脑内胶质细胞源性神经营养因子及其受体复合物研究进展

胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)在脑内广泛分布,通过其受体复合物介导激活细胞内信号转导通路,发挥维持神经元功能和损伤修复等作用。胶质细胞源性神经营养因子家族受体α(GD-NF family receptorα,GFRα)和RET是其受体复合物的主要成员。GDNF和其受体复合物可能参与多种脑部病变的病理生理过程,是潜在的治疗靶点之一。     

Purification of bioactive glycosylated recombinant glial cell line-derived neurotrophic factor

Glial cell line-derived neurotrophic factor (GDNF) neuroprotective effect on dopaminergic neurons has been described in vitro and in vivo, turning up as a promising drug for the treatment of Parkinson's disease. Unglycosylated bacteria-obtained GDNF has already been successfully delivered for a long period of time through an infusion pump directly to the putamen of Parkinsonian patients. Nevertheless, improved distribution and safety issues need to be solved and alternative strategies to long-term delivery seem necessary. The use of glycosylated GDNF could eliminate some safety concerns regarding the presence of antibodies against exogenous unglycosylated GDNF used for the treatment. Therefore, we have chosen a mammalian expression system as a source of glycosylated GDNF. In the present work, we describe the purification of recombinant rat GDNF from the culture media of baby hamster kidney (BHK) cells through several purification steps. Highly pure N-glycosylated recombinant GDNF has been obtained similar to the endogenous protein. Furthermore, the purified protein is biologically active when tested its ability to induce PC12 neurite outgrowth.     

Decreased expression of glial cell line-derived neurotrophic factor signaling in rat models of neuropathic pain

1. In an attempt to clarify whether glial cell line-derived neurotrophic factor (GDNF), a survival factor for subpopulations of primary afferent neurons, is involved in the states of neuropathic pain, we observed changes in the expressions of GDNF and its signal-transducing receptor Ret after nerve injury in two rat models of neuropathic pain. 2. In the rats treated with sciatic nerve ligation (chronic constrictive injury (CCI) model) or spinal nerve ligation at L5 (SNL model), the thresholds of paw withdrawal in response to mechanical or heat stimuli began to decrease on the injured side within the first week after the operation and the decreases in the thresholds persisted for more than 2 weeks. 3. In CCI-treated rats, the GDNF contents in L4 and L5 dorsal root ganglia (DRGs) on the injured side were markedly decreased at day 7 after the operation and stayed at low levels at day 14. In SNL-treated rats, comparable reductions of GDNF levels in L4 and L5 DRGs on the injured side were observed at 14 postoperative days. 4. Significant decreases of the percentages of DRG neurons expressing Ret were also observed at L4 DRGs in CCI-treated rats at 7 and 14 postoperative days and in SNL-treated rats at 14 days. 5. In CCI- or SNL-treated rats, continuous intrathecal administration of GDNF (12 microg day-1) using an osmotic pump suppressed the increased sensitivities to nociceptive stimuli to control levels. 6. The present results suggested that the dysfunction of GDNF signaling in the nociceptive afferent system may contribute to the development and/or maintenance of neuropathic pain states.     

白果内酯刺激大鼠星形胶质细胞GDNF和VEGF表达(英文)

目的:观察白果内酯对大鼠星形胶质细胞GDNF和VEGF表达的影响。方法:应用半定量PCR了解大鼠星形胶质细胞分别经不同浓度白果内酯(0,5,15,50,100μmol·L~(-1))作用12、24和48h后细胞内GDNF和VEGF mRNA的变化,同时应用免疫组化了解经50μmol·L~(-1)作用24h后细胞内GDNF和VEGF蛋白的变化。结果:大鼠星形胶质细胞经50μmol·L~(-1)白果内酯作用12h后,细胞内GDNF和VEGF mRNA水平开始显著增高,经50μmol·L~(-1)作用24h后细胞胞浆内可见明显的VEGF和GDNF蛋白表达。结论:白果内酯可能通过诱导星形胶质细胞GDNF和VEGF的表达对神经细胞起保护作用。     

胶质细胞源性神经营养因子在人脑胶质瘤的表达及意义

目的从基因表达的水平探讨胶质细胞源性神经营养因子(GDNF)与人脑胶质细胞瘤发生发展的关系。方法采用原位杂交的方法用特异性探针结合59例胶质瘤标本和20例正常脑组织中的GDNFmRNA,观察GDNFmRNA的表达情况。结果GDNFmRNA在正常脑组织和胶质瘤标本中均有表达,但胶质瘤中的表达明显高于正常脑组织,GDNFmRNA在不同级别胶质瘤中的表达也存在显著性差异,恶性程度越高,GDNFmRNA表达强度越强。结论GDNF可能作为一种重要的因子参与胶质瘤的恶性增殖过程,影响胶质瘤的发生、发展及其他生物学特性。     

