Glial Cell Line-derived Neurotrophic Factor mRNA Levels Research Articles

Activation of mGlu3 Receptors Stimulates the Production of GDNF in Striatal Neurons

Metabotropic glutamate (mGlu) receptors have been considered potential targets for the therapy of experimental parkinsonism. One hypothetical advantage associated with the use of mGlu receptor ligands is the lack of the adverse effects typically induced by ionotropic glutamate receptor antagonists, such as sedation, ataxia, and severe learning impairment. Low doses of the mGlu2/3 metabotropic glutamate receptor agonist, LY379268 (0.25–3 mg/kg, i.p.) increased glial cell line-derived neurotrophic factor (GDNF) mRNA and protein levels in the mouse brain, as assessed by in situ hybridization, real-time PCR, immunoblotting, and immunohistochemistry. This increase was prominent in the striatum, but was also observed in the cerebral cortex. GDNF mRNA levels peaked at 3 h and declined afterwards, whereas GDNF protein levels progressively increased from 24 to 72 h following LY379268 injection. The action of LY379268 was abrogated by the mGlu2/3 receptor antagonist, LY341495 (1 mg/kg, i.p.), and was lost in mGlu3 receptor knockout mice, but not in mGlu2 receptor knockout mice. In pure cultures of striatal neurons, the increase in GDNF induced by LY379268 required the activation of the mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathways, as shown by the use of specific inhibitors of the two pathways. Both in vivo and in vitro studies led to the conclusion that neurons were the only source of GDNF in response to mGlu3 receptor activation. Remarkably, acute or repeated injections of LY379268 at doses that enhanced striatal GDNF levels (0.25 or 3 mg/kg, i.p.) were highly protective against nigro-striatal damage induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice, as assessed by stereological counting of tyrosine hydroxylase-positive neurons in the pars compacta of the substantia nigra. We speculate that selective mGlu3 receptor agonists or enhancers are potential candidates as neuroprotective agents in Parkinson's disease, and their use might circumvent the limitations associated with the administration of exogenous GDNF.

Effect of nigrostriatal damage induced by 6-hydroxydopamine on the expression of glial cell line–derived neurotrophic factor in the striatum of the rat

Several types of brain injuries have been associated with alterations in the striatal expression of neurotrophic factors, including glial cell line–derived neurotrophic factor (GDNF). However, contradictory results on the striatal expression of GDNF have been reported in different animal models of Parkinson's disease. For this reason, we examined the effect of nigrostriatal damage on the mRNA and protein expression levels of GDNF in the striatum as a function of time following a striatal or medial forebrain bundle 6-hydroxydopamine lesion. At different time points after the administration of 6-hydroxydopamine, striatal expression levels of GDNF were analyzed with semi-quantitative Western blotting. No significant changes in GDNF expression levels were observed within the 35-day observation period, either between the denervated and the intact striatum of medial forebrain bundle and striatally lesioned rats or between the striata of lesioned animals and those of control animals. In order to reinforce these results, striata of lesioned rats, sacrificed 18 days after lesioning, were analyzed with enzyme-linked immunosorbent assay and real-time polymerase chain reaction. At this time point, both techniques confirmed the results of the Western blot analysis, detecting no changes in striatal expression of GDNF, either at the protein level, or at the mRNA level. These data show that nigrostriatal damage induced by 6-hydroxydopamine has no effect on the striatal expression of GDNF.

