Glial Cell Line-derived Neurotrophic Factor Function Research Articles

Advances in glial cell line-derived neurotrophic factor

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor with significant research value due to its profound effects on dopaminergic, cholinergic, and motor neurons. Initially identified for its neuroprotective role in dopaminergic neurons, GDNF has been found to support various neuronal populations and plays a crucial role in neural development, maintenance, and repair. This review provides a comprehensive overview of the molecular characteristics, tissue distribution, physiological function of GDNF and its protective effect on a series of diseases, emphasizing its potential therapeutic applications. Meanwhile, this study discusses the challenges in delivering GDNF across the blood-brain barrier and explores current strategies to enhance its clinical efficacy, including the use of gene therapy and innovative delivery methods. In summary, the review underscores the promise of GDNF as a therapeutic agent for neurodegenerative diseases and nerve injuries, highlighting the need for further research to translate these findings into clinical practice.

Opposing Spatially Segregated Function of Endogenous GDNF-RET Signaling in Cocaine Addiction.

Cocaine addiction is a serious condition with potentially lethal complications and no current pharmacological approaches towards treatment. Perturbations of the mesolimbic dopamine system are crucial to the establishment of cocaine-induced conditioned place preference and reward. As a potent neurotrophic factor modulating the function of dopamine neurons, glial cell line-derived neurotrophic factor (GDNF) acting through its receptor RET on dopamine neurons may provide a novel therapeutic avenue towards psychostimulant addiction. However, current knowledge on endogenous GDNF and RET function after the onset of addiction is scarce. Here, we utilized a conditional knockout approach to reduce the expression of the GDNF receptor tyrosine kinase RET from dopamine neurons in the ventral tegmental area (VTA) after the onset of cocaine-induced conditioned place preference. Similarly, after establishing cocaine-induced conditioned place preference, we studied the effect of conditionally reducing GDNF in the ventral striatum nucleus accumbens (NAc), the target of mesolimbic dopaminergic innervation. We find that the reduction of RET within the VTA hastens cocaine-induced conditioned place preference extinction and reduces reinstatement, while the reduction of GDNF within the NAc does the opposite: prolongs cocaine-induced conditioned place preference and increases preference during reinstatement. In addition, the brain-derived neurotrophic factor (BDNF) was increased and key dopamine-related genes were reduced in the GDNF cKO mutant animals after cocaine administration. Thus, RET antagonism in the VTA coupled with intact or enhanced accumbal GDNF function may provide a new approach towards cocaine addiction treatment.

Glial cell line-derived neurotrophic factor contributes to alcoholic-induced liver injury by regulating the NF-κB pathway.

Alcoholic liver disease (ALD) is associated with high morbidity and mortality worldwide. The pathogenesis of ALD is not completely understood. Although accumulating evidence suggests an important role of glial cell line-derived neurotrophic factor (GDNF) in several diseases, there are no data concerning its role in ALD. This study compared patients with ALD with control subjects and used a mouse model and a cell culture model to investigate the function of GDNF in ALD and its mechanism of action in hepatocyte injury. Serum levels of GDNF were measured in 25 patients with ALD and 25 healthy control subjects. A 4-week Lieber-DeCarli ethanol (EtOH) liquid diet combined with the Gao-Binge model was used in the mouse study. Mouse primary hepatocytes and Huh-7 cells were used for cell experiments. The parameters of liver injury, inflammatory cytokines, and lipid metabolism were measured. Patients with alcoholic hepatitis had higher serum GDNF than control subjects. Expression of GDNF mRNA and protein was markedly increased in mice in the chronic-plus-binge ALD mouse model. The level of GDNF mRNA was upregulated in primary hepatic stellate cells isolated from ethanol-fed mouse liver. Ethanol induced GDNF expression in LX2 cells. The levels of inflammatory cytokines (tumor necrosis factor α, interleukin 1β, and monocyte chemotactic protein 1) were significantly increased after GDNF stimulation in primary hepatocytes and Huh-7 cells. After GDNF stimulation, levels of both p-AKT and p-NF-κB were significantly increased in primary hepatocytes and Huh-7 cells. The NF-κB activity induced by GDNF was significantly decreased by an NF-κB inhibitor, which limited hepatocyte injury and inflammation. The concentration of GDNF is increased in the circulation of ALD patients. GDNF promotes alcohol-induced liver injury and inflammation via the activation of NF-κB, which mediates hepatocyte injury and inflammatory cytokine expression. Based on these findings, GDNF is a potential therapeutic target for preventing or ameliorating liver injury in ALD.

