Exogenous Glial Cell Line-derived Neurotrophic Factor Research Articles

Glial-Cell-Line-Derived Neurotrophic Factor Promotes Glioblastoma Cell Migration and Invasion via the SMAD2/3-SERPINE1-Signaling Axis.

Glial-cell-line-derived neurotrophic factor (GDNF) is highly expressed and is involved in the malignant phenotype in glioblastomas (GBMs). However, uncovering its underlying mechanism for promoting GBM progression is still a challenging work. In this study, we found that serine protease inhibitor family E member 1 (SERPINE1) was a potential downstream gene of GDNF. Further experiments confirmed that SERPINE1 was highly expressed in GBM tissues and cells, and its levels of expression and secretion were enhanced by exogenous GDNF. SERPINE1 knockdown inhibited the migration and invasion of GBM cells promoted by GDNF. Mechanistically, GDNF increased SERPINE1 by promoting the phosphorylation of SMAD2/3. In vivo experiments demonstrated that GDNF facilitated GBM growth and the expressions of proteins related to migration and invasion via SERPINE1. Collectively, our findings revealed that GDNF upregulated SERPINE1 via the SMAD2/3-signaling pathway, thereby accelerating GBM cell migration and invasion. The present work presents a new mechanism of GDNF, supporting GBM development.

FK962 protects retinal ganglion cell under hypoxia/reoxygenation: Possible involvement of glial cell line-derived neurotrophic factor signaling pathway

Loss of retinal ganglion cells (RGCs) is the cause of visual impairment and blindness in glaucoma. Previously, our studies showed that FK962 (N-[1-acetylpiperidin-4-yl]-4-fluorobenzamide) promoted neurite elongation in rat RGCs and trigeminal ganglion (TG) cells. In TG cells, glial cell line-derived neurotrophic factor (GDNF) is known to be involved in the mechanism. The purpose of the present study is to investigate whether, 1) FK962 shows an RGC-protective effect under hypoxia/reoxygenation (H/R) and 2) GDNF is involved in the neuroprotective mechanism of FK962. Rat primary retinal cells were cultured under 24-h hypoxia/24-h reoxygenation conditions, with or without FK962, recombinant GDNF, GDNF antibody and RET receptor tyrosine kinase inhibitor, GSK3179106. Cells were co-immunostained with RBPMS and Neurofilament 200 as a RGC marker, and the number of survived RGCs was counted. Results showed H/R treatment decreased the number of survived RGCs. FK962 promoted RGC survival under H/R by a bell-shaped dose response, with the highest RGC-protective effect of 10−8 M. The protective effect was the same level with 10−12 M exogenous GDNF. Addition of GDNF antibody or GSK3179106 counteracted the neuroprotective effect of FK962. From these results, it is suggested that FK962 ameliorates RGC death under H/R, possibly via a GDNF signaling pathway.

The role of Sertoli cells-secreted factors in different stages of germ cells development in mice exposed to BDE-209

Decabromodiphenyl ether (BDE-209), a frequently used brominated flame retardant, readily enters the environment and is difficult to degrade with bioaccumulation. BDE-209 could cause male reproductive toxicity, but the regulatory functions of Sertoli cells-secreted factors remain uncertain. In present study, male mice were treated with 75 mg/kg BDE-209 and then stopped exposure for 50 days. Exogenous Glial cell line-derived neurotrophic factor (GDNF), a Sertoli cell-secreted factor, was injected into testes of mice treated with BDE-209 for 50 days to explore the role of GDNF in BDE-209-induced reproductive toxicity. The mouse spermatogonia cell line GC-1 spg was used in vitro to further verify regulatory effects of Sertoli cells-secreted factors on meiotic initiation. The results showed that BDE-209 inhibited expressions of the self-renewal pathway GFRα-1/RAS/ERK1/2 in spermatogonial stem cells (SSCs), and reduced expressions of spermatogonia proliferation-related pathway NRG3/ERBB4 and meiosis initiation factor Stra8. Furthermore, BDE-209 decreased the levels of both GDNF and retinoic acid (RA) secreted by Sertoli cells in testes. Importantly, the alterations of above indicators induced by BDE-209 did not recover after 50-day recovery period. After exogenous GDNF injection, the decreased expression of GFRα-1/RAS/ERK in SSCs was reversed. However, the level of RA and expressions of NRG3/ERBB4/Stra8 were not restored. The in vitro experimental results showed that exogenous RA reversed the reductions in NRG3/ERBB4/Stra8 and ameliorated inhibition of GC-1 spg cells proliferation induced by BDE-209. These results suggested that Sertoli cells-secreted factors play roles in regulating various stages of germ cell development. Specifically, BDE-209 affected the self-renewal of SSCs by decreasing GDNF secretion resulting in the inhibition of GFRα-1/RAS/ERK pathway; BDE-209 hindered the proliferation of spermatogonia and initiation of meiosis by inhibiting the secretion of RA and preventing RA from binding to RARα, resulting in the suppression of NRG3/ERBB4/Stra8 pathway. As a consequence, spermatogenesis was compromised, leading to persistent male reproductive toxicity.