Long‐term changes in neurotrophic factor expression in distal nerve stump following denervation and reinnervation with motor or sensory nerve

Several factors have been proposed to account for poor motor recovery after prolonged denervation, including motor neuron cell death and incomplete or poor regeneration of motor fibers into the muscle. Both may result from failure of the muscle and the distal motor nerve stump to continue expression of neurotrophic factors following delayed muscle reinnervation. This study investigated whether regenerating motor or sensory axons modulate distal nerve neurotrophic factor expression. We found that transected distal tibial nerve up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) mRNA, down-regulated neurotrophin-3 and ciliary neurotrophic factor mRNA, and that although these levels returned to normal with regeneration, the chronically denervated distal nerve stump continued to express these neurotrophic factors for at least 6 months following injury. A sensory nerve (the cutaneous saphenous nerve) sutured to distal tibial nerve lowered injury-induced BDNF and GDNF mRNA levels in distal stump, but repair with a mixed nerve (peroneal, containing muscle and cutaneous axons) was more effective. Repair with sensory or mixed nerves did not affect nerve growth factor or neurotrophin-3 expression. Thus, distal nerve contributed to a neurotrophic environment for nerve regeneration for at least 6 months, and sensory nerve repair helped normalize distal nerve neurotrophic factor mRNA expression following denervation. Furthermore, as BDNF and GDNF levels in distal stump increased following denervation and returned to control levels following reinnervation, their levels serve as markers for the status of regeneration by either motor or sensory nerve.

Selective injury to dopaminergic neurons up-regulates GDNF in substantia nigra postnatal cell cultures: Role of neuron–glia crosstalk

The effect of selective injury to dopaminergic neurons on the expression of glial cell line-derived neurotrophic factor (GDNF) was examined in substantia nigra cell cultures. H(2)O(2), mimicking increased oxidative stress, or l-DOPA, the main symptomatic treatment for Parkinson's disease, increased GDNF mRNA and protein levels in a time-dependent mode in neuron-glia mixed cultures. The concentration dependence indicated that mild, but not extensive, injury induced GDNF up-regulation. GDNF neutralization with an antibody decreased dopaminergic cell viability in H(2)O(2)-treated cultures, showing that up-regulation of GDNF was protecting dopaminergic neurons. Neither H(2)O(2) nor l-DOPA directly affected GDNF expression in astrocyte cultures, but conditioned media from challenged mixed cultures increased GDNF mRNA and protein levels in astrocyte cultures, indicating that GDNF up-regulation was mediated by neuronal factors. Since pretreatment with 6-OHDA completely abolished H(2)O(2)-induced GDNF up-regulation, we propose that GDNF up-regulation is triggered by failing dopaminergic neurons that signal astrocytes to increase GDNF expression.

Gene expression patterns for GDNF and its receptors in the human putamen affected by Parkinson's disease: A real-time PCR study

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-β superfamily, is a potent trophic factor for dopaminergic neurons of the ventral midbrain, which are known to degenerate during Parkinson's disease (PD). The neuroprotective, neurorestorative, and stimulatory properties of GDNF has prompted numerous suggestions that this trophic factor may be a potential therapeutic tool to treat PD, and it has also been widely speculated that altered GDNF expression levels may be involved in the pathophysiology of the disease. In this study, we have investigated if mRNA expression levels for GDNF and/or its receptors are altered during PD in the human putamen, a target area for dopamine neurons of the substantia nigra compacta. Expression levels were analyzed with quantitative real-time reverse transcriptase polymerase reaction (RT qPCR) in post-mortem tissues from PD patients and aged matched controls. Primer pairs specific for GDNF (isoforms I and II), and its receptor molecules, GFRα1 and cRET were utilized. GDNF, cRET and GFRα1 mRNA expression was clearly detected in the putamen of control and Parkinson's disease patients. A modest but significant upregulation of GDNF mRNA levels (Isoform I) was observed in the putamen of Parkinson's disease patients with a marked loss of nigral neurons. No significant changes were observed for the expression of cRet and GFRa1. These data suggest that the extensive loss of dopaminergic neurons in the substantia nigra, and concomitant loss of striatal dopamine, may induce compensatory changes in the expression of target derived GDNF, but not its receptor system.

Specific regulation of rat glial cell line‐derived neurotrophic factor gene expression by riluzole in C6 glioma cells

Contrasting with its robust expression during embryogenesis, the glial cell line-derived neurotrophic factor (GDNF) is repressed in the adult organism. However, rapid induction of this neuronal growth factor is observed following diverse neuronal insults and it is now widely accepted that the control of its expression could constitute a powerful target in neuropharmacology. We investigated the effects of the neuroprotective drug, riluzole, on the GDNF gene expression in glial cells. Exposure of C6 glioma cells to riluzole (1 microM) significantly increased GDNF protein and mRNA levels. Using luciferase reporter gene constructs encoding fragments of the 5' untranslated region of the rat GDNF gene, we demonstrated that riluzole mediates its effect at the transcription level. Furthermore, luciferase assays revealed the presence of a negative regulatory region within the +343/+587 region of exon 1. This region was shown to contribute to the high sensitivity and specificity of the induction mediated by riluzole in the C6 glioma cell line at pharmacologically relevant concentrations. The effects of riluzole were inhibited by the mitogen-activated protein kinase extracellular signal-related kinase (MEK) inhibitor PD 98059. Together, these results indicated that the induction of GDNF release by riluzole in the C6 glioma cells results from the activation of its corresponding gene promoter through a signalling pathway involving MEK activity. This study suggests that the regulation of GDNF gene transcription in glial cells could contribute to the pharmacological properties of riluzole and possibly other neuroprotective drugs.

Amantadine and memantine induce the expression of the glial cell line-derived neurotrophic factor in C6 glioma cells

Aminoadamantanes are commonly used in the treatment of Parkinson's and Alzheimer's diseases. While these drugs are shown to antagonise ionotropic glutamate receptors on neuronal cells, additional mechanisms could contribute to their neuroprotective properties. The aim of the present study was to investigate the effect of aminoadamantanes on the production of the glial cell line-derived neurotrophic factor (GDNF) in glial cells. For this purpose, we measured the modulation of GDNF release in C6 glioma cell cultures treated for 24 h with amantadine and memantine. Both drugs dose-dependently increased GDNF level in the culture medium with similar potency (submicromolar range) and efficacy (three to four-fold induction). RT-PCR studies revealed that both compounds also increased GDNF mRNA levels and their influence on the GDNF gene transcription was further evidenced using a rat GDNF promoter luciferase reporter assay. Together, these results demonstrate that the neuroprotective effect of amantadine and memantine could involve the regulation of GDNF production by glial cells.

Down-regulation of GFRα-1 expression by antisense oligodeoxynucleotide aggravates thermal hyperalgesia in a rat model of neuropathic pain

Glial cell line-derived neurotrophic factor (GDNF) has been hypothesized to play an important role in the modulation of nociceptive signals especially during neuropathic pain. The present study examined the expression of GDNF and GFRα-1 (the high-affinity receptor of GDNF) in dorsal root ganglions (DRG) in a rat model of neuropathic pain induced by chronic constriction injury (CCI) to the sciatic nerve. In order to address the role of GDNF and GFRα-1 in neuropathic pain, antisense oligodeoxynucleotide (ODN) specifically against GFRα-1 was intrathecally administered to result in down-regulation of GFRα-1 expression. The results showed that both the protein and mRNA levels of GDNF and GFRα-1 were significantly increased after CCI, while the thermal hyperalgesia of neuropathic pain rats could be significantly aggravated by antisense ODN treatment, but not by normal saline (NS) or mismatch ODN treatment. The present study demonstrated that endogenous GDNF and GFRα-1 might play an anti-hyperalgesic role in neuropathic pain of rats. In addition, we found a down-regulation of somatostatin (SOM) in DRG and spinal dorsal horn after expression of GFRα-1 was knocked down, which suggested the possible relationship between the anti-hyperalgesic effect of GDNF and GFRα-1 on neuropathic pain and endogenous SOM.