Increased Endogenous GDNF in Mice Protects Against Age-Related Decline in Neuronal Cholinergic Markers.

Gradual decline in cholinergic transmission and cognitive function occurs during normal aging, whereas pathological loss of cholinergic function is a hallmark of different types of dementia, including Alzheimer’s disease (AD), Lewy body dementia (LBD), and Parkinson’s disease dementia (PDD). Glial cell line-derived neurotrophic factor (GDNF) is known to modulate and enhance the dopamine system. However, how endogenous GDNF influences brain cholinergic transmission has remained elusive. In this study, we explored the effect of a twofold increase in endogenous GDNF (Gdnf hypermorphic mice, Gdnfwt/hyper) on cholinergic markers and cognitive function upon aging. We found that Gdnfwt/hyper mice resisted an overall age-associated decline in the cholinergic index observed in the brain of Gdnfwt/wt animals. Biochemical analysis revealed that the level of nerve growth factor (NGF), which is important for survival and function of central cholinergic neurons, was significantly increased in several brain areas of old Gdnfwt/hyper mice. Analysis of expression of genes involved in cholinergic transmission in the cortex and striatum confirmed modulation of cholinergic pathways by GDNF upon aging. In line with these findings, Gdnfwt/hyper mice did not undergo an age-related decline in cognitive function in the Y-maze test, as observed in the wild type littermates. Our results identify endogenous GDNF as a potential modulator of cholinergic transmission and call for future studies on endogenous GDNF function in neurodegenerative disorders characterized by cognitive impairments, including AD, LBD, and PDD.

GDNF/RET signaling in dopamine neurons in vivo.

The glial cell line-derived neurotrophic factor (GDNF) and its canonical receptor Ret can signal both in tandem and separately to exert many vital functions in the midbrain dopamine system. It is known that Ret has effects on maintenance, physiology, protection and regeneration in the midbrain dopamine system, with the physiological functions of GDNF still somewhat unclear. Notwithstanding, Ret ligands, such as GDNF, are considered as promising candidates for neuroprotection and/or regeneration in Parkinson's disease, although data from clinical trials are so far inconclusive. In this review, we discuss the current knowledge of GDNF/Ret signaling in the dopamine system in vivo as well as crosstalk with pathology-associated proteins and their signaling in mammals.

Possible function of GDNF and Schwann cells in wound healing of periodontal tissue.

The purpose of this study was to evaluate the function of Schwann cells in wound healing of periodontal tissue. In our previous study, glial cell line-derived neurotrophic factor (GDNF) promoted the migration of human periodontal ligament (PDL) cells and that GDNF expression increased in wounded periodontal tissue. GDNF reportedly induces the migration of Schwann cell precursors. Schwann cells play a crucial role in the regeneration of peripheral tissues, including bone tissue. However, the role of Schwann cells on periodontal tissue regeneration remains unclear. A transwell assay and a WST-1 (water-soluble tetrazolium compound-1) proliferation assay were used to determine whether GDNF promotes the migration and proliferation of Schwann cells, respectively. Quantitative RT-PCR and Alizarin Red S staining were performed to examine the effect of these cells on the differentiation of human preosteoblast (Saos2 cells) using conditioned medium from YST-1 (YST-1-CM). Western blotting analysis was performed to determine whether YST-1-CM activates ERK signaling pathway in Saos2 cells. The expression of Schwann cell markers, S100 calcium-binding protein B (S100-B) and growth associated protein 43 (GAP-43), was determined in normal and wounded periodontal tissue by immunofluorescent staining. Glial cell line-derived neurotrophic factor promoted the migration of YST-1 cells but did not affect the proliferation of YST-1 cells. Saos2 cells cultured with YST-1-CM increased the expression of osteoblastic markers and mineralization. YST-1-CM also induced phosphorylation of ERK1/2 in Saos2 cells. The number of S100-B-immunoreactive cells which also expressed GAP-43 was increased in rat wounded periodontal tissue during healing process. The accumulation of Schwann cells in wounded periodontal tissue suggests that they play a significant role in wound healing of this tissue, especially alveolar bone tissue.

Hippo/YAP Pathway Plays a Critical Role in Effect of GDNF Against Aβ-Induced Inflammation in Microglial Cells.

Neuroinflammation is a critical mechanism responsible for the progression of Alzheimer's disease (AD). Recent studies reveal that Hippo/Yes-associated protein (YAP) signaling pathway is highly associated with a series of inflammation-related disorders. Glial cell line-derived neurotrophic factor (GDNF), with its neurotrophic and anti-apoptotic functions for nervous system, has been demonstrated to decrease the expression of proinflammatory mediators. Here we investigated whether Hippo/YAP signaling may affect amyloid-β (Aβ)-induced proinflammatory cytokine production in microglial cells and explored its relationship with the anti-inflammation function of GDNF. The results showed that Aβ induced a decrease in the expression of YAP in microglia cells. YAP agonist XMU-MP-1 or its overexpression in microglial cells caused decreased expression of proinflammatory cytokines, whereas YAP antagonist Verteporfin or knockdown of YAP had the opposite effect. Treatment with GDNF resulted in upregulation of YAP expression and reduced the production of proinflammatory cytokines. Meanwhile YAP knockdown weakened the function of GDNF in microglial cells. In conclusion, Hippo/YAP pathway plays a critical role in effect of GDNF against Aβ-induced inflammatory response in microglia. Targeting GDNF or Hippo/YAP signaling may be promising therapeutic approach for the treatment of AD.

GDNF induces RET-SRC-HER2-dependent growth in trastuzumab-sensitive but SRC-independent growth in resistant breast tumor cells.

We investigated the role of glial cell line-derived neurotrophic factor (GDNF) in compensating trastuzumab (TZMB)-induced apoptosis in HER2+ breast cancer (BC) cells using xenograft tumors. We generated BC xenografts in nude mice using samples from three patients selected based on their HER2 status and response to TZMB therapy. TZMB treatment resulted in shrinkage of the HER2+ TZMB-sensitive xenograft tumor but not the HER2- or HER2+ TZMB-resistant ones. GDNF neutralized TZMB activity and induced growth in all tumors. Three distinct cell lines were derived from these tumors and named, respectively, TZMB-sensitive (TSTC), HER2- (HNTC), and TZMB-resistant (TRTC). Over 50% of TRTC but 1% of TSTC cells expressed CD44, whereas 84% of TSTC were CD24+ compared to only 1% of TRTC, despite comparable levels of HER2 detected in both. TZMB induced profound morphological changes toward apoptosis in TSTC but not in TRTC or HNTC. However, GDNF significantly compensated TZMB-mediated TSTC cell loss and promoted growth by 37 and 50%, respectively, in TSTC and TRTC. Inhibition of SRC by Saracatinib (SARC) blocked GDNF function and accelerated TZMB-mediated cell death in TSTC, but GDNF continued promoting TRTC growth. These changes paralleled with expression levels of the key molecules involved in growth and apoptosis. Collectively, we found in our xenograft samples that firstly SRC mediates GDNF pro-survival functions by bridging RET-HER2 crosstalk in TZMB-responsive BC tumors. Secondly, SARC-TZMB interactions can synergistically eradicate such tumor cells; and thirdly, GDNF can support antibody resistance by acting independent from SRC in tumors with poor HER2 response to TZMB therapy.

GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function.

Degeneration of nigrostriatal dopaminergic system is the principal lesion in Parkinson’s disease. Because glial cell line-derived neurotrophic factor (GDNF) promotes survival of dopamine neurons in vitro and in vivo, intracranial delivery of GDNF has been attempted for Parkinson’s disease treatment but with variable success. For improving GDNF-based therapies, knowledge on physiological role of endogenous GDNF at the sites of its expression is important. However, due to limitations of existing genetic model systems, such knowledge is scarce. Here, we report that prevention of transcription of Gdnf 3’UTR in Gdnf endogenous locus yields GDNF hypermorphic mice with increased, but spatially unchanged GDNF expression, enabling analysis of postnatal GDNF function. We found that increased level of GDNF in the central nervous system increases the number of adult dopamine neurons in the substantia nigra pars compacta and the number of dopaminergic terminals in the dorsal striatum. At the functional level, GDNF levels increased striatal tissue dopamine levels and augmented striatal dopamine release and re-uptake. In a proteasome inhibitor lactacystin-induced model of Parkinson’s disease GDNF hypermorphic mice were protected from the reduction in striatal dopamine and failure of dopaminergic system function. Importantly, adverse phenotypic effects associated with spatially unregulated GDNF applications were not observed. Enhanced GDNF levels up-regulated striatal dopamine transporter activity by at least five fold resulting in enhanced susceptibility to 6-OHDA, a toxin transported into dopamine neurons by DAT. Further, we report how GDNF levels regulate kidney development and identify microRNAs miR-9, miR-96, miR-133, and miR-146a as negative regulators of GDNF expression via interaction with Gdnf 3’UTR in vitro. Our results reveal the role of GDNF in nigrostriatal dopamine system postnatal development and adult function, and highlight the importance of correct spatial expression of GDNF. Furthermore, our results suggest that 3’UTR targeting may constitute a useful tool in analyzing gene function.

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.

GDNF Signaling Levels Control Migration and Neuronal Differentiation of Enteric Ganglion Precursors

Pleiotropic growth factors play a number of critical roles in continuous processes of embryonic development; however, the mechanisms by which a single regulatory factor is able to orchestrate diverse developmental events remain imperfectly understood. In the development of the enteric nervous system (ENS), myenteric ganglia (MGs) form initially, after which the submucosal ganglia (SMGs) develop by radial inward migration of immature ENS precursors from the myenteric layer. Here, we demonstrate that glial cell line-derived neurotrophic factor (GDNF) is essential for the formation not only of the MGs, but the SMGs as well, establishing GDNF as a long-term acting neurotrophic factor for ENS development in a mouse model. GDNF promotes radial migration of SMG precursors. Interestingly, premigratory SMG precursors in the myenteric layer were distinguished from the surrounding neuronally differentiating cells by their lower activation of the GDNF-mediated MAPK pathway, suggesting that low activation of GDNF downstream pathways is required for the maintenance of the immature state. ENS precursors devoid of GDNF signaling during midgestation halt their migration, survive, and remain in an undifferentiated state over the long-term in vivo. Reactivation of GDNF signaling in these dormant precursors restores their migration and neuronal differentiation in gut organ culture. These findings suggest that pleiotropic function of GDNF is at least in part governed by modulating levels of intracellular activation of GDNF downstream pathways; high activation triggers neuronal differentiation, whereas low activation is crucial for the maintenance of progenitor state.

Glial cell-line derived neurotrophic factor is essential for electroconvulsive shock-induced neuroprotection in an animal model of Parkinson's disease

Sustained motor improvement in human patients with idiopathic Parkinson's disease has been described following electroconvulsive shock (ECS) treatment. In rats, ECS stimulates the expression of various trophic factors (TFs), some of which have been proposed to exert neuroprotective actions. We previously reported that ECS protects the integrity of the rat nigrostriatal dopaminergic system against 6-hydroxydopamine (6-OHDA)-induced toxicity; in order to shed light into its neuroprotective mechanism, we studied glial cell-line derived neurotrophic factor (GDNF) levels (the most efficient TF for dopaminergic neurons) in the substantia nigra (SN) and striatum of 6-OHDA-injected animals with or without ECS treatment. 6-OHDA injection decreased GDNF levels in the SN control animals, but not in those receiving chronic ECS, suggesting that changes in GDNF expression may participate in the ECS neuroprotective mechanism. To evaluate this possibility, we inhibit GDNF by infusion of GDNF function blocking antibodies in the SN of 6-OHDA-injected animals treated with ECS (or sham ECS). Animals were sacrificed 7 days after 6-OHDA infusion, and the integrity of the nigrostriatal system was studied by tyrosine hydroxylase immunohistochemistry and Cresyl Violet staining. Neuroprotection observed in ECS-treated animals was inhibited by GDNF antibodies in the SN. These results robustly demonstrate that GDNF is essential for the ECS neuroprotective effect observed in 6-OHDA-injected animals.

