophins

We compared the effects of glial cell line-derived neurotrophic factor (GDNF) on dorsal root ganglion (DRG) sensory neurons to that of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3). All of these factors were retrogradely transported to sub-populations of sensory neuron cell bodies in the L4/L5 DRG of neonatal rats. The size distribution of ¹²⁵I-GDNF-labeled neurons was variable and consisted of both small and large DRG neurons (mean of 506.60 μm²). ¹²⁵I-NGF was preferentially taken up by small neurons with a mean cross-sectional area of 383.03 μm². Iodinated BDNF and NT-3 were transported by medium to large neurons with mean sizes of 501.48 and 529.27 μm², respectively. A neonatal, sciatic nerve axotomy-induced cell death model was used to determine whether any of these factors could influence DRG neuron survival in vivo. GDNF and NGF rescued nearly 100% of the sensory neurons. BDNF and NT-3 did not promote any detectable level of neuronal survival despite the fact that they underwent retrograde transport. We examined the in vitro survival-promoting ability of these factors on neonatal DRG neuronal cultures derived from neonatal rats. GDNF, NGF, and NT-3 were effective in vitro, while BDNF was not. The range of effects seen in the models described here underscores the importance of testing neuronal responsiveness in more than one model. The biological responsiveness of DRG neurons to GDNF in multiple models suggests that this factor may play a role in the development and maintenance of sensory neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 22–32, 1997.     

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of axotomized retinal ganglion cells in adult rats: comparison to and combination with brain-derived neurotrophic factor (BDNF)

Adult rat retinal ganglion cells (RGC) undergo degeneration after optic nerve transection. Studies have shown that exogenously applied neurotrophic factors such as brain-derived neurotrophic factor (BDNF) can attenuate axotomy-induced as well as developmental RGC death. Here, we examined whether glial cell line-derived neurotrophic factor (GDNF), a known neurotrophic factor for dopaminergic neurons and motor neurons, could provide neurotrophic support to RGC in adult rats. We determined whether RGC could retrogradely transport GDNF from their target tissue. After injection into the superior colliculus of adult rats, 125I-GDNF was retrogradely transported to contralateral eyes but not to ipsilateral eyes. The transport of 125I-GDNF could be blocked by coinjection of excess unlabeled GDNF, indicating that it was receptor mediated. We tested whether intravitreally applied GDNF could prevent axotomy-induced RGC degeneration. The RGC were prelabeled with Fluorogold (FG) and axotomized by intraorbital optic nerve transection. GDNF, BDNF (positive control), cytochrome c (negative control), or a GDNF/BDNF combination was injected intravitreally on days 0 and 7. On day 14, FG-labeled RGC were counted from whole-mount retinas. We found that, similar to BDNF, GDNF could significantly attenuate the degeneration of RGC in a dose-dependent fashion. Furthermore, the combination treatment of GDNF and BDNF showed better protection than either factor used individually. Our data indicate that GDNF is a neurotrophic factor for the adult rat RGC. GDNF, like BDNF, may be useful for the treatment of human RGC degenerative diseases.     

Bone marrow stromal cells produce nerve growth factor and glial cell line-derived neurotrophic factors

Bone marrow stromal cells (BMSC) have attracted interest through their possible use for cell therapy in neurological diseases. Recent reports demonstrated that these cells are able to migrate and have potential for neuronal differentiation after transplantation into brain parenchyma. The objective of this work was determine whether rat BMSC express NGF and GDNF, in order to study its potential application for treatment of neurodegenerative diseases. BMSC were harvested from male rats and cultured in DMEM supplemented with 20% fetal bovine serum. At passage 6 the total RNA was isolated using TriZol reactive. RT-PCRs to evaluate the expression of NGF and GDNF using specific primers were carried out. Our results indicate that rat BMSC have potential to produce NGF and GDNF. We have not found any report in favor of GDNF or NGF production from rat BMSC.     

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.     