Endogenous GDNF Is Unable to Halt Dopaminergic Injury Triggered by Microglial Activation.

Overactivation of microglial cells seems to play a crucial role in the degeneration of dopaminergic neurons occurring in Parkinson's disease. We have previously demonstrated that glial cell line-derived neurotrophic factor (GDNF) present in astrocytes secretome modulates microglial responses induced by an inflammatory insult. Therefore, astrocyte-derived soluble factors may include relevant molecular players of therapeutic interest in the control of excessive neuroinflammatory responses. However, in vivo, the control of neuroinflammation is more complex as it depends on the interaction between different types of cells other than microglia and astrocytes. Whether neurons may interfere in the astrocyte-microglia crosstalk, affecting the control of microglial reactivity exerted by astrocytes, is unclear. Therefore, the present work aimed to disclose if the control of microglial responses mediated by astrocyte-derived factors, including GDNF, could be affected by the crosstalk with neurons, impacting GDNF's ability to protect dopaminergic neurons exposed to a pro-inflammatory environment. Also, we aimed to disclose if the protection of dopaminergic neurons by GDNF involves the modulation of microglial cells. Our results show that the neuroprotective effect of GDNF was mediated, at least in part, by a direct action on microglial cells through the GDNF family receptor α-1. However, this protective effect seems to be impaired by other mediators released in response to the neuron-astrocyte crosstalk since neuron-astrocyte secretome, in contrast to astrocytes secretome, was unable to protect dopaminergic neurons from the injury triggered by lipopolysaccharide-activated microglia. Supplementation with exogenous GDNF was needed to afford protection of dopaminergic neurons exposed to the inflammatory environment. In conclusion, our results revealed that dopaminergic protective effects promoted by GDNF involve the control of microglial reactivity. However, endogenous GDNF is insufficient to confer dopaminergic neuron protection against an inflammatory insult. This reinforces the importance of further developing new therapeutic strategies aiming at providing GDNF or enhancing its expression in the brain regions affected by Parkinson's disease.

GDNF triggers proliferation of rat C6 glioma cells via the NF-κB/CXCL1 signaling pathway.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor that is characterized by its high proliferative and migratory potential, leading to a high invasiveness of this tumor type. However, the underlying mechanism of GBM proliferation and migration has not been fully elucidated. In this study, at first, we used RNA-seq together with bioinformatics technology to screen for C-X-C motif ligand 1 (CXCL1) as a proliferation-related gene. And exogenous glial cell line-derived neurotrophic factor (GDNF) induced proliferation and up-regulated the level of CXCL1 in rat C6 glioma cells determined by sqPCR and ELISA. Then, we manipulated the CXCL1 expression by using a lentiviral vector (CXCL1-RNAi) approach. By this, the proliferation of C6 cells was decreased, suggesting that CXCL1 plays a key role in proliferation in these cells. We hypothesized that exogenous GDNF promoted NF-κB nuclear translocation and therefore, analyzed the interaction of CXCL1 with NF-κB by Western Blot and immunofluorescence. Additionally, we used BAY 11-7082, a phosphorylation inhibitor of NF-κB, to elucidate NF-κB mediated the effect of GDNF on CXCL1. These results demonstrated that GDNF enhanced the proliferation of rat C6 glioma cells through activating the NF-κB/CXCL1 signaling pathway. In summary, these studies not only revealed the mechanism of action of exogenous GDNF in promoting the proliferation of C6 glioma cells but may also provide a new biological target for the treatment of malignant glioma.

