Glial Cell Line-derived Neurotrophic Factor mRNA Research Articles

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.

Rehmannia glutinosa induces glial cell line-derived neurotrophic factor gene expression in astroglial cells via cPKC and ERK1/2 pathways independently

Among four herbs of traditional Chinese medicines (TCMs) used in the therapy of dementia, Rehmannia glutinosa (RG) was found to induce the gene expression of glial cell line-derived neurotrophic factor (GDNF) in C6 glioblastoma cells and primary cultured astrocytes. The RG-induced GDNF mRNA up-regulation in C6 glioblastoma cells was completely attenuated by the presence of a pan-specific protein kinase C (PKC) inhibitor (Ro-31-8220) and a MAPK/ERK kinase 1 (MEK1) inhibitor (U0126). A conventional PKC inhibitor (Gö6976) also significantly decreased GDNF gene induction. On the other hand, RG treatment was found to stimulate phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), which preceded GDNF mRNA induction in C6 glioblastoma cells. However, none of the PKC inhibitors significantly changed RG-stimulated ERK1/2 phosphorylation. Therefore, RG-stimulated GDNF gene expression could be independently up-regulated through cPKC and ERK 1/2 pathways in C6 glioblastoma cells.

An Analog of a Dipeptide-Like Structure of FK506 Increases Glial Cell Line-Derived Neurotrophic Factor Expression through cAMP Response Element-Binding Protein Activated by Heat Shock Protein 90/Akt Signaling Pathway

Glial cell line-derived neurotrophic factor (GDNF) is an important neurotrophic factor that has therapeutic implications for neurodegenerative disorders. We previously showed that leucine-isoleucine (Leu-Ile), an analog of a dipeptide-like structure of FK506 (tacrolimus), induces GDNF expression both in vivo and in vitro. In this investigation, we sought to clarify the cellular mechanisms underlying the GDNF-inducing effect of this dipeptide. Leu-Ile transport was investigated using fluorescein isothiocyanate-Leu-Ile in cultured neurons, and the results showed the transmembrane mobility of this dipeptide. By liquid chromatography-mass spectrometry and quartz crystal microbalance assay, we identified heat shock cognate protein 70 as a protein binding specifically to Leu-Ile, and molecular modeling showed that the ATPase domain is the predicted binding site. Leu-Ile stimulated Akt phosphorylation, which was attenuated significantly by heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA). Moreover, enhanced interaction between phosphorylated Akt and Hsp90 was detected by immunoprecipitation. Leu-Ile elicited an increase in cAMP response element binding protein (CREB) phosphorylation, which was inhibited by GA, indicating that CREB is a downstream target of Hsp90/Akt signaling. Leu-Ile elevated the levels of GDNF mRNA and protein expression, whereas inhibition of CREB blocked such effects. Leu-Ile promoted the binding activity of phosphorylated CREB with cAMP response element. These findings show that CREB plays a key role in transcriptional regulation of GDNF expression induced by Leu-Ile. In conclusion, Leu-Ile activates Hsp90/Akt/CREB signaling, which contributes to the upregulation of GDNF expression. It may represent a novel lead compound for the treatment of dopaminergic neurons or motoneuron diseases.

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.

Adenoviral gene transfer of BDNF and GDNF synergistically prevent motoneuron loss in the nucleus ambiguus

We have previously shown that neuroprotective effects of an adenoviral glial cell line-derived neurotrophic factor (GDNF) gene transfer on the lesioned adult rat motoneurons in the nucleus ambiguus. In the present study, we examined neuroprotective effects of adenoviral gene transfer of brain-derived neurotrophic factor (BDNF) or/and GDNF to motoneurons in nucleus ambiguus using an adult rat vagal nerve avulsion model. The animals avulsed and inoculated with adenoviral vectors encoding BDNF (AxCAmBDNFME) or/and GDNF (AxCAhGDNF) showed immunolabeling for BDNF or/and GDNF in the nucleus ambiguus on the treated side, respectively, and expression of virus-induced BDNF or/and GDNF mRNA transcripts in the brainstem tissue that contained the nucleus ambiguus of the treated side. The treatment with AxCAhGDNF or AxCAmBDNFME significantly prevented the loss of vagal motoneurons in comparison to the control; the protective effect of AxCAmBDNFME was greater than that of AxCAhGDNF. The combined treatment with AxCAmBDNFME and AxCAhGDNF acted synergistically and significantly larger number of vagal motoneurons was preserved as compared to either AxCAmBDNFME treatment or AxCAhGDNF treatment. The treatment with AxCAmBDNFME or/and AxCAhGDNF after avulsion also suppressed the activity of nitric oxide synthase in lesioned motoneurons in the nucleus ambiguus. These results indicate that adenovirus-mediated BDNF and GDNF gene transfer may prevent the degeneration of motoneurons in humans after either vagal nerve injury or recurrent laryngeal nerve injury.