Endogenous GDNF in ventral tegmental area and nucleus accumbens does not play a role in the incubation of heroin craving

Glial cell line-derived neurotrophic factor (GDNF) activity in ventral tegmental area (VTA) mediates the time-dependent increases in cue-induced cocaine-seeking after withdrawal (incubation of cocaine craving). Here, we studied the generality of these findings to incubation of heroin craving. Rats were trained to self-administer heroin for 10 days (6 hours/day; 0.075 mg/kg/infusion; infusions were paired with a tone-light cue) and tested for cue-induced heroin-seeking in extinction tests after 1, 11 or 30 withdrawal days. Cue-induced heroin seeking was higher after 11 or 30 days than after 1 day (incubation of heroin craving), and the time-dependent increases in extinction responding were associated with time-dependent changes in GDNF mRNA expression in VTA and nucleus accumbens. Additionally, acute accumbens (but not VTA) GDNF injections (12.5 µg/side) administered 1-3 hours after the last heroin self-administration training session enhanced the time-dependent increases in extinction responding after withdrawal. However, the time-dependent increases in extinction responding after withdrawal were not associated with changes in GDNF protein expression in VTA and accumbens. Additionally, interfering with endogenous GDNF function by chronic delivery of anti-GDNF monoclonal neutralizing antibodies (600 ng/side/day) into VTA or accumbens had no effect on the time-dependent increases in extinction responding. In summary, heroin self-administration and withdrawal regulate VTA and accumbens GDNF mRNA expression in a time-dependent manner, and exogenous GDNF administration into accumbens but not VTA potentiates cue-induced heroin seeking. However, based on the GDNF protein expression and the anti-GDNF monoclonal neutralizing antibodies manipulation data, we conclude that neither accumbens nor VTA endogenous GDNF mediates the incubation of heroin craving.

Characterization of the Intracellular Localization, Processing, and Secretion of Two Glial Cell Line-Derived Neurotrophic Factor Splice Isoforms

Endocrine and neuronal cells have highly developed secretion mechanisms, and the secretion can be either constitutive or regulated by physiological stimuli. In the constitutive pathway, intracellular transport vesicles undergo immediate fusion reactions after arrival at the target. In regulated secretion, vesicles accumulate near the target membrane until triggered to fuse, typically by a local rise in free Ca(2+). In the present study, we characterize the processing and secretion mechanisms of the glial cell line-derived neurotrophic factor (GDNF). Although the function of GDNF has been extensively studied, very little is known about the basic cell biology of GDNF and its precursor forms (alpha)pro-GDNF and (beta)pro-GDNF that have different pro-regions. Our results show that both (alpha)pro-GDNF and (beta)pro-GDNF are secreted. We demonstrate that KCl-induced depolarization increases the secretion of (beta)pro-GDNF and corresponding mature GDNF, but not (alpha)pro-GDNF and corresponding mature GDNF, to the cell medium in a Ca(2+)-dependent manner. In parallel with this, immunofluorescence analysis of cells show that (alpha)pro-GDNF/GDNF is localized mostly in the Golgi complex, whereas (beta)pro-GDNF/GDNF is localized primarily in secretogranin II and Rab3A-positive vesicles of the regulated secretory pathway. In addition, we find that matrix metalloproteinases and plasmin that cleave pro-BDNF and pro-NGF are not responsible for the cleavage of pro-GDNF, whereas furin endoproteinase, PACE4, and proprotein convertases PC5A, PC5B, and PC7 can cleave pro-GDNF into mature GDNF. Thus, the processing and secretion mechanisms of GDNF are different from those of BDNF and NGF.

Role of Ventral Tegmental Area Glial Cell Line–Derived Neurotrophic Factor in Incubation of Cocaine Craving

Ventral tegmental area (VTA) brain-derived neurotrophic factor (BDNF) contributes to time-dependent increases in cue-induced cocaine seeking after withdrawal (incubation of cocaine craving). Here, we studied the role of glial cell line-derived neurotrophic factor (GDNF) in incubation of cocaine craving because, like BDNF, GDNF provides trophic support to midbrain dopamine neurons. We first trained rats to self-administer intravenous cocaine for 10 days (6 hours/d, cocaine injections were paired with a tone-light cue). We then manipulated VTA GDNF function and assessed cue-induced cocaine seeking in extinction tests after withdrawal from cocaine. VTA injections of an adeno-associated virus (AAV) vector containing rat GDNF cDNA (5 x 10(8) viral genomes) on withdrawal Day 1 increased cue-induced cocaine seeking on withdrawal days 11 and 31; this effect was not observed after VTA injections of an AAV viral vector containing red fluorescent protein (RFP). Additionally, VTA, but not substantial nigra (SN), GDNF injections (1.25 microg or 12.5 microg/side) immediately after the last cocaine self-administration session increased cue-induced drug seeking on withdrawal days 3 and 10; this effect was reversed by VTA injections of U0126, which inhibits the activity of extracellular signal-regulated kinases (ERK). Finally, interfering with VTA GDNF function by chronic delivery of anti-GDNF monoclonal neutralizing antibodies via minipumps (600 ng/side/d) during withdrawal Days 1-14 prevented the time-dependent increases in cue-induced cocaine seeking on withdrawal days 11 and 31. Our results indicate that during the first weeks of withdrawal from cocaine self-administration, GDNF-dependent neuroadaptations in midbrain VTA neurons play an important role in the development of incubation of cocaine craving.

GDNF isoform affects intracellular trafficking and secretion of GDNF in neuronal cells

Glial cell line derived neurotrophic factor (GDNF) plays a critical role in central and peripheral neuron survival and function. In human and rodents, GDNF exists in an alternative spliced isoform (GDNFΔ78), which has a 78 bp deletion in the pro-region of the GDNF encoding sequence. Whether the GDNF isoform affects GDNF function is unknown. Here, we investigated the secretion and intracellular localization of the GDNFΔ78 isoform in neuronal cell populations. Our data indicate that a decreased secretion and an abnormal intracellular distribution of GDNFΔ78 occurred in neuronal cells. The colocalization studies revealed much more localization of GDNFΔ78 with Golgi marker-TGN38, which indicates that the accumulation of GDNFΔ78 in the Golgi apparatus might in part account for its intracellular trafficking and secretion deficit. To our knowledge, it is reported for the first time that the GDNFΔ78 isoform has a deficit in GDNF intracellular trafficking and secretion.

GDNF prevents TGF-β-induced damage of the plasma membrane in cerebellar granule neurons by suppressing activation of p38-MAPK via the phosphatidylinositol 3-kinase pathway

Transforming growth factor-beta (TGF-beta) and glial-cell-line-derived neurotrophic factor (GDNF) have been shown to synergize in several paradigms of neuronal survival. We have previously shown that cerebellar granule neurons (CGN) degenerate in low potassium via ERK1/2 (extra-cellular-regulated kinase)-dependent plasma membrane (PM) damage and caspase-3-dependent DNA fragmentation. Here, we have investigated the putative synergistic function of GDNF and TGF-beta in CGN degeneration. GDNF alone prevents low-potassium-induced caspase-3 activation and DNA fragmentation but does not affect either low-potassium-induced ERK activation or PM damage. TGF-beta alone does not affect low-potassium-induced DNA fragmentation but potentiates low-potassium-induced PM damage. This effect of TGF-beta is independent of ERK1/2 activation but dependent on p38-MAPK (mitogen-activated protein kinase) activation. When co-applied with TGF-beta, GDNF paradoxically antagonizes TGF-beta-induced potentiation of PM damage by inhibiting TGF-beta-induced p38-MAPK activation. In addition, PI3K (phosphatidylinositol 3-kinase) inhibitors abolish the GDNF effect. This study thus demonstrates a differential mechanism of action of GDNF and TGF-beta on CGN degeneration. GDNF inhibits caspase-3-dependent DNA fragmentation but does not affect ERK-dependent PM damage. However, GDNF can attenuate TGF-beta-induced p38-MAPK-dependent PM damage via the PI3K pathway.

Branching morphogenesis of the ureteric epithelium during kidney development is coordinated by the opposing functions of GDNF and Sprouty1

Branching of ureteric bud-derived epithelial tubes is a key morphogenetic process that shapes development of the kidney. Glial cell line-derived neurotrophic factor (GDNF) initiates ureteric bud formation and promotes subsequent branching morphogenesis. Exactly how GDNF coordinates branching morphogenesis is unclear. Here we show that the absence of the receptor tyrosine kinase antagonist Sprouty1 (Spry1) results in irregular branching morphogenesis characterized by both increased number and size of ureteric bud tips. Deletion of Spry1 specifically in the epithelium is associated with increased epithelial Wnt11 expression as well as increased mesenchymal Gdnf expression. We propose that Spry1 regulates a Gdnf/Ret/Wnt11-positive feedback loop that coordinates mesenchymal–epithelial dialogue during branching morphogenesis. Genetic experiments indicate that the positive (GDNF) and inhibitory (Sprouty1) signals have to be finely balanced throughout renal development to prevent hypoplasia or cystic hyperplasia. Epithelial cysts develop in Spry1-deficient kidneys that share several molecular characteristics with those observed in human disease, suggesting that Spry1 null mice may be useful animal models for cystic hyperplasia.

Doxycycline-regulated co-expression of GDNF and TH in PC12 cells

Current gene therapy models for Parkinson's disease (PD) have adapted two treatment strategies. One is to restore dopamine (DA) production by delivering the genes of DA-synthesizing enzymes such as tyrosine hydroxylase (TH) to the striatum to relieve motor symptoms of PD. Another is to block or slow down progressive degenerative changes by delivering neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) to protect the remained neurons. To test the assumption that the combination of the two strategies may have a compound or synergistic effect, we had constructed tetracycline-inducible (tet-off) AAV vector carrying GDNF and TH. After co-transfection of PC12 cells with this vector and the inducer plasmid, the expression of GDNF and TH protected these cells from 1-methyl-4-phenyl-pyridinium-induced injury, and significantly increased the content of dopamine in GDNF/TH-expressing cells compared with the control. Furthermore, mRNA expression of GDNF and TH could be effectively and reversibly regulated by doxycycline (Dox) and the function of GDNF and TH could be repressed by Dox. These results suggest that the tet-off AAV vector carrying GDNF and TH may be a useful tool for gene therapy in the treatment of PD.

Alterations in the expressions of mRNA for GDNF and its receptors in the ventral midbrain of rats exposed to subchronic phencyclidine

Phencyclidine (PCP) produces schizophrenia-like symptoms in normal humans. This suggests that the dysfunction of glutamatergic neurotransmission may play an important role in the pathology of schizophrenia. However, PCP also exerts its effect on the mesolimbic dopamine (DA) system and modulates DA function in the brain, the abnormality of which is proposed to be a main pathology of schizophrenia. Recently, glial cell-line derived neurotrophic factor (GDNF) has been shown to play a protective role for DA neurons against neurotoxic injuries and maintaining DA function in the brain. We hypothesized that subchronic PCP may alter the function of GDNF in the ventral midbrain, where DA cell bodies are localized. Male Wistar rats were injected intraperitoneally with PCP daily for 10 days at 5 or 10 mg/kg, and their brains were removed 24 h after the last injection. The expressions of GDNF and its receptor (GFRα-1 and c-ret) mRNAs in the substantia nigra compacta (SNC) and ventral tegmental area (VTA) were determined by non-radioactive in situ hybridization, and those of GDNF and c-ret mRNA were found to be increased after the PCP subchronic administration. No significant changes, however, were observed in the expressions of GFRα-1 and basic fibroblast growth factor. These results suggest that subchronic PCP may modulate the function of the GDNF system, which exerts a trophic action on DA neurons in the ventral midbrain.