GDNF to the rescue: GDNF delivery effects on motor neurons and nerves, and muscle re-innervation after peripheral nerve injuries.

Peripheral nerve injuries commonly occur due to trauma, like a traffic accident. Peripheral nerves get severed, causing motor neuron death and potential muscle atrophy. The current golden standard to treat peripheral nerve lesions, especially lesions with large (≥ 3 cm) nerve gaps, is the use of a nerve autograft or reimplantation in cases where nerve root avulsions occur. If not tended early, degeneration of motor neurons and loss of axon regeneration can occur, leading to loss of function. Although surgical procedures exist, patients often do not fully recover, and quality of life deteriorates. Peripheral nerves have limited regeneration, and it is usually mediated by Schwann cells and neurotrophic factors, like glial cell line-derived neurotrophic factor, as seen in Wallerian degeneration. Glial cell line-derived neurotrophic factor is a neurotrophic factor known to promote motor neuron survival and neurite outgrowth. Glial cell line-derived neurotrophic factor is upregulated in different forms of nerve injuries like axotomy, sciatic nerve crush, and compression, thus creating great interest to explore this protein as a potential treatment for peripheral nerve injuries. Exogenous glial cell line-derived neurotrophic factor has shown positive effects in regeneration and functional recovery when applied in experimental models of peripheral nerve injuries. In this review, we discuss the mechanism of repair provided by Schwann cells and upregulation of glial cell line-derived neurotrophic factor, the latest findings on the effects of glial cell line-derived neurotrophic factor in different types of peripheral nerve injuries, delivery systems, and complementary treatments (electrical muscle stimulation and exercise). Understanding and overcoming the challenges of proper timing and glial cell line-derived neurotrophic factor delivery is paramount to creating novel treatments to tend to peripheral nerve injuries to improve patients’ quality of life.

Cross-link regulation of precursor N-cadherin and FGFR1 by GDNF increases U251MG cell viability.

Glial cell line-derived neurotrophic factor (GDNF) is considered to be involved in the development of glioma. However, uncovering the underlying mechanism of the proliferation of glioma cells is a challenging work in progress. We have identified the binding of the precursor of N-cadherin (proN-cadherin) and GDNF on the cell membrane in previous studies. In the present study, we observed increased U251 Malignant glioma (U251MG) cell viability by exogenous GDNF (50ng/ml). We also confirmed that the high expression of the proN-cadherin was stimulated by exogenous GDNF. Concurrently, we affirmed that lower expression of proN-cadherin correlated with reduced glioma cell viability. Additionally, we observed glioma cell U251MG viability as the phosphorylation level of FGFR1 at Y653 and Y654 was increased after exogenous GDNF treatment, which led to increased interaction between proN-cadherin and FGFR1 (pY653+Y654). Our experiments presented a new mechanism adopted by GDNF supporting glioma development and indicated a possible therapeutic potential via the inhibition of proN-cadherin/FGFR1 interaction.

Glial cell line-derived neurotrophic factor supplementation promotes bovine in vitro oocyte maturation and early embryo development

Paracrine factors such as glial cell line-derived neurotrophic factor (GDNF), which was originally derived from the supernatants of a rat glioma cell line, play pivotal roles in oocyte maturation and early embryo development in mammals, such as mice, rats, pigs, sheep, and even humans. However, whether GDNF facilitates in vitro oocyte maturation or early embryo development in bovines is not yet known. We show for the first time that GDNF and its receptor, GDNF family receptor alpha-1 (GFRA1), are presented in ovarian follicles at different stages as well as during oocyte maturation and early embryo development. Immunostaining results revealed the subcellular localizations of GDNF and GFRA1 in oocytes throughout follicle development, first in germinal vesicles and during blastocyst embryo stages. The ability of exogenously applied GDNF to promote oocyte maturation and early embryo development was evaluated in culture, where we found that an optimal concentration of 50 ng/mL promotes the maturation of cumulus-oocyte complexes and the nuclei of denuded oocytes as well as the development of embryos after IVF. To further investigate the potential mechanism by which GDNF promotes oocyte maturation, bovine oocytes were treated with morpholinos targeting Gfra1. The suppression of GFRA1 presence blocked endogenous and exogenous GDNF functions, indicating that the effects of GDNF that are essential and beneficial for bovine oocyte maturation and early embryo development occur through this receptor. Furthermore, we show that supplementation with GDNF improves the efficiency of bovine IVF embryo production.

Glial Cell Line Derived Neurotrophic Factor Protein Secretion by Skeletal Muscles is Altered by Activity of L‐Type Calcium Channels

Glial cell line‐derived neurotrophic factor (GDNF) is an important signaling molecule throughout the peripheral nervous system. GDNF protein is produced and secreted by skeletal muscle cells and helps maintain motor neuron innervation at the neuromuscular junction. Treatment with exogenous GDNF prevents denervation which occurs with aging and neurodegenerative disease. The therapeutic potential of GDNF cannot be fully explored without understanding the mechanisms by which GDNF protein production is regulated. Although the specific cellular events necessary for regulation of GDNF secretion remains to be elucidated, previous studies from our laboratory have shown that increased skeletal muscle activity significantly alters GDNF protein levels in vivo and in vitro. The primary objective of this study was to determine the role of calcium in regulating GDNF protein secretion by skeletal muscle cells. Skeletal muscle cells (C2C12) were grown in culture and allowed to differentiate into myotubes which display a contractile phenotype. The following treatments were given, Nifedipine to block the voltage‐gated L‐type calcium channels, and Bay K8644 to enhance calcium influx through L‐type calcium channels. Both Western Blot and ELISA were employed for protein identification and GDNF protein quantification respectively. Preliminary results indicate that treatment of myotubes with Bay K8644 (100μM) caused an increase in GDNF secretion to 170% of that in untreated controls. Treatment of cells with Nifedipine blocked the stimulatory effect of Bay K8644, and treatment with Nifedipine alone (10μM and 100μM) inhibited GDNF secretion to 77% of that in control cells. The results suggest that secretion of GDNF protein by C2C12 myotubes is regulated by intracellular calcium levels. Acquiring a greater understanding of the role that calcium plays in regulating GDNF production may help to identify potential sites for therapeutic intervention to increase or decrease GDNF production.Support or Funding InformationNSF Grant DBI‐1062883 and NIH Grant 1R15AG022908‐01A2

GDNF-based therapies, GDNF-producing interneurons, and trophic support of the dopaminergic nigrostriatal pathway. Implications for Parkinson's disease.

The glial cell line-derived neurotrophic factor (GDNF) is a well-established trophic agent for dopaminergic (DA) neurons in vitro and in vivo. GDNF is necessary for maintenance of neuronal morphological and neurochemical phenotype and protects DA neurons from toxic damage. Numerous studies on animal models of Parkinson’s disease (PD) have reported beneficial effects of GDNF on nigrostriatal DA neuron survival. However, translation of these observations to the clinical setting has been hampered so far by side effects associated with the chronic continuous intra-striatal infusion of recombinant GDNF. In addition, double blind and placebo-controlled clinical trials have not reported any clinically relevant effect of GDNF on PD patients. In the past few years, experiments with conditional Gdnf knockout mice have suggested that GDNF is necessary for maintenance of DA neurons in adulthood. In parallel, new methodologies for exogenous GDNF delivery have been developed. Recently, it has been shown that a small population of scattered, electrically interconnected, parvalbumin positive (PV+) GABAergic interneurons is responsible for most of the GDNF produced in the rodent striatum. In addition, cholinergic striatal interneurons appear to be also involved in the modulation of striatal GDNF. In this review, we summarize current knowledge on brain GDNF delivery, homeostasis, and its effects on nigrostriatal DA neurons. Special attention is paid to the therapeutic potential of endogenous GDNF stimulation in PD.

Targeting exogenous GDNF gene to the bovine somatic cell beta-casein locus for the production of transgenic bovine animals.

