Effects of GDNF on 6-OHDA-induced death in a dopaminergic cell line: modulation by inhibitors of PI3 kinase and MEK

Parkinson's disease is a neurodegenerative disorder associated with the selective death of dopaminergic neurons. Glial cell line-derived neurotrophic factor (GDNF) can protect dopaminergic neurons in several parkinsonian models. We used the dopaminergic cell line MN9D to explore the mechanisms underlying GDNF-mediated protection against the neurotoxin 6-hydroxydopamine (6-OHDA). MN9D cell viability was decreased 24 hr after a 15-min exposure to 6-OHDA (50-1000 microM) as revealed by staining with Hoechst reagent and Trypan blue. The addition of GDNF (10 ng/ml) before, during, and after exposure to 6-OHDA significantly increased the number of viable cells as assessed by Hoechst staining. In contrast, 6-OHDA-induced cell membrane damage was unaffected as measured by Trypan blue exclusion. The PI3K specific inhibitor LY294002 (10-50 microM) blocked GDNF-mediated protection against nuclear condensation, as did the MAPK kinase (MEK) inhibitor U0126 (5- 20 microM). These studies suggest that GDNF can protect dopaminergic cells against some but not all aspects of 6-OHDA-induced toxicity by acting through both PI3K and MAPK signaling pathways.     

TGFbeta trophic factors differentially modulate motor axon outgrowth and protection from excitotoxicity

Transforming growth factor (TGF) beta-like trophic factors have been shown to be protective in acute neuronal injury paradigms. In the current study, we analyzed and compared members of this growing family, including glial cell line-derived neurotrophic factor (GDNF), neurturin, nodal, persephin, and TGFbeta1, for protection against chronic glutamate toxicity. In parallel, we developed a organotypic spinal cord culture system to study the ability of these factors to promote motor axon outgrowth across white matter. Using these systems, we were able to differentiate the neuroprotective effect of the TGFbeta-like factors from their motor axon outgrowth-promoting activity. GDNF, neurturin, persephin, nodal, and TGFbeta1 all protected against excitotoxic motor neuron degeneration. Low amounts of GDNF (1 ng/ml) and high concentrations of neurturin induced vigorous motor axon outgrowth. In contrast, nodal, persephin, and TGFbeta1 did not induce motor axon outgrowth. Both GDNF and neurturin bind to Ret receptor complexes and were capable of activating the MAP kinase pathway. A specific inhibitor of MAP kinase kinase, PD98059, inhibited the motor axon outgrowth-promoting activity of the GDNF but not the neuroprotective activity. Similarly, the specific PI3K inhibitors, LY294002 and wortmannin, were able to inhibit the promotion of motor axon outgrowth by GDNF, but did not affect neuroprotective activity. Our results suggest that the neurite outgrowth-promoting effect of GDNF is mediated through the PI3K and MAP kinase pathways. The neuroprotective effect of GDNF appears to be through a separate pathway.     

腺病毒介导的GDNF基因转移体外表达及生物学活性研究

为利用重组腺病毒介导的胶质细胞源性神经营养因子（GDNF）基因转移治疗帕金森病（PD）提供依据。方法：采用免疫组化、RT－PCR及ELISA定量分析观察人GDNF腺病毒（Ad-GDNF)在大鼠星形胶质 PC12细胞的表达,通过观察病毒直接感染及病毒感染的PC12细胞上清对中脑原代培养细胞中的TH阳性细胞（DA能神经元）生存能力和形态分化的影响来验证其生物学活性。结果Ad-GDNF在星形胶质细胞、PC12细胞及大鼠中脑原代培养细胞均可有效表达,其表达产物对中脑DNA能神经元的生存和形态分化均有显著的促进作用。结论：腺病毒介导的GDNF基因转移可在体外有效表达,且表达产物具有生物学活性,提示该手段在PD治疗方面具有良好的应用前景。     

TGFβ Trophic Factors Differentially Modulate Motor Axon Outgrowth and Protection from Excitotoxicity

Transforming growth factor (TGF) β-like trophic factors have been shown to be protective in acute neuronal injury paradigms. In the current study, we analyzed and compared members of this growing family, including glial cell line-derived neurotrophic factor (GDNF), neurturin, nodal, persephin, and TGFβ1, for protection against chronic glutamate toxicity. In parallel, we developed a organotypic spinal cord culture system to study the ability of these factors to promote motor axon outgrowth across white matter. Using these systems, we were able to differentiate the neuroprotective effect of the TGFβ-like factors from their motor axon outgrowth-promoting activity. GDNF, neurturin, persephin, nodal, and TGFβ1 all protected against excitotoxic motor neuron degeneration. Low amounts of GDNF (1 ng/ml) and high concentrations of neurturin induced vigorous motor axon outgrowth. In contrast, nodal, persephin, and TGFβ1 did not induce motor axon outgrowth. Both GDNF and neurturin bind to Ret receptor complexes and were capable of activating the MAP kinase pathway. A specific inhibitor of MAP kinase kinase, PD98059, inhibited the motor axon outgrowth-promoting activity of the GDNF but not the neuroprotective activity. Similarly, the specific PI3K inhibitors, LY294002 and wortmannin, were able to inhibit the promotion of motor axon outgrowth by GDNF, but did not affect neuroprotective activity. Our results suggest that the neurite outgrowth-promoting effect of GDNF is mediated through the PI3K and MAP kinase pathways. The neuroprotective effect of GDNF appears to be through a separate pathway.     

Glial-derived neurotrophic factor (GDNF) prevents ethanol (EtOH) induced B92 glial cell death by both PI3K/AKT and MEK/ERK signaling pathways

We investigated the neuroprotective effect of glial-derived neurotrophic factor (GDNF) upon alcohol-exposed B92 cultures, as well as the role of the cytoskeleton and mitogen-activated protein kinase (MAPK) pathways in this effect. Ethanol (EtOH) was added to cultures, either alone or in combination with 30 ng/ml GDNF. Exposure to EtOH (86 and 172 mM; 60 and 120 min) increased the frequency of apoptotic cells identified by nuclear DNA staining with 4,6-diamidino-2-phenylindole (DAPI). Cultures treated with GDNF showed a decrease in ethanol-induced apoptosis. A jun N-terminal kinase (JNK) pathway is activated by EtOH and their pharmacological inhibition (by SP600125) neutralized ethanol-induced apoptosis, suggesting a role for JNK in EtOH neurotoxicity. Immunocytochemically detected phospho-JNK (p-JNK) showed an unusual filamental expression, and localized together with actin stress fibers. Examination of the cytoskeleton showed that EtOH depolymerized actin filaments, inducing p-JNK dissociation and translocation to the nucleus, which suggests that released p-JNK may contribute to glial cell death after EtOH exposure. Treatment with GDNF, in turn, may neutralize the ethanol-induced cell death pathway. Either a phosphatidylinositol 3-kinase (PI3K)/AKT pathway inhibitor (LY294002) or an inhibitor of the extracellular signal-regulated kinase (ERK) 1, 2 pathways (UO126) failed to neutralize GDNF protective effects. However, the simultaneous use of both inhibitors blocked the protective effect of GDNF, suggesting a role for both signaling cascades in the GDNF protection. These findings provide further insight into the mechanism involved in ethanol-induced apoptosis and the neurotrophic protection of glial cells.     

Expression of the GDNF family members and their receptors in the mature rat cochlea

The GDNF family comprises glial cell line-derived neurotrophic factor (GDNF) and the related proteins neurturin, artemin and persephin, which form a subgroup of the TGF-beta superfamily of growth factors. All four neurotrophic factors provide neuronal cell protection and cell survival. GDNF expression was found in the cochlea, and GDNF has been shown to be effective for inner ear protection from drugs and noise-induced insults. As the other members of the GDNF family also provide protective effects on neuronal cells, they may play important roles in the inner ear. We used RT-PCR to examine the expression of GDNF, neurturin, artemin, persephin and their receptors GFRalpha-1, GFRalpha-2, GFRalpha-3 and c-ret in whole rat cochlea as well as in functionally different subfractions (modiolus and sensorineural epithelium/lateral wall) and compared the levels of neurotrophin and receptor mRNAs in the cochlea to those in substantia nigra brain region. Our results demonstrate the expression of all GDNF family members and their receptors in cochlea and substantia nigra. However, the relative levels of mRNA were different for several genes tested in subfractions of the cochlea and/or compared to expression levels in substantia nigra. The presence of mRNA for all four members of the GDNF family and their preferred receptors in the rat cochlea suggests potential functional importance of these neurotrophic factors as protection and survival factors in the inner ear.     

Neuroprotection by neurotrophins and GDNF family members in the excitotoxic model of Huntington's disease

Huntington's disease is a neurodegenerative disorder characterized by a selective degeneration of striatal projection neurons, which deal with choreic movements. Neuroprotective therapy could be achieved with the knowledge of the specific trophic requirements of these neuronal populations. Thus, the induction of endogenous trophic response or the exogenous administration of neurotrophic factors may help to prevent or stop the progression of the illness. Excitotoxicity has been implicated in the etiology of Huntington's disease, because intrastriatal injection of glutamate receptor agonists reproduces some of the neuropathological features of this disorder. Activation of glutamate receptors in the striatum differentially regulates the expression of neurotrophins, glial cell line-derived neurotrophic factor (GDNF), neurturin, and their receptors in the striatum and in its connections, cortex, and substantia nigra, showing a selective trophic response against excitotoxic insults. Transplantation of cells genetically engineered to release neurotrophic factors in the striatum has been used to study the neuroprotective effects of neurotrophin and GDNF family members in the excitotoxic model of Huntington's disease. Neurotrophins (brain-derived neurotrophic factor [BDNF], neurotrophin-3, and neurotrophin-4) protected striatal projection neurons against quinolinic or kainic acid treatment. However, GDNF family members showed a more specific action. Neurturin only protected gamma-aminobutyric acid (GABA)/enkephalinergic neurons that project to the external segment of the globus pallidus, whereas GDNF exerts its effects on GABA/substance P positive neurons, which project to the substantia nigra pars compacta and the internal segment of the globus pallidus. In conclusion, the trophic requirements of each population of striatal projection neurons are due to a complex interaction between several neurotrophic factors, such as neurotrophins and GDNF family members, which can be modified, in different pathological conditions. Moreover, these neurotrophic factors may be able to provide selective protection for basal ganglia circuits, which are affected in striatonigral degenerative disorders, such as Huntington's disease or multisystem atrophy.     

The MAPK pathway is required for depolarization-induced "promiscuous" immediate-early gene expression but not for depolarization-restricted immediate-early gene expression in neurons

Depolarization, growth factors, neurotrophins, and other stimuli induce expression of immediate early genes (IEGs) in neurons. We identified a subset of IEGs, IPD-IEGs, which are induced preferentially by depolarization, but not by neurotrophins or growth factors, in PC12 cells. The "promiscuous" IEGs Egr1 and c-fos, induced by growth factors and neurotrophins, in addition to depolarization, require activation of the MAP kinase signaling pathway for induction in response to KCl depolarization in PC12 cells; MEK1/2 inhibitors block KCl-induced Egr1 and c-fos expression. In contrast, MEK1/2 inhibition has no effect on KCl-induced expression of the known IPD-IEGs in PC12 cells. Additional "candidate" IDP-IEGs were identified by a microarray comparison of genes induced by KCl in the presence vs. the absence of an MEK1/2 inhibitor in PC12 cells. Northern blot analyses demonstrated that representative newly identified candidate IPD-IEGs, as with the known IPD-IEGs, are also induced by a MAP kinase- independent pathway in response to depolarization, both in PC12 cells and in rat primary cortical neurons. Nerve growth factor and epidermal growth factor are unable to induce the expression of the Crem/Icer, Nur77, Nor1, Rgs2, Dusp1 (Mkp1), and Dscr1 genes in PC12 cells, validating their identification as IPD-IEGs. Inhibiting calcium/calmodulin-dependent kinase II (CaMKII), calcineurin, or protein kinase A (PKA) activity prevents KCl-induced IPD-IEG mRNA accumulation, suggesting that the IPD-IEG genes are induced by depolarization in neurons via a combination of calcineurin/PKA- and CaMKII-dependent pathways.     

Dental pulp stem cells stimulate neuronal differentiation of PC12 cells

Dental pulp stem cells (DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium (DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor (NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2 (MAP-2) and cytoskeletal marker βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and βIII-tubulin were analysed by quantitative polymerase chain reaction (qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors (NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and βIII-tubulin immunostaining. Furthermore, DPSCs-CM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls; however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.