Nerve conduit filled with GDNF gene-modified schwann cells enhances regeneration of the peripheral nerve

A recombinant retrovirus vector containing the glial cell line-derived neurotrophic factor (GDNF) gene was constructed and transfected into Schwann cells (SCs) to investigate the possibility of GDNF transfection and functional expression of transfected SCs, including GDNF secretion and its mRNA expression. We found that transfection of the GDNF gene into SCs led to significantly enhanced expression of GDNF mRNA. The rate of GDNF secretion by GDNF-SCs was also increased. Functionally, more surviving motoneurons were seen when they were cocultured in GDNF-SC-conditioned medium than when they were in normal SC-conditioned medium. When bridging a rat sciatic nerve defect with a conduit filled with GDNF-transfected SCs, nerve regeneration was better than that of the control. In conclusion, transfection of SCs with the GDNF gene could enhance SC function. Application of genetically modified SCs could be a better way to promote nerve regeneration.

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.

Glial cell line derived neurotrophic factor is expressed by epithelia of human renal dysplasia.

Differentiation of the metanephros is abnormal in cases of renal dysplasia, resulting in abnormal kidney organization. In vitro and in vivo studies indicate that glial cell line derived neurotrophic factor (GDNF) is a major regulator of kidney development and ureteral arborization. Therefore, we investigated the pattern of GDNF gene expression in human dysplastic kidneys. Specimens of whole tissues of human normal and dysplastic kidneys associated with obstructive uropathy were analyzed for GDNF mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Immunohistochemistry with GDNF antibody and laser capture microdissection plus RT-PCR were done to identify cells producing GDNF. Apoptosis, BCL-2 and Ki67 were also studied. There were few if any GDNF transcripts in normal kidneys, whereas GDNF was over expressed in renal dysplasia specimens. Strong GDNF expression was found in the dysplastic tubules of dysplastic kidneys, whereas peritubular mesenchyma expressed no GDNF protein. Laser capture microdissection/RT-PCR detected GDNF mRNA in epithelial cells isolated from dysplastic tubules but not in cells from the surrounding mesenchyma, which was confirmed by sequence analysis. GDNF expression by epithelial cells was associated with high proliferation, BCL-2 expression and rare apoptosis. GDNF gene expression is restricted to the tubular epithelium of dysplastic human kidneys. Our results strongly suggest that GDNF not only influences kidney morphogenesis, but is also implicated in abnormal kidney development.

Adenoviral GDNF gene transfer enhances neurofunctional recovery after recurrent laryngeal nerve injury

To assess the possibility of gene therapy for recurrent laryngeal nerve (RLN) injury, we examined functional and histological recovery after glial cell line-derived neurotrophic factor (GDNF) gene transfer in a rat RLN crush model. Adenoviral vector encoding beta-galactosidase gene (AxCALacZ) or human GDNF gene (AxCAhGDNF) was injected into the crush site of the RLN. Neurons in the nucleus ambiguus on the crushed side were labeled with X-gal or GDNF immnohistochemistry after AxCALacZ or AxCAhGDNF injection. Reverse transcription-polymerase chain reaction analysis revealed expression of human GDNF mRNA transcripts in brainstem tissue containing the nucleus ambiguus on the crushed side after AxCAhGDNF injection. Animals injected with AxCAhGDNF displayed significantly improved motor nerve conduction velocity of the RLN and recovery rate of vocal fold movement at 2 and 4 weeks after treatment as compared to controls. AxCAhGDNF-injected animals showed a significantly larger axonal diameter and improved remyelination in crushed RLN as compared to controls. Adenoviral GDNF gene transfer may thus promote laryngeal function recovery after RLN injury. Inoculation of adenoviral vector containing the GDNF gene at the site of damage soon after nerve injury may assist patients with laryngeal paralysis caused by nerve injury during head and neck surgery.