Considerable attention is currently being directed toward methods for producing recombinant human proteins in the mammary glands of genetically modified transgenic livestock. However, the expression of inserted genes in transgenic animals is variable and often very low because of the randomness of the site of transgene integration. One possible strategy to avoid the expression problem associated with random integration is to use site-specific integration by targeting integration to a high expression locus and, thereby, to improve expression of the transferred gene. In the present study, we focused on glial cell line-derived neurotrophic factor (GDNF), a novel type of neurotrophic factor first cloned in 1993. Research has shown that GDNF may have potential applications in the treatment of Parkinson's disease and other diseases of the central nervous system since it acts as a protective factor for central dopaminergic neurons. Here, we constructed a gene targeting vector to knock-in the human GDNF gene at the bovine beta-casein gene locus as a first step to producing transgenic animals with a high level of expression of human GDNF protein in their mammary glands. Bovine fetal fibroblast cells were transfected with linearized pNRTCNbG by electroporation. Three cell clones were identified with successful targeting to the beta-casein locus; and were confirmed using both polymerase chain reaction analysis and sequencing. Gene-targeted cells were used as nuclear donors; a total of 161 embryos were reconstructed, 23 of which developed to the blastocyst stage. These blastocysts were transferred to 8 recipient cows, but no offspring were obtained.

Glial cell line-derived neurotrophic factor induces cell proliferation in the mouse urogenital sinus.

Glial cell line-derived neurotrophic factor (GDNF) is a TGFβ family member, and GDNF signals through a glycosyl-phosphatidylinositol-linked cell surface receptor (GFRα1) and RET receptor tyrosine kinase. GDNF signaling plays crucial roles in urogenital processes, ranging from cell fate decisions in germline progenitors to ureteric bud outgrowth and renal branching morphogenesis. Gene ablation studies in mice have revealed essential roles for GDNF signaling in urogenital development, although its role in prostate development is unclear. We investigated the functional role of GDNF signaling in the urogenital sinus (UGS) and the developing prostate of mice. GDNF, GFRα1, and RET show time-specific and cell-specific expression during prostate development in vivo. In the UGS, GDNF and GFRα1 are expressed in the urethral mesenchyme (UrM) and epithelium (UrE), whereas RET is restricted to the UrM. In each lobe of the developing prostate, GDNF and GFRα1 expression declines in the epithelium and becomes restricted to the stroma. Using a well-established organ culture system, we determined that exogenous GDNF increases proliferation of UrM and UrE cells, altering UGS morphology. With regard to mechanism, GDNF signaling in the UrM increased RET expression and phosphorylation of ERK1/2. Furthermore, inhibition of RET kinase activity or ERK kinases suppressed GDNF-induced proliferation of UrM cells but not UrE cells. We therefore propose that GDNF signaling in the UGS increases proliferation of UrM and UrE cells by different mechanisms, which are distinguished by the role of RET receptor tyrosine kinase and ERK kinase signaling, thus implicating GDNF signaling in prostate development and growth.

Glial cell line-derived neurotrophic factor promotes increased phenotypic marker expression in femoral sensory and motor-derived Schwann cell cultures

Schwann cells (SCs) secrete growth factors and extracellular matrix molecules that promote neuronal survival and help guide axons during regeneration. Transplantation of SCs is a promising strategy for enhancing peripheral nerve regeneration. However, we and others have shown that after long-term in vitro expansion, SCs revert to a de-differentiated state similar to the phenotype observed after injury. In vivo, glial cell-line derived neurotrophic factor (GDNF) may guide the differentiation of SCs to remyelinate regenerating axons. Therefore, we hypothesized that exogenous GDNF may guide the differentiation of SCs into their native phenotypes in vitro through stimulation of GDNF family receptor (GFR)α-1. When activated in SCs, GFRα-1 promotes phosphorylation of Fyn, a Src family tyrosine kinase responsible for mediating downstream signaling for differentiation and proliferation. In this study, SCs harvested from the sensory and motor branches of rat femoral nerve were expanded in vitro and then cultured with 50 or 100ng/mL of GDNF. The exogenous GDNF promoted differentiation of sensory and motor-derived SCs back to their native phenotypes, as demonstrated by decreased proliferation after 7days and increased expression of S100Ββ and phenotype-specific markers. Furthermore, inhibiting Fyn with Src family kinase inhibitors, PP2 and SU6656, and siRNA-mediated knockdown of Fyn reduced GDNF-stimulated differentiation of sensory and motor-derived SCs. These results demonstrate that activating Fyn is necessary for GDNF-stimulated differentiation of femoral nerve-derived SCs into their native phenotypes in vitro. Therefore GDNF could be incorporated into SC-based therapies to promote differentiation of SCs into their native phenotype to improve functional nerve regeneration.

Depletion of Glial Cell Line-Derived Neurotrophic Factor by Disuse Muscle Atrophy Exacerbates the Degeneration of Alpha Motor Neurons in Caudal Regions Remote from the Spinal Cord Injury

We have been previously reported that disuse muscle atrophy exacerbates both motor neuron (MN) degeneration in caudal regions remote from a spinal cord injury, and decrease in glial cell line-derived neurotrophic factor (GDNF) protein level in paralyzed muscle. In this study we found that disuse muscle atrophy exacerbated the decrease in GDNF protein level in the L4/5 spinal cord, which was not immunopositive for GDNF. Our results were consistent with the fact that in the lumbar spinal cord of rats with mid-thoracic contusion, GDNF expression was not detected, while expression of GDNF receptors (GFRα1 and RET) was. Our study showed that administration of exogenous recombinant GDNF into the atrophic muscle partially rescued α-MN degeneration in the L4/5 spinal cord. These results suggest that the depletion of GDNF protein by muscle atrophy exacerbates α-MN degeneration in caudal regions remote from the injury.

6-Hydroxydopamine induces distinct alterations in GDF5 and GDNF mRNA expression in the rat nigrostriatal system in vivo

Growth/differentiation factor (GDF)5 and glial cell line-derived neurotrophic factor (GDNF) are neurotrophic factors that promote the survival of midbrain dopaminergic neurons in vitro and in vivo. Both factors have potent neurotrophic and neuroprotective effects in rat models of Parkinson's disease (PD) and represent promising new therapies for PD. The aim of this study was to investigate the expression of GDF5, GDNF and their receptors in the nigrostriatal dopaminergic system in rat models of PD. It found that endogenous GDF5, GDNF and their receptors are differentially expressed in two 6-hydroxydopamine lesion models of PD. In both striatal and medial forebrain bundle (MFB) lesion models, striatal levels of GDF5 mRNA increased at 10 days post-lesion, while GDNF mRNA levels in the nigrostriatal system decreased after 10 and 28 days. Midbrain mRNA levels for both GDF5 receptors transiently increased after striatal lesion, whereas those of two GDNF receptors decreased at later time-points in both models. Despite the fact that exogenous GDF5 and GDNF have comparable effects on dopaminergic neurons in vitro and in vivo, their endogenous responses to neurotoxic injury are different. This highlights the importance of studying neurotrophic factor expression at distinct disease stages and in various animal models of PD.

Lentiviral Vector-Mediated Gradients of GDNF in the Injured Peripheral Nerve: Effects on Nerve Coil Formation, Schwann Cell Maturation and Myelination

Although the peripheral nerve is capable of regeneration, only a small minority of patients regain normal function after surgical reconstruction of a major peripheral nerve lesion, resulting in a severe and lasting negative impact on the quality of life. Glial cell-line derived neurotrophic factor (GDNF) has potent survival- and outgrowth-promoting effects on motoneurons, but locally elevated levels of GDNF cause trapping of regenerating axons and the formation of nerve coils. This phenomenon has been called the “candy store” effect. In this study we created gradients of GDNF in the sciatic nerve after a ventral root avulsion. This approach also allowed us to study the effect of increasing concentrations of GDNF on Schwann cell proliferation and morphology in the injured peripheral nerve. We demonstrate that lentiviral vectors can be used to create a 4 cm long GDNF gradient in the intact and lesioned rat sciatic nerve. Nerve coils were formed throughout the gradient and the number and size of the nerve coils increased with increasing GDNF levels in the nerve. In the nerve coils, Schwann cell density is increased, their morphology is disrupted and myelination of axons is severely impaired. The total number of regenerated and surviving motoneurons is not enhanced after the distal application of a GDNF gradient, but increased sprouting does result in higher number of motor axon in the distal segment of the sciatic nerve. These results show that lentiviral vector mediated overexpression of GDNF exerts multiple effects on both Schwann cells and axons and that nerve coil formation already occurs at relatively low concentrations of exogenous GDNF. Controlled expression of GDNF, by using a viral vector with regulatable GDNF expression, may be required to avoid motor axon trapping and to prevent the effects on Schwann cell proliferation and myelination.