Cabergoline stimulates synthesis and secretion of nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor by mouse astrocytes in primary culture

Neuroprotection is the primary concern in patients with newly diagnosed Parkinson's disease. The D2/weak D1 dopamine agonist cabergoline elicits neuroprotection by antioxidation and scavenging free radicals, and may protect neurons by up-regulating endogenous neurotrophic factors synthesis in the brain. In primary cultured mouse astrocytes, cabergoline 37 micromol/l immediately elevated concentrations of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor (GDNF) in culture medium, reaching 9.9-, 2.6- and 30-fold, respectively, of control levels at 16 h. Relative mRNA levels were 3.0-, 1.5- and 1.9-fold, respectively, of controls at 3 h. These effects may be mediated partly by the dopamine D2 receptor. Cabergoline may be a good candidate for an inducer of GDNF, which may have neuroprotective and neurorestorative properties in dopaminergic nigral neurons.     

Lithium treatment alters brain concentrations of nerve growth factor,brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor in a rat model of depression

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) are proteins involved in neuronal survival and plasticity of dopaminergic, cholinergic and serotonergic neurons in the central nervous system. Since decreased size and impaired function of some neuronal populations may be relevant in depression it has been hypothesized that these molecules may have a functional role in the pathophysiology as well as treatment of depression. Using an animal model of depression, the Flinders Sensitive Line (FSL) rats and their controls, the Flinders Resistant Line (FRL), we investigated the effects of chronic lithium treatment on brain NGF, BDNF and GDNF. Lithium was administered as food supplementation during 6 wk. NGF, BDNF and GDNF measurements were performed by enzyme-linked immunosorbent assay (ELISA). Lithium altered the brain concentrations of neurotrophic factors in the hippocampus, frontal cortex, occipital cortex and striatum. Moreover, the changes were different in the two rat strains. Our data support the notion that neurotrophic factors play a role in depression and in the mechanism of the action of lithium.     

Glia as targets for antidepressants: an involvement in glial cell line-derived neurotrophic factor]

Recently, clinical and animal studies have shown that neuronal and glial plasticity are important for the therapeutic action of antidepressants. Thus, it has been suggested that neurotrophic factors or growth factors, which are potent regulators for neuronal and glial plasticity, might be involved in the effect of antidepressants. Post-mortem studies provide evidence for glial reduction in different brain areas in mood disorders. Therefore, we focused on glial cell line-derived neurotrophic factor (GDNF) in mood disorders, because GDNF plays an important role in neurogenesis and high-ordered brain function, such as learning and memory. GDNF family ligands have shown promise of efficacy for neurodegenerative disorders such as Parkinson's disease, suggesting that GDNF family ligands exist in the closest position to clinical development for treatment of diseases of the central nervous system. We reported that total GDNF levels in whole blood in patients with mood disorders were significantly lower than those in healthy control subjects (Takebayashi et al, 2006), and antidepressants increased GDNF production through monoamine-independent activation of protein tyrosine kinase (PTK) and extracellular signal-regulated kinase (ERK) in glial cells (Hisaoka et al, 2007). Clarifying the monoamine-independent novel target of antidepressants in glia might contribute to the development of more efficient therapeutics for depression.     

Neuroprotective mechanism of glial cell line-derived neurotrophic factor on dopamine neurons: role of antioxidation.

Recombinant human GDNF was infused into the rat striatum either acutely or subchronically. Its effects and its interactions with MPP+ on antioxidant enzyme activities were examined. Results indicated that acute GDNF infusion significantly increased glutathione peroxidase, superoxide dismutase and catalase activities. Subchronic GDNF treatment decreased the DA level and enhanced DA turnover. Pre-treatment with GDNF markedly protected DA neurons against MPP+-induced toxicity. These results suggest that GDNF protects DA neurons through its activation of the antioxidant enzyme systems.     

胶质细胞源性神经营养因子与神经病理性疼痛

胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)是神经营养因子家族成员之一,GD-NF对中枢和周围神经系统多种神经元的生长、发育、分化、维持和损伤修复起重要作用。另外,GDNF还参与中枢和外周水平神经病理性疼痛的形成和发展。该文主要就GDNF和神经病理性疼痛的研究作一简要综述。     

临床孤立综合征患者血清和脑脊液中脑源性与胶质细胞源性神经营养因子水平及其神经保护作用研究

目的 探讨多发性硬化（MS）的早期表现--临床孤立综合征（CIS）患者血清及脑脊液中脑源性神经营养因子（BDNF）、胶质细胞源性神经营养因子（GDNF）水平及其神经保护作用.方法 对27例CIS患者及21例对照者进行研究,CIS患者发作期进行扩展残疾状态量表（EDSS）评分、寡克隆带测定及MRI检查,液相芯片分析技术检测血清及脑脊液BDNF、GDNF浓度.结果 CIS患者发作期血清及脑脊液BDNF[分别为（5.981±0.995）和（0.178±0.008）μg/L]、GDNF浓度[分别为（0.039±0.007）和（0.082±0.011）μg/L]与对照组[血清：（4.374±0.501）、（0.042±0.007）μg/L;脑脊液：（0.152±0.011）、（0.065±0.013）μg/L]比较差异均无统计学意义（均P＞0.05）;脑脊液BDNF与GDNF浓度呈正相关（r=0.777,P=0.000）,血清BDNF与GDNF浓度无相关性（r=-0.375,P=0.126）.血清及脑脊液BDNF、GDNF浓度与EDSS评分、血脑屏障指数、Delpech指数、Tourtellotte合成率及脑萎缩无明显相关性（P＞0.05）.结论 CIS患者体内BDNF与GDNF水平相关,二者可能具有协同的神经保护作用.BDNF及GDNF与CIS患者血脑屏障破坏及中枢神经系统内IgG合成无关,与神经功能残疾及脑萎缩的关系仍需研究.     

Glial cell line-derived neurotrophic factor induces cell migration and matrix metalloproteinase-13 expression in glioma cells

Malignant gliomas are the most common primary brain tumors in adults and the second most common tumor in children. Gliomas are associated high morbidity and mortality because these tumors are highly invasive into surrounding brain tissue, making complete surgical resection impossible. Glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent neurotrophic factor in a variety of neuronal cell populations. However, the molecular mechanisms and pathologic roles underlying GDNF-induced glioma migration remain unclear. In this study, we found that application of recombinant human GDNF enhances the migration of U87 and U251 cells but not C6 cells. In addition, we found that the expression of matrix metalloproteinase-13 (MMP-13) mRNA, protein and secretion increase in response to GDNF stimulation. The GDNF-induced increase in cell migration was antagonized by MMP-13 neutralizing antibody or silencing MMP-13. We then examined the involvement of mitogen-activated protein kinases (MAPKs) in glioma cell migration induced by GDNF. GDNF-induced MMP-13 expression and glioma migration were attenuated by MEK/extracellular signal-regulating kinase (ERK) and c-Jun N-terminal protein kinase (JNK) inhibitors, as well as ERK and JNK dominant-negative mutants. Treatment with GDNF-induced MEK/ERK and JNK/c-Jun activation and increased AP-1 DNA binding activity in a time-dependent manner. Treatment with AP-1 inhibitors (tanshinone IIA and curcumin) also reduced GDNF-induced glioma cell migration. In migration-prone sublines, cells with greater migration ability had higher GDNF expression. These results indicate that GDNF enhances migration of glioma cells through the increase of MMP-13 production and is mainly regulated by the MEK/ERK and JNK, c-Jun and AP-1 pathways.     

A simple and efficient method for the production of human glycosylated glial cell line-derived neurotrophic factor using a Semliki Forest virus expression system

Human glial cell line-derived neurotrophic factor (hGDNF) is a very promising protein for the treatment of Parkinson's disease and other neurodegenerative disorders. The present work describes a quick and simple method to obtain a high amount of purified hGDNF using a mammalian cell-derived system. The method is based on the high expression level provided by a Semliki Forest virus vector and its ability to induce a strong shut-off of host-cell protein synthesis in mammalian cells. As a result, hGDNF is the only protein present in the supernatant and can be efficiently purified by a single chromatographic step. Using this system it was possible to eliminate other secreted proteins from the culture medium, like insulin-like growth factor-5, which are hard to remove using other hGDNF production methods. Purified hGDNF presents a complex glycosylation pattern typical of mammalian expression systems and is biologically active. This protocol could be extended to other secreted proteins and could be easily scaled up for industrial purposes.     

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 (G?6976) 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.     

Decreased levels of whole blood glial cell line-derived neurotrophic factor (GDNF) in remitted patients with mood disorders

Recent post-mortem and imaging studies provide evidence for a glial reduction in different brain areas in mood disorders. This study was aimed to test whether glial cell line-derived neurotrophic factor (GDNF), a member of transforming growth factor (TGF)-beta superfamily, in blood levels was associated with mood disorders. We measured GDNF and TGF-beta levels in whole blood in remitted patients with mood disorders [n=56; major depressive disorders (MDD) 39, bipolar disorders (BD) 17] and control subjects (n=56). GDNF and TGF-beta were assayed with the sandwich ELISA method. Total GDNF levels were significantly lower in MDD and in BD than in control subjects (MDD, p=0.0003; BD, p=0.018), while no significant difference in total TGF-beta1 or total TGF-beta2 levels was found in these groups. Our study suggests that lower GDNF levels might be involved in the pathophysiology of mood disorders, although this preliminary study has several limitations.     

Preservation of biological activity of glial cell line-derived neurotrophic factor (GDNF) after microencapsulation and sterilization by gamma irradiation

A main issue in controlled delivery of biotechnological products from injectable biodegradable microspheres is to preserve their integrity and functional activity after the microencapsulation process and final sterilization. The present experimental work tested different technological approaches to maintain the biological activity of an encapsulated biotechnological product within PLGA [poly (lactic-co-glycolic acid)] microspheres (MS) after their sterilization by gamma irradiation. GDNF (glial cell line-derived neurotrophic factor), useful in the treatment of several neurodegenerative diseases, was chosen as a labile model protein. In the particular case of optic nerve degeneration, GDNF has been demonstrated to improve the damaged retinal ganglion cells (RGC) survival. GDNF was encapsulated in its molecular state by the water-in-oil-in-water (W/O/W) technique or as solid according to the solid-in-oil-in-water (S/O/W) method. Based on the S/O/W technique, GDNF was included in the PLGA microspheres alone (S/O/W 1) or in combination with an antioxidant (vitamin E, Vit E) (S/O/W 2). Microspheres were sterilized by gamma-irradiation (dose of 25kGy) at room and low (-78°C) temperatures. Functional activity of GDNF released from the different microspheres was evaluated both before and after sterilization in their potential target cells (retinal cells). Although none of the systems proposed achieved with the goal of totally retain the structural stability of the GDNF-dimer, the protein released from the S/O/W 2 microspheres was clearly the most biologically active, showing significantly less retinal cell death than that released from either W/O/W or S/O/W 1 particles, even in low amounts of the neurotrophic factor. According to the results presented in this work, the biological activity of biotechnological products after microencapsulation and sterilization can be further preserved by the inclusion of the active molecule in its solid state in combination with antioxidants and using low temperature (-78°C) during gamma irradiation exposure.     

Role of PI3-K/Akt pathway and its effect on glial cell line-derived neurotrophic factor in midbrain dopamine cells

AIM: To explore the intracellular mechanisms underlying the survival/differentiation effect of the glial cell line-derived neurotrophic factor (GDNF) on dopamine (DA) cells. METHODS: Midbrain slice culture and primary cell culture were established, and the cultures were divided into 3 groups: control group, GDNF group, and the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) pathway-inhibited group. Then the expression of tyrosine hydroxylase (TH) was detected by immunostaining as well as Western blotting. RESULTS: GDNF treatment induced an increase in the number of TH-immunoreactive (ir) cells and the neurite number of TH-ir cells, as well as in the level of TH expression in cultures (Number of TH-ir cells in the slice culture: control group, 8.76+/-0.75; GDNF group, 18.63+/-0.95. Number of TH-ir cells and neurite number of TH-ir cells in cell culture: control group, 3.65+/-0.88 and 2.49+/-0.42; GDNF group, 6.01+/-0.43 and 4.89+/-0.46). Meanwhile, the stimulation of cultured cells with GDNF increased the phosphorylation of Akt, which is a downstream effector of PI3-K/Akt. The effects of GDNF were specifically blocked by the inhibitor of the PI3-K/Akt pathway, wortmannin (Number of TH-ir cells in slice culture: PI3-K/Akt pathway-inhibited group, 6.98+/-0.58. Number of TH-ir cells and neurite number of TH-ir cells in cell culture: PI3-K/Akt pathway-inhibited group, 3.79+/-0.62 and 2.50+/-0.25, respectively). CONCLUSION: The PI3-K/Akt pathway mediates the survival/differentiation effect of GDNF on DA cells.     

Protective effect of the glial cell line-derived neurotrophic factor (GDNF) on human mesencephalic neuron-derived cells against neurotoxicity induced by paraquat

Paraquat is a cationic herbicide that causes acute cell injury by undergoing redox cycling. Oxidative stress is thought to be the crucial mechanism invoked by this redox-cycling compound. The cytotoxicity of paraquat was examined in an immortalized human mesencephalic neuron-derived cell line. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction activity was examined as cytotoxicity indicator. Cells were seeded with densities at inoculation of 5 × 10(4)cells/ml and 10 × 10(4)cells/ml, and paraquat was added 24h later to give final concentrations from 10 to 500 μM. At 24 and 48 h of treatment, mitochondrial activity was determined with the MTT assay. To further understand the effect of paraquat exposure on human mesencephalic neuron-derived cells, the cells were differentiated and similar experiments were carried out. Supplementation of culture medium with dibutyryl cyclic AMP and GDNF significantly increased the resistance of the cultures to the paraquat-mediated cytotoxicity. These results confirm that GDNF confers protection against paraquat-mediated cytotoxicity and show that immortalized human mesencephalic neuron-derived cells are an adequate in vitro system for evaluating the cytoprotective effects of GDNF on oxidative injury caused by xenobiotics.