Novel targets for valproic acid: up‐regulation of melatonin receptors and neurotrophic factors in C6 glioma cells

Valproic acid (VPA) is a potent anti-epileptic and effective mood stabilizer. It is known that VPA enhances central GABAergic activity and activates the mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-ERK) pathway. It can also inhibit various isoforms of the enzyme, histone deacetylase (HDAC), which is associated with modulation of gene transcription. Recent in vivo studies indicate a neuroprotective role for VPA, which has been found to up-regulate the expression of brain-derived neurotrophic factor (BDNF) in the rat brain. Given the interaction between the pineal hormone, melatonin, and GABAergic systems in the central nervous system, the effects of VPA on the expression of the mammalian melatonin receptor subtypes, MT1 and MT2, were examined in rat C6 glioma cells. The effects of VPA on the expression of glial cell line-derived neurotrophic factor (GDNF) and BDNF were also examined. RT-PCR studies revealed a significant induction of melatonin MT1 receptor mRNA in C6 cells following treatment with 3 or 5 mm VPA for 24 h or 5 mm VPA for 48 h. Western analysis and immunocytochemical detection confirmed that the VPA-induced increase in MT1 mRNA results in up-regulation of MT1 protein expression. Blockade of the MAPK-ERK pathway by PD98059 enhanced the effect of VPA on MT1 expression, suggesting a negative role for this pathway in MT1 receptor regulation. In addition, significant increases in BDNF, GDNF and HDAC mRNA expression were observed after treatment with VPA for 24 or 48 h. Taken together, the present findings suggest that the neuroprotective properties of VPA involve modulation of neurotrophic factors and receptors for melatonin, which is also thought to play a role in neuroprotection. Moreover, the foregoing suggests that combinations of VPA and melatonin could provide novel therapeutic strategies in neurological and psychiatric disorders.

Anatomical basis of glial cell line-derived neurotrophic factor expression in the striatum and related basal ganglia during postnatal development of the rat.

There is increasing evidence that glial cell line-derived neurotrophic factor (GDNF) plays a role as a limiting, striatal target-derived neurotrophic factor for dopamine neurons of the substantia nigra pars compacta (SNpc) by regulating the magnitude of the first phase of postnatal natural cell death which occurs in these neurons. While it has been shown that GDNF mRNA is relatively abundant in postnatal striatum, the cellular basis of its expression has been unknown. We therefore used nonradioactive in situ hybridization and immunohistochemistry to examine the cellular basis of GDNF mRNA and protein expression, respectively, in postnatal striatum and related structures. We found that GDNF mRNA is expressed within medium-sized striatal neurons. Expression in glia was not observed. At the protein level, regionally, GDNF expression in striatum was observed in striosomal patches, as previously described. At a cellular level a few neurons were observed, but they do not account for the striosomal pattern. This pattern is predominantly due to GDNF-positive neuropil. Some of this neuropil arises from tyrosine hydroxylase-positive nigro-striatal dopaminergic afferents. Astrocytic processes do not appear to contribute to the striosomal pattern. GDNF-positive fibers are identified not only within intrinsic striatal neuropil, but also in fibers within the major striatal efferent targets: the globus pallidus, the entopeduncular nucleus, and the SN pars reticulata. We conclude that during normal postnatal development, medium-sized neurons are the principal source of GDNF within the striatum.

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.

Expression of GDNF in spinal cord injury and its repression by ONO-1714

ONO-1714 is a newly developed specific inhibitor for inducible nitric oxide synthase (iNOS). We have shown that ONO-1714 has some neuroprotective effects. In this report, we investigated the effects of ONO-1714 in injured spinal cords, and analyzed the expression of glial cell-line-derived neurotrophic factor (GDNF) after injury. Male Sprague-Dawley rats were subjected to contusive spinal cord injury and administrated 0.1 mg/kg ONO-1714. The injured spinal cords were isolated at appropriate time points and GDNF mRNA was determined by semi-quantitative RT-PCR. GDNF-positive cells were also counted after immunohistochemical stainings. ONO-1714 diminished the early stage production of GDNF after injury as well as it reduced the production of nitric oxide produced by iNOS and apoptotic cells.

Stimulation of production of glial cell line-derived neurotrophic factor and nitric oxide by lipopolysaccharide with different dose-responsiveness in cultured rat macrophages

To understand the molecular basis of inflammation-induced neurotrophic influences, we investigated the effects of lipopolysaccharide (LPS) on production of glial cell line-derived neurotrophic factor (GDNF) in the injured rat spinal cord or in cultured rat macrophages in comparison with the effects on synthesis/secretion of inducible nitric oxide synthase (iNOS) and nitric oxide (NO). We found that GDNF mRNA expression lasted longer than that of iNOS mRNA in the injured spinal cord after injection of the high-dose LPS that had improved locomotor function, suggesting that the GDNF expression and its balance with NO generation were critical for injury regeneration. Therefore, we next investigated the effects of LPS on cultured macrophages. Levels of iNOS mRNA and secreted NO were enhanced by LPS at lower concentrations (10 ng/mL and above), whereas mRNA expression and secretion of GDNF were elevated only at higher concentrations (100 ng/mL and above). The culture medium of macrophages treated with 10 ng/mL of LPS was actually neurotoxic against cultured cortical neurons, whereas that conditioned at 1000 ng/mL was not. These observations suggest that neurotoxicity partly based on NO is induced by a lower degree of inflammation, whereas neurotrophic effects based on GDNF are manifested at a higher degree of inflammatory activity.

Expression and function of GDNF family ligands and receptors in the carotid body

The carotid body is a neural crest-derived neuroendocrine organ that detects the oxygen level in blood and regulates ventilation. Unlike many other neural crest derivatives, the trophic factors mediating survival and differentiation of neuroendocrine cells of the carotid body are unknown. Given that many neural crest derivatives rely on the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) for survival and function, we undertook an analysis of the carotid body as a potential site of GFL action. RET and GDNF family receptor alphas (GFRα) 1–3 are expressed in the developing carotid body as detected by RT-PCR and immunocytochemistry. mRNA for GDNF, and artemin (ARTN) were also present. In vitro, treatment with GDNF, neurturin (NRTN), or ARTN, individually or in combination, produced an increase in the number and length of processes of the Type-I glomus cells of the carotid body [embryonic day-17 (E17) rats]. However, GFLs alone or in combination had no effect on glomus cell survival in either postnatal day-1 (P1) or E17 carotid body cultures. These results suggest that one or more GFLs may have a role in carotid body function. In addition, the results of this study suggest that endogenous or exogenous GFLs may enhance target innervation by carotid body transplants.

Regulated Delivery of Glial Cell Line-Derived Neurotrophic Factor into Rat Striatum, Using a Tetracycline-Dependent Lentiviral Vector

In this study, a tetracycline-regulated lentiviral vector system, based on the tetracycline-dependent transactivator rtTA2(S)-M2, was developed for controlled expression of glial cell line-derived neurotrophic factor (GDNF) in the rat brain. Expression of the marker gene green fluorescent protein (GFP) and GDNF was tightly regulated in a dose-dependent manner in neural cell lines in vitro. Injection of high-titer lentiviral vectors into the rat striatum resulted in a 7-fold induction of GDNF tissue levels (1060 pg/mg tissue), when doxycycline (a tetracycline analog) was added to the drinking water. However, low levels of GDNF (150 pg/mg tissue) were also detected in animals that did not receive doxycycline, indicating a significant background leakage from the vector system in vivo. The level of basal expression was markedly reduced when a 10-fold lower dose of the tetracycline-regulated GDNF vector was injected into the striatum (3-11 pg/mg tissue), and doxycycline-induced GDNF tissue levels obtained in these animals were about 190 pg/mg tissue. Doxycycline-induced expression of GDNF resulted in a significant downregulation of the tyrosine hydroxylase (TH) protein in the intact striatum. Removal of doxycycline from the drinking water rapidly (within 3 days) turned off transgenic GDNF mRNA expression and GDNF protein levels in the tissue were completely reduced by 2 weeks, demonstrating the dynamics of the system in vivo. Accordingly, TH protein expression returned to normal by 2-8 weeks after removal of doxycycline, indicating that GDNF-induced downregulation of TH is a reversible event.