GDNF is required for neural colonization of the pancreas

The mammalian pancreas is densely innervated by both the sympathetic and parasympathetic nervous systems, which control exocrine and endocrine secretion. During embryonic development, neural crest cells migrating in a rostrocaudal direction populate the gut, giving rise to neural progenitor cells. Recent studies in mice have shown that neural crest cells enter the pancreatic epithelium at E11.5. However, the cues that guide the migration of neural progenitors into the pancreas are poorly defined. In this study we identify glial cell line-derived neurotrophic factor (GDNF) as a key player in this process. GDNF displays a dynamic expression pattern during embryonic development that parallels the chronology of migration and differentiation of neural crest derivatives in the pancreas. Conditional inactivation of Gdnf in the pancreatic epithelium results in a dramatic loss of neuronal and glial cells and in reduced parasympathetic innervation in the pancreas. Importantly, the innervation of other regions of the gut remains unaffected. Analysis of Gdnf mutant mouse embryos and ex vivo experiments indicate that GDNF produced in the pancreas acts as a neurotrophic factor for gut-resident neural progenitor cells. Our data further show that exogenous GDNF promotes the proliferation of pancreatic progenitor cells in organ culture. In summary, our results point to GDNF as crucial for the development of the intrinsic innervation of the pancreas.

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.

Expression of Glial Cell Line‐Derived Neurotrophic Factor and its Receptors in Cultured Retinal Müller Cells Under High Glucose Circumstance

This study aimed to explore the effect of high glucose concentration on the expression of glial cell line-derived neurotrophic factor (GDNF) and its family ligand receptors (GFRs) GFRα1 and GFRα2 in Müller cells and the protective role of GDNF in cultured Müller cells under high glucose circumstance. Cultured Müller cells (untreated or treated with 200 ng/mL of GDNF) were exposed to high glucose conditions (20 mmol/L glucose). We found that the expression levels of GDNF and GFRα1 mRNA and protein increased gradually over time under high glucose and exogenous GDNF-treated conditions, whereas the upregulation in GFRα2 expression was observed only in the early stage of high glucose conditions. Exogenous GDNF not only decreased apoptosis in cultured Müller cells under high glucose circumstance, but also accelerated the levels and speed of synthesis of GDNF and GFRα1 proteins in Müller cells. These results suggest that Müller cells can synthesize GDNF and GFRs under high glucose conditions, and GDNF may play important role in protecting Müller cells during the early stage of diabetic retinopathy. The difference in GFRs expression indicated that GDNF and neurturin may exert different effects on Müller cells under high glucose circumstance.

The GDNF Target Vsnl1 Marks the Ureteric Tip

Glial cell line-derived neurotrophic factor (GDNF) is indispensable for ureteric budding and branching. If applied exogenously, GDNF promotes ectopic ureteric buds from the Wolffian duct. Although several downstream effectors of GDNF are known, the identification of early response genes is incomplete. Here, microarray screening detected several GDNF-regulated genes in the Wolffian duct, including Visinin like 1 (Vsnl1), which encodes a neuronal calcium-sensor protein. We observed renal Vsnl1 expression exclusively in the ureteric epithelium, but not in Gdnf-null kidneys. In the tissue culture of Gdnf-deficient kidney primordium, exogenous GDNF and alternative bud inducers (FGF7 and follistatin) restored Vsnl1 expression. Hence, Vsnl1 characterizes the tip of the ureteric bud epithelium regardless of the inducer. In the tips, Vsnl1 showed a mosaic expression pattern that was mutually exclusive with β-catenin transcriptional activation. Vsnl1 was downregulated in both β-catenin-stabilized and β-catenin-deficient kidneys. Moreover, in a mouse collecting duct cell line, Vsnl1 compromised β-catenin stability, suggesting a counteracting relationship between Vsnl1 and β-catenin. In summary, Vsnl1 marks ureteric bud tips in embryonic kidneys, and its mosaic pattern demonstrates a heterogeneity of cell types that may be critical for normal ureteric branching.