Chinese Library Classification No. R459.9; R364; R622     

Complete and long-term rescue of lesioned adult motoneurons by lentiviral-mediated expression of glial cell line-derived neurotrophic factor in the facial nucleus.

To date, delivery of neurotrophic factors has only allowed to transiently protect axotomized facial motoneurons against cell death. In the present report, long-term protection of these neurons was evaluated by continuously expressing the neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) within the facial nucleus using a lentiviral vector system. The viral vector was injected unilaterally into the facial nucleus of 4-month-old Balb/C mice. In contrast to axotomy in other adult rodents, facial nerve lesion in these animals leads to a progressive and sustained loss and/or atrophy of >50% of the motoneurons. This model thus represents an attractive model to evaluate potential protective effects of neurotrophic factors for adult-onset motoneuron diseases, such as amyotrophic lateral sclerosis. One month after unilateral lentiviral vector injection, the facial nerve was sectioned, and the animals were killed 3 months later. Viral delivery of the GDNF gene led to long-term expression and extensive diffusion of GDNF within the brainstem. In addition, axotomized motoneurons were completely protected against cell death, because 95% of the motoneurons were present as demonstrated by both Nissl staining and choline acetyltransferase immunoreactivity. Furthermore, GDNF prevented lesion-induced neuronal atrophy and maintained proximal motoneuron axons, despite the absence of target cell reinnervation. This is the first evidence that viral-mediated delivery of GDNF close to the motoneuron cell bodies of the facial nucleus of adult mice can lead to complete and long-term protection against lesion-induced cell death.     

重组大鼠质粒pEGFP-GDNF的构建和在脊髓内表达的实验研究

采用RT PCR方法从大鼠胎脑组织总RNA中扩增出该基因的全序列cDNA ,并以融合蛋白方式克隆到增强型绿色荧光蛋白 (EGFP)报告基因的真核表达载体中 ,成功构建了重组质粒 pEGFP GDNF在体表达载体。采用基因注射法将阳离子脂质体DC Chol和pEGFP GDNF基因混合后转染至大鼠胸段脊髓组织中 ,观察GDNF在大鼠脊髓中的表达。RT PCR结果表明注射局部GDNFmRNA表达增加。荧光显微镜下观察发现 ,注射局部灰质和白质神经细胞内均有较多绿色荧光蛋白表达。本研究结果提示 ,阳离子脂质体介导GDNF体内转基因的方法是可行的 ,EGFP可作为报告基因观察GDNF在体内的表达。     

Comparison of the capability of GDNF, BDNF, or both, to protect nigrostriatal neurons in a rat model of Parkinson's disease

Both glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) can protect nigrostriatal dopaminergic neurons from neurotoxins in rodent and monkey models of Parkinson's disease (PD). These two neurotrophic factors are usually tested individually. This study was designed to compare GDNF, BDNF, or both, for their capabilities to correct behavioral deficits and protect nigrostriatal dopaminergic neurons in a rat model of PD. Gene transfer used a helper virus-free Herpes Simplex Virus (HSV-1) vector system and a modified neurofilament heavy gene promoter that supports long-term expression in forebrain neurons. Rats received unilateral intrastriatal injections of HSV-1 vectors that express either GDNF or BDNF, or both vectors, followed by intrastriatal injections of 6-hydroxydopamine (6-OHDA). Recombinant GDNF or BDNF was detected in striatal neurons in rats sacrificed at 7 months after gene transfer. Of note, GDNF was significantly more effective than BDNF for both correcting behavioral deficits and protecting nigrostriatal dopaminergic neurons. Expression of both neurotrophic factors was no more effective than expression of only GDNF. These results suggest that GDNF is more effective than BDNF for correcting the rat model of PD, and that there are no detectable benefits from expressing both of these neurotrophic factors.     

Serotonin increases glial cell line-derived neurotrophic factor release in rat C6 glioblastoma cells

Antidepressants, which increase monoamine levels, induce glial cell line-derived neurotrophic factor (GDNF) release in C6 cells. Thus, we examined whether monoamines affect on GDNF release in C6 cells. We found that serotonin (5-HT) specifically increased GDNF mRNA expression and GDNF release in a dose- and time-dependent manner. The 5-HT-induced GDNF release was mediated through the MEK/mitogen-activated protein kinase (MAPK) pathway and, at least, 5-HT(2A) receptors. The action of 5-HT on GDNF release may provide important insights into the mechanism of antidepressants.     