Neuroprotective mechanism of glial cell line-derived neurotrophic factor in mesencephalic neurons

Glial cell line-derived neurotrophic factor (GDNF) provides neuroprotection, but its neuroprotective mechanism has not been resolved. We investigated the neuroprotective mechanism of GDNF using primary culture of the rat mesencephalon. Bleomycin sulfate (BLM) and L-buthionine-[S,R]-sulfoximine (BSO) caused apoptosis in both dopaminergic and nondopaminergic neurons, as revealed by the presence of chromatin condensation, and positive staining by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL). GDNF preincubation blocked the neurotoxicity and reduced the number of the TUNEL-positive cells caused by BLM and BSO exposure. In contrast, GDNF did not provide neuroprotection against glutamate toxicity, which was not accompanied by these apoptotic features. The neuroprotection was mediated by phosphatidylinositol 3-kinase, an effector downstream from c-Ret, because it was blocked by LY294002. GDNF pretreatment caused up-regulation of Bcl-2 and Bcl-x. Furthermore, GDNF suppressed oxygen radical accumulation caused by BLM. Apoptosis induced by BLM and BSO was blocked by a caspase-3 inhibitor. Caspase-3 activity was elevated by BLM and suppressed by GDNF pretreatment. These findings indicate that GDNF has no effect on necrosis but exerts protection against apoptosis by activation of phosphatidylinositol 3-kinase and the subsequent up-regulation of Bcl-2 and Bcl-x, which suppresses accumulation of oxygen radicals followed by caspase-3 activation.     

Glial cell line-derived neurotrophic factor is expressed in penis of adult rat and retrogradely transported in penile parasympathetic and sensory nerves

Glial cell line-derived neurotrophic factor (GDNF), 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. In the present study, expression of GDNF mRNA in the shaft of adult rat penis is demonstrated. In situ hybridization revealed GDNF mRNA expression in cells lying in the narrow zone between the tunica albuginea and the cavernous tissue. Most subtunical cells exhibited immunoreactivity for vimentin and S100 beta, but they did not stain for smooth muscle alpha actin or PGP9.5. This suggests that the GDNF mRNA-expressing cells may have a mesenchymal origin. Also retrograde axonal transport of intracavernously injected 125I-labeled GDNF in penile parasympathetic and sensory neurons is shown. The transport was inhibited by excess unlabeled GDNF, whereas excess cytochrome c had no effect. This is in agreement with the view that the transport was mediated by binding to specific receptors located on axon terminals. In addition, this study demonstrates expression of GDNF family receptor-alpha 3 (GFR alpha 3) mRNA in most adrenergic, but only in a minor part (5.3%) of the penis-projecting adult rat major pelvic ganglion neurons, as well as in almost half (45.6%) of the penile S1 dorsal root ganglion neurons. In conclusion, the present data suggest that GDNF may act as a neurotrophic factor for subpopulations of adult rat penile parasympathetic and sensory neurons.     

Glial cell line-derived neurotrophic factor reverses ethanol-mediated increases in tyrosine hydroxylase immunoreactivity via altering the activity of heat shock protein 90

We previously found that glial cell line-derived neurotrophic factor (GDNF) in the midbrain ventral tegmental area (VTA) negatively regulates alcohol drinking (He, D. Y., McGough, N. N., Ravindranathan, A., Jeanblanc, J., Logrip, M. L., Phamluong, K., Janak, P. H., and Ron, D. (2005) J. Neurosci. 25, 619-628). Several studies suggest a role for GDNF in the regulation of tyrosine hydroxylase (TH) levels in the midbrain (Georgievska, B., Kirik, D., and Bjorklund, A. (2004) J. Neurosci. 24, 6437-6445). Up-regulation of TH levels has been reported as a hallmark of biochemical adaptations to in vivo chronic exposure to drugs of abuse, including ethanol (Ortiz, J., Fitzgerald, L. W., Charlton, M., Lane, S., Trevisan, L., Guitart, X., Shoemaker, W., Duman, R. S., and Nestler, E. J. (1995) Synapse 21, 289-298). We hypothesized that GDNF plays an important role in regulating prolonged ethanol-mediated increases in TH protein levels. Using the SH-SY5Y dopaminergic-like cell line, we found that the increase in TH levels in the presence of ethanol required the activation of the cAMP/PKA pathway and was reversed by GDNF. Ethanol treatment did not alter the mRNA level or protein translation of TH, but enhanced the stability of the protein that was decreased by GDNF. Interestingly, we observed that ethanol treatment resulted in an increase in TH association with the chaperone heat shock protein (HSP90) that was mediated by the cAMP/PKA pathway and inhibited by GDNF. Taken together, these data suggest that prolonged ethanol exposure leads to increased association of TH and HSP90 via the cAMP/PKA pathway, resulting in the stabilization and subsequent accumulation of TH. GDNF reverses this ethanol-mediated adaptation by inhibiting the interaction of TH with HSP90.