Changes of expression of glial cell line-derived neurotrophic factor and its receptor in dorsal root ganglions and spinal dorsal horn during electroacupuncture treatment in neuropathic pain rats

Injury to the nervous system occasionally leads to intense and persistent neuropathic pain, which is resistant to conventional analgesic methods. It was reported that electroacupuncture (EA) had potent analgesic effect on neuropathic pain by activating various endogenous transmitters such as the opioid peptides. Glial cell line-derived neurotrophic factor (GDNF) has been hypothesized to play an important role in modulation of nociceptive signals especially during neuropathic pain state. Using immunohistochemistry, Western blot, and RT-PCR analysis techniques, the present study observed the effects of EA on the expression of GDNF and GDNF family receptor α-1 (GFRα-1, the high-affinity receptor of GDNF) in neuropathic pain rats. The results showed that both protein and mRNA levels of GDNF and GFRα-1 in the dorsal root ganglions (DRG), as well as GDNF protein in the spinal dorsal horn, were significantly increased after chronic constriction injury (CCI) of the rats’ sciatic nerve and could be further enhanced by EA treatment. The present data demonstrated that EA could activate endogenous GDNF and GFRα-1 system of neuropathic pain rats and this might underlie the effectiveness of EA in the treatment of neuropathic pain.

Involvement of glial cell line‐derived neurotrophic factor in activation processes of rodent macrophages

The physiological roles of glial cell line-derived neurotrophic factor (GDNF) expressed in the microglia/macrophages of the injured spinal cord have not yet been clarified. mRNA expression of chemokines, including monocyte chemoattractant protein (MCP)-1, was evoked within 1 hr after transection of the spinal cord, and GDNF mRNA expression was similarly up-regulated. Immunohistochemical analysis showed that GDNF was coexpressed with MCP-1 in the CD11b-positive cells. Therefore, we examined further the effects of GDNF on cultured rat peritoneal macrophages. GDNF enhanced the phagocytic activity of the macrophages via GFRalpha-1, glycosylphosphatidylinositol-anchored specific binding site of GDNF, in a c-Ret-independent manner. The influence of autocrine and/or paracrine GDNF synthesis was evaluated by performing activation experiments using macrophages cultured from heterozygous (+/-) GDNF gene-deficient mice or wild-type (+/+) mice. There were no morphological differences dependent on genetic types or stimulators. However, the GDNF mRNA level, but not the MCP-1 or GFRalpha-1 mRNA level, was substantially lower in the mutant macrophages than in the +/+ cells irrespective of stimulation with MCP-1 or lipopolysaccharide (LPS). The phagocytic activity enhanced by MCP-1 or LPS was significantly lower in the mutant cells (+/-) than in the +/+ ones, demonstrating the involvement of endogenous GDNF in the activation processes of macrophages in vitro and suggesting that not only neuroprotective function but also activation of macrophages is effected by the GDNF produced after a spinal cord injury.

GDNF, Ret, GFRα1 and 2 in the adult rat retino-tectal system after optic nerve transection

In the present study, we have studied the expression of glial cell line-derived neurotrophic factor (GDNF) and its receptors Ret, GFRα1, and GFRα2 in the retino-tectal system before and after optic nerve transection. Using retrograde neuronal tracing in combination with in situ hybridization, we found that Ret and GFRα1 are expressed by 13–14% of the retinal ganglion cells (RGCs). These Ret-expressing RGCs could not be identified as belonging to any particular of the RG A, RG B, and RG C sub types. Ret is co-expressed with the brain-derived neurotrophic factor receptor TrkB in these RGCs. Optic nerve transection resulted in reduced Ret mRNA levels in retina, while the levels of GDNF, GFRα1, and 2 mRNA increased. Administration of GDNF protein supported the axotomized RGCs. Analysis of normal superior colliculus (SC) did not show any expression of GDNF mRNA, yet GDNF mRNA levels in SC increased after injury. Together, these findings identify a portion of RGCs as being possible targets for pharmacological treatment with GDNF in a direct mode of action. The absence of detectable GDNF mRNA in normal SC questions the role for GDNF as being a target-derived factor produced in the SC for adult RGCs. The results support a function for GDNF locally in the retina and as part of an injury-induced system that may act to enhance neuroprotective and neuroregenerative responses both to endogenous GDNF ligands and those administered exogenously.

Endothelin-1 stimulates glial cell line-derived neurotrophic factor expression in cultured rat astrocytes

Effects of endothelin-1 (ET-1) on glial cell line-derived neurotrophic factor (GDNF) production in cultured astrocytes were examined. Treatment of cultured astrocytes with ET-1 (100 nM) increased mRNA levels of GDNF in 1–6 h. The effect of ET-1 was inhibited by BQ788, an ET B receptor antagonist, but not by FR139317, an ET A receptor antagonist. ET-1 stimulated release of GDNF into culture medium. Dexamethasone (1 μM) and pyrrolidine dithiocarbamate (PDTC, 100 μM), which inhibit activation of NFκB, prevented the increases in GDNF mRNA by H 2O 2. In contrast, the effect of ET-1 was not affected by dexamethasone and PDTC. The increase of astrocytic GDNF mRNA by ET-1 was inhibited by BAPTA/AM (30 μM) and PD98059 (50 μM), but not by calphostin C, staurosporine, and cyclosporine A. These results suggest that ET-1 stimulated expression of astrocytic GDNF through ET B receptor-mediated increases in cytosolic Ca 2+ and ERK activation.

Pain-related Differential Expression of NGF, GDNF, IL-6, and Their Receptors in Human Vasculitic Neuropathies

Pain-related differential expressions of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF) and interleukin-6 (IL-6), and their receptors were investigated in human vasculitic neuropathies. The mRNA levels of pain-related neurotrophic factors, NGF, GDNF and IL-6, were examined in the sural nerves of 22 painful and non-painful patients with acute necrotizing vasculitic neuropathies, together with their concomitant soluble receptors (p75, GFR(alpha)-1 and IL-6R(alpha)). The mRNAs for these factors and receptors in the lesioned nerves were up-regulated to a variable extent in both groups. NGF mRNA expression was more closely correlated with that of p75 in painful neuropathy with relatively preserved large fiber density, compared with non-painful neuropathy, though the NGF mRNA level in painful neuropathy was lower than that in non-painful neuropathy. GDNF was closely associated with GFR(alpha)-1 in mRNA levels regardless of the pain state, but IL-6 was not associated with IL-6R(alpha). The differential expression of neurotrophic factors and their cognate soluble receptors in human vasculitic neuropathy suggests that NGF, which was effectively transferred to sensory axons with p75, may induce pain.

1,25-Dihydroxyvitamin D 3 increases striatal GDNF mRNA and protein expression in adult rats

Glial cell line-derived neurotrophic factor (GDNF) has been postulated as a possible candidate for therapeutic treatment in Parkinson’s disease (PD). Recent in vitro data suggest that 1,25-dihydroxyvitamin D3 [1,25(OH) 2D 3] treatment may enhance GDNF mRNA expression. In the present study, using semiquantitative RT-PCR and Western blot, we have shown that 1,25(OH) 2D 3 administration intraperitoneally, significantly increases GDNF mRNA and protein levels in the striatum of adult rats.

Induction of GDNF mRNA expression by melatonin in rat C6 glioma cells.

In order to determine the physiological effect of melatonin on glial cell line-derived neurotrophic factor (GDNF), which is reportedly up-regulated by high doses of this hormone, concentration-dependent studies were carried out in cultured cells. RT-PCR studies indicated that, in addition to GDNF, rat C6 glioma cells express both of the G protein-coupled melatonin receptor subtypes, MT1 and MT2. When C6 cells were treated with physiological (0.05-1 nM) or higher (10 and 100 nM) concentrations of melatonin for 24 h, a significant induction of relative GDNF mRNA levels (n = 4) was detected by semi-quantitative RT-PCR. These findings suggest that induction of GDNF is involved in physiological neuroprotection by melatonin. Given the potency of GDNF in maintaining nigrostriatal dopaminergic integrity, understanding the mechanisms of its induction by melatonin could provide novel therapies for Parkinson's disease.

Overexpression of GDNF Induces and Maintains Hyperinnervation of Muscle Fibers and Multiple End-Plate Formation

This study examined the role of glial cell line-derived neurotrophic factor (GDNF) in synaptic plasticity at the developing neuromuscular junction. Transgenic mice overexpressing GDNF in skeletal muscle under the myosin light chain-1 promoter were isolated. Northern blot and ELISA at 6 weeks of age indicated that GDNF mRNA and protein levels were elevated threefold in the lateral gastrocnemius muscle (LGM) of the GDNF-transgenic animals. Histochemical examination of LGM tissue sections at 6 weeks of age revealed a 70% increase in the number of cholinesterase-positive end plates without changes in end-plate area. Multiple end plates on a single muscle fiber were also observed, in addition to multiple axonal processes terminating on individual end plates. No change in the number of spinal motoneurons, overall LGM size, or muscle type composition was observed. Finally, overexpression of GDNF in muscle caused hypertrophy of neuronal somata in dorsal root ganglia without affecting their number. These findings demonstrate that overexpression of a single neurotrophic factor in skeletal muscle induces multiple end-plate formation and maintains hyperinnervation well beyond the normal developmental period. We suggest that GDNF, a muscle-derived motoneuron neurotrophic factor, serves an important role in the regulation of synaptic plasticity in the developing and adult neuromuscular junction.

Riluzole stimulates nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor synthesis in cultured mouse astrocytes

Riluzole is an antiexcitotoxic agent used for the treatment of amyotrophic lateral sclerosis, and reported to have neuroprotective effects in animal models of Parkinson's disease, Huntington's disease and brain ischemia. We investigated the effects of riluzole on synthesis of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in cultured mouse astrocytes. The protein and mRNA levels were measured by enzyme-linked immunosorbent assay and semiquantitative reverse transcription-polymerase chain reaction, respectively. Treatment with riluzole at 100 μg/ml (426 μM) for 24 h increased the contents of NGF, BDNF, and GDNF in the culture medium 109-fold, 2.0-fold and 3.1-fold over the control, respectively. The drug-induced relative mRNA levels of NGF, BDNF, and GDNF were 7.3-fold at 2 h, 2.1-fold at 4 h, and 1.9-fold at 2 h, respectively. These results indicate that riluzole stimulates synthesis of NGF, BDNF and GDNF in cultured astrocytes. Riluzole might exert neuroprotective effects, at least in part, via stimulation of neurotrophic factors.

Neurotrophic Factors and Receptors in the Immature and Adult Spinal Cord after Mechanical Injury or Kainic Acid

Delivery of neurotrophic factors to the injured spinal cord has been shown to stimulate neuronal survival and regeneration. This indicates that a lack of sufficient trophic support is one factor contributing to the absence of spontaneous regeneration in the mammalian spinal cord. Regulation of the expression of neurotrophic factors and receptors after spinal cord injury has not been studied in detail. We investigated levels of mRNA-encoding neurotrophins, glial cell line-derived neurotrophic factor (GDNF) family members and related receptors, ciliary neurotrophic factor (CNTF), and c-fos in normal and injured spinal cord. Injuries in adult rats included weight-drop, transection, and excitotoxic kainic acid delivery; in newborn rats, partial transection was performed. The regulation of expression patterns in the adult spinal cord was compared with that in the PNS and the neonate spinal cord. After mechanical injury of the adult rat spinal cord, upregulations of NGF and GDNF mRNA occurred in meningeal cells adjacent to the lesion. BDNF and p75 mRNA increased in neurons, GDNF mRNA increased in astrocytes close to the lesion, and GFRalpha-1 and truncated TrkB mRNA increased in astrocytes of degenerating white matter. The relatively limited upregulation of neurotrophic factors in the spinal cord contrasted with the response of affected nerve roots, in which marked increases of NGF and GDNF mRNA levels were observed in Schwann cells. The difference between the ability of the PNS and CNS to provide trophic support correlates with their different abilities to regenerate. Kainic acid delivery led to only weak upregulations of BDNF and CNTF mRNA. Compared with several brain regions, the overall response of the spinal cord tissue to kainic acid was weak. The relative sparseness of upregulations of endogenous neurotrophic factors after injury strengthens the hypothesis that lack of regeneration in the spinal cord is attributable at least partly to lack of trophic support.