Intrathecal gene delivery of glial cell line-derived neurotrophic factor ameliorated paraplegia in rats after spinal ischemia

Paraplegia is a catastrophic complication of thoracic aortic surgery. At present, there is no effective mean to prevent the ischemia-induced spinal cord trauma. Gene delivery of neurotrophic factors may hold promises for prevention of spinal injury. In the present study, we evaluated the effect of glial cell line-derived neurotrophic factor (GDNF) gene delivery on prevention of the pathological changes due to spinal ischemia. Recombinant adenovirus vectors encoding GDNF (Ad-GDNF) and green fluorescent protein (Ad-GFP) were used for gene transfer studies. Treatment with cobalt chloride induced dose-dependent bcl-2 and synaptophysin downregulation in spinal neuronal cells, which could be effectively reversed by GDNF gene transfer. Intrathecal injection of Ad-GDNF led to maximal GDNF expression in spinal cord within 2-7 days. Thus, after intrathecal administration of adenovirus vectors for 3 days, Sprague-Dawley rats received transient aortic occlusion to induce spinal ischemia and were monitored for behavior deficits. The Ad-GDNF-treated rats showed significantly lower paraplegia rate (40%) than that of Ad-GFP- or saline-treated groups (75-85%; P<0.01). In addition, the Ad-GDNF-treated rats exhibited significantly improved locomotor function comparing with rats of control groups (P<0.001). Histological analysis revealed that GDNF gene delivery profoundly attenuated the infiltration of leukocytes in spinal cord after ischemic insults. Furthermore, GDNF gene delivery prominently attenuated the ischemia-induced neuronal loss in dorsal horn lamina VI-VIII and reduction in synaptophysin expression in spinal cords. In conclusion, GDNF gene transfer confers protection to the neuronal cells and synapses networks, thereby alleviated the paraplegia due to spinal ischemia.     

Brain-derived growth factor and glial cell line-derived growth factor use distinct intracellular signaling pathways to protect PD cybrids from H2O2-induced neuronal death

The cause of idiopathic PD is obscure, and most cases are sporadic. Oxidative stress and deficiency of various neurotrophic factors (NTFs) could be factors triggering neurodegeneration in the substantia nigra (SN). Cytoplasmic hybrid cells (cybrids) made from mitochondrial DNA of idiopathic PD subjects have reduced glutathione (GSH) levels and increased vulnerability to H2O2. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) rescue PD cybrids from H2O2-induced cell death. GDNF mediated effects require Src kinase and phosphatidylinositol 3-kinase (PI3K)/Akt activation. Inhibiting either PI3K/Akt or ERK pathways blocks the effects of BDNF. Inhibiting p38MAPK and c-Jun N-terminal kinase (JNK) pathways enhances the neuroprotective effects of both NTFs. These results demonstrate that expression of PD mitochondrial genes in cybrids increases vulnerability to oxidative stress that is ameliorated by both BDNF and GDNF, which utilize distinct signaling cascades to increase intracellular GSH and enhance survival-promoting cell signaling.     

Involvement of protein kinase C in glutamate release from cultured microglia

Glutamate release from microglial cells may cause neuronal damage. To elucidate the mechanism of glutamate release, we examined the possible regulation by nitric oxide and protein kinase C. Cultured microglia prepared from the whole brains of newborn rats released glutamate by the stimulation with lipopolysaccharide (LPS) dose dependently. The time course study revealed that glutamate release showed a long lag time about 6 h after LPS stimulation, whereas about 3 h lag time was observed in LPS-induced NO production. An inhibitor for NO synthase, N(G)-nitro-L-arginine, could effectively inhibit the glutamate release. Glutamate release induced by LPS was enhanced by 1 nM phorbol myristate acetate (PMA). Furthermore, high concentrations of PMA (>10 nM) induced glutamate release even without LPS stimulation. Glutamate release stimulated either by 100 ng/ml LPS or 100 nM PMA was inhibited by staurosporine, and also by alpha-aminoadipate. These results provide insight into the pathways regulating microglial pathological activation by protein kinase C and may be a base for the protection against microglia-evoked neurotoxicity.