Axonal regrowth downregulates the synthesis of glial cell line-derived neurotrophic factor in the lesioned rat sciatic nerve

The effect of axonal regeneration on de novo synthesis of glial cell line-derived neurotrophic factor (GDNF) in rat sciatic nerves was examined. Transection of the sciatic nerve caused a prominent increase in the GDNF content in the distal segments within 1 week. The high level was sustained until 4 weeks in the animal model in which the nerve ends were ligated with thread (non-regeneration group); however, it was reduced to the original level within 2 or 4 weeks after the transection only in the segments invaded by regenerating axons in the models in which the nerve ends were coaptated (regeneration group). Expression of both GDNF protein and mRNA was decreased with a reciprocal increase in the density of neurofilaments, used as a marker of axonal ingrowth in distal segments of the regeneration group, suggesting that axonal contact turned off the GDNF-mediated nerve regeneration activity.

Increase by FK960, a novel cognitive enhancer, in glial cell line-derived neurotrophic factor production in cultured rat astrocytes

We examined the effect of N-(4-acetyl-1-piperazinyl)- p-fluorobenzamide monohydrate (FK960), a novel anti-dementia drug, on neurotrophic factor production in cultured rat astrocytes. FK960 (100 nM) increased mRNA and protein levels of glial cell line-derived neurotrophic factor (GDNF). FK960 did not affect mRNA levels of neurotrophic factors other than GDNF. The effect of FK960 was not affected by antagonists of dopamine and α7-nicotinic acetylcholine receptors. FK960 stimulated phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase (ERK) without any effect on phosphoryolation of p38 and c-Jun N-terminal kinase. FK960 increased the levels of c-Fos and phosphorylation of cAMP responsive element binding protein (CREB). The effect of FK960 on c-Fos was inhibited by PD98059 (10 μM), an ERK kinase inhibitor, and cycloheximide (1 μg/ml), a transcription inhibitor, and the effect of FK960 on CREB phosphorylation was blocked by PD98059. The effect of FK960 on GDNF mRNA expression was attenuated by PD98059, curcumin (10 μM), an activator protein-1 inhibitor, cycloheximide and actinomycin D (10 μg/ml). These results suggest that FK960 stimulates GDNF production in c-Fos- and CREB-dependent mechanisms in cultured astrocytes and that ERK signal is responsible for both c-Fos expression and CREB phosphorylation in the cascades.

Increase by FK960, a novel cognitive enhancer, in glial cell line-derived neurotrophic factor production in cultured rat astrocytes

We examined the effect of N-(4-acetyl-1-piperazinyl)-p-fluorobenzamide monohydrate (FK960), a novel anti-dementia drug, on neurotrophic factor production in cultured rat astrocytes. FK960 (100 nM) increased mRNA and protein levels of glial cell line-derived neurotrophic factor (GDNF). FK960 did not affect mRNA levels of neurotrophic factors other than GDNF. The effect of FK960 was not affected by antagonists of dopamine and α7-nicotinic acetylcholine receptors. FK960 stimulated phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase (ERK) without any effect on phosphoryolation of p38 and c-Jun N-terminal kinase. FK960 increased the levels of c-Fos and phosphorylation of cAMP responsive element binding protein (CREB). The effect of FK960 on c-Fos was inhibited by PD98059 (10 μM), an ERK kinase inhibitor, and cycloheximide (1 μg/ml), a transcription inhibitor, and the effect of FK960 on CREB phosphorylation was blocked by PD98059. The effect of FK960 on GDNF mRNA expression was attenuated by PD98059, curcumin (10 μM), an activator protein-1 inhibitor, cycloheximide and actinomycin D (10 μg/ml). These results suggest that FK960 stimulates GDNF production in c-Fos- and CREB-dependent mechanisms in cultured astrocytes and that ERK signal is responsible for both c-Fos expression and CREB phosphorylation in the cascades.