Differential regulation of GDNF, neurturin, and their receptors in primary cultures of rat glial cells

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) bind to GFRα-1 and GFRα-2 receptors, respectively, and their neurotrophic activity is mediated by the tyrosine kinase receptor, Ret. All these molecules were found to be expressed in primary cultures of rat glial cells, which were largely composed of astrocytes and maintained in serum-free medium. Although GDNF, NTN and Ret mRNA levels were at the limit of detection, RNase protection assays revealed relatively high amounts of GFRα-1 and GFRα transcripts. To characterize signals controlling their expression, glial cells were exposed to serum or treated with hormones acting through nuclear receptors and by activators of the cAMP or protein kinase C (PKC)-dependent pathways. Retinoic acid or 1,25-dihydroxyvitamin D3 appeared ineffective. In contrast, the 5-fold increase in GFRα-2 mRNA after 24 hr of treatment with 10−10 M of tri-iodothyronine, suggests a physiological role of thyroid hormone in the regulation of this receptor in vivo. The serum induced a 7-fold increase in GFRα-1 mRNA levels. These changes may be mediated by the cAMP or PKC pathways because both forskolin and TPA up-regulated the GFRα-1 gene. Interestingly, only TPA led to a coordinated increase in the levels of GDNF, GFRα-1 and GFRα-2 mRNAs. On the other hand, NTN transcripts remained constant, irrespective of the culture conditions. Taken together, these results indicate that GDNF family ligands and their receptors are regulated in glial cells by common or independent transductional pathways, which could modulate their specific expression during brain development or in the case of trauma. J. Neurosci. Res. 64:242–251, 2001. © 2001 Wiley-Liss, Inc.

Differential regulation of GDNF, neurturin, and their receptors in primary cultures of rat glial cells.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) bind to GFR alpha-1 and GFR alpha-2 receptors, respectively, and their neurotrophic activity is mediated by the tyrosine kinase receptor, Ret. All these molecules were found to be expressed in primary cultures of rat glial cells, which were largely composed of astrocytes and maintained in serum-free medium. Although GDNF, NTN and Ret mRNA levels were at the limit of detection, RNase protection assays revealed relatively high amounts of GFR alpha-1 and GFR alpha transcripts. To characterize signals controlling their expression, glial cells were exposed to serum or treated with hormones acting through nuclear receptors and by activators of the cAMP or protein kinase C (PKC)-dependent pathways. Retinoic acid or 1,25-dihydroxyvitamin D3 appeared ineffective. In contrast, the 5-fold increase in GFR alpha-2 mRNA after 24 hr of treatment with 10(-10) M of tri-iodothyronine, suggests a physiological role of thyroid hormone in the regulation of this receptor in vivo. The serum induced a 7-fold increase in GFR alpha-1 mRNA levels. These changes may be mediated by the cAMP or PKC pathways because both forskolin and TPA up-regulated the GFR alpha-1 gene. Interestingly, only TPA led to a coordinated increase in the levels of GDNF, GFR alpha-1 and GFR alpha-2 mRNAs. On the other hand, NTN transcripts remained constant, irrespective of the culture conditions. Taken together, these results indicate that GDNF family ligands and their receptors are regulated in glial cells by common or independent transductional pathways, which could modulate their specific expression during brain development or in the case of trauma.