胶质细胞源性神经营养因子的表达、功能和在药物依赖中的作用

1993年,Lin等[1]从大鼠胶质细胞株B49中提纯到一种可促进胚胎中脑多巴胺能神经元存活的神经营养因子,并命名为胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF).GDNF和后来发现的neurturin(NRTN)、persephin(PSPN)、artemin(ARTN)在结构和功能上有很大的相似性,共同构成一个家族,称为GDNF家族.     

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。     

简便快速获取鼠胶质细胞源性神经营养因子基因的RT-PCR方法

目的：建立一种简便快速地获取鼠胶质细胞源性神经营养因子（GDNF）基因的RT-PCR方法。方法：Gene Bank中调出鼠的GDNF基因全序列设计引物，提取鼠基因组总RNA做模板进行RT-PCR，经酶切和凝胶电泳鉴定产物。结果：扩增出650bp鼠的GDNF基因，经EcoR Ⅰ酶切可见预期的650bp片段，证实获得正确的产物。结论：成功建立一种简便快速地从鼠基因组总RNA获取鼠GDNF基因的RT-PCR。     

褪黑素对SD大鼠胚胎中脑神经干细胞增殖和分化的影响

目的：脑内神经发生受多种病理生理因素调节，既往的研究表明褪黑素（Melatonin，MT）具有广泛的神经营养和神经保护作用，本研究观察MT对SD大鼠胚胎中脑神经干细胞（NSCs）增殖和分化的影响。方法：分离培养孕13-14天SD大鼠胚胎的中脑NSCs，用MTS方法测定MT对NSCs增殖的影响；通过RT-PCR、Western blot和免疫荧光方法检测MT对NSCs分化方向的调节作用；RT-PCR和Western blot方法检测NSCs上MT受体及脑源神经营养因子（BDNF）和胶质细胞源性神经营养因子（GDNF）的表达。结果：MT（0．05、0．1和1nM）均可促进NSCs的增殖，与对照组相比具有显著性差异（P〈0．05或P〈O．01）；而且，MT明显促进中脑NSCs分化时多巴胺能神经元标志蛋白酪氨酸羟化酶（TH）表达（P〈O．05），抑制胶质纤维酸性蛋白（GFAP）的表达；此外，MT可使NSCs内BDNF和GDNF的表达水平显著提高（P〈0．01）；同时我们还发现MT受体的两个亚型MTl和MT2在中脑NSCs均有表达，提示可能参与了对NSCs的调节作用。结论：本研究发现，MT不仅可以促进中脑NSCs的增殖，还显著提高NSCs向多巴胺能神经元方向的分化，而抑制其向星形胶质细胞的分化，这可能与MT上调BDNF和GDNF的表达有关。以上结果可为MT临床治疗某些老年性神经疾患并改善部分临床症状的作用机理提供线索。     

非痴呆型血管性认知障碍患者血清BDNF、GDNF水平的测定及其临床意义

目的探讨血清脑源性神经营养因子(BDNF)、胶质源性神经营养因子(GDNF)水平与非痴呆型血管性认知障碍(VCIND)的关系。方法选取45例VCIND患者作为VCIND组,另选48例同期非认知障碍脑梗死(NDCI)患者作为NDCI组。采用酶联免疫吸附法(ELISA)对两组患者的血清BDNF、GDNF水平进行测定并分析。结果 VCIND组血清BDNF、GDNF水平分别为(3.23±1.35)ng/ml、(5.40±1.77)pg/ml,均低于NDCI组的(3.96±1.12)ng/ml、(9.47±1.98)pg/ml,差异有统计学意义(P<0.05)。结论高血压可能是VCNID的危险因素, BDNF、GDNF可能在脑梗死后发展为血管性认知障碍的病理生理过程中发挥作用。     

三七三醇皂苷对糖尿病大鼠学习记忆能力及星形胶质细胞活性的影响

目的 探讨三七三醇皂苷(PTS)对糖尿病大鼠学习记忆的改善作用,并从星形胶质细胞角度揭示其机制。方法 24只SD大鼠随机分为对照组、糖尿病组及PTS治疗组,每组8只。于SD大鼠腹腔注射链脲佐菌素诱导糖尿病模型,对照组为正常SD大鼠,PTS治疗组为糖尿病大鼠给予PTS治疗。检测大鼠血糖和体质量,3个月后,水迷宫实验观察各组大鼠的学习记忆功能,免疫组化染色检测脑海马区星形胶质细胞形态,Western blot法检测神经胶质纤维酸性蛋白(GFAP)及胶质细胞源性神经营养因子(GDNF)表达。结果 糖尿病组较对照组学习记忆能力减退,脑海马区星形胶质细胞胞体萎缩、突起变细,脑海马区GFAP及GDNF表达明显减少(均P<0.05)。PTS组较糖尿病组学习记忆能力改善,脑海马区星形胶质细胞形态改善、GFAP及GDNF表达明显恢复(均P<0.05)。结论 PTS能恢复糖尿病大鼠脑海马区星形胶质细胞活性,恢复GDNF表达,提高糖尿病大鼠学习记忆功能。     

GDNF与物质依赖行为的相互作用及机制

<正>物质依赖是一种慢性、复发性脑疾病,给个人健康及社会带来严重的危害[1-2]。从首次使用依赖物质发展到依赖,其中的机制非常复杂,涉及脑内多个神经递质、受体系统,如DA、GABA系统。DA能神经元的存活和功能、学习记忆、情绪及突触可塑性与神经胶质细胞源性神经营养因子(Glial cell lineDerived Neurotrophic Factor,GDNF)关系密切[3]。     

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。     

糖尿病大鼠海马和大脑皮层GDNF蛋白表达的变化

目的 研究糖尿病大鼠不同血糖水平下脑内胶质细胞源性神经营养因子(GDNF)表达的变化.方法 将30只Wistar大鼠随机均分为糖尿病血糖未控制(DMI)组、糖尿病血糖控制(DM2)组和对照(NC)组.12周后检测血糖与糖化血红蛋白(HbA1c)水平,免疫组织化学(SP)法检测GDNF蛋白表达,HE染色观察海马及大脑皮层神经元细胞计数和形态学变化.结果 海马CA区和大脑皮层的GDNF蛋白表达均按NC组＞DM2组＞DM1组依次减弱(P＜0.05),且均与HbA1c水平之间呈负相关(r分别为-0.961和-0.963,P＜0.05).与NC组相比,DM1组和DM2组海马CA区神经元无明显减少,而海马及大脑皮层神经元呈明显退形性形态学改变.结论 长期慢性高血糖可下调海马和大脑皮层GDNF蛋白表达,从而可能引起中枢神经病变.     

bFGF，GDNF，PDGF，IGF-I在大鼠脊髓中的定位表达

目的　碱性成纤维细胞生长因子 (bFGF)、胶质细胞源性神经营养因子 (GDNF)、血小板源性生长因子 (PDGF)和胰岛素样生长因子 -I (IGF -I)对神经细胞起特殊的营养作用 ,但它们在脊髓中的定位分布和生物学作用还不十分清楚。本研究旨在探索这一问题 ,方法　取正常成年SD大鼠脊髓分别用抗bFGF ,GDNF ,PDGF ,IGF -I抗体行免疫组化染色 ,观察上述 4种神经营养因子免疫阳性反应物在大鼠脊髓中的分布。结果　bFGF ,GDNF ,PDGF ,IGF -I的免疫阳性反应物主要见于脊髓灰质内各板层神经元 ,腹角和背角深部明显 ,背角浅层较少见。但II板层还可见bFGF阳性细胞和GDNF阳性膨体 ,中央管可见PDGF、bFGF阳性的室管膜细胞。结论　bFGF ,GDNF ,PDGF ,IGF -I在维持脊髓神经元的生理功能中可能起重要作用     

Effect of GDNF-releasing biodegradable microspheres on the function and the survival of intrastriatal fetal ventral mesencephalic cell grafts.

The transplantation of fetal ventral mesencephalic (FVM) cell suspensions into the brain striatal system is an alternative approach for the treatment of Parkinson's disease (PD). However, one objection to this procedure is the relatively poor survival of implanted cells. Attempts have been made to improve the survival of grafted dopaminergic neurons using glial cell line-derived neurotrophic factor (GDNF). Nevertheless, the clinical application of GDNF is limited, due to the difficulties in administering a protein to the brain tissue and due to the ubiquity of its receptor, thus leading to neurological side effects. A strategy to deliver GDNF in the brain based on the intracerebral implantation of biodegradable poly(D,L-lactic acid-co-glycolic acid) sustained release microspheres has been developed. Such microparticles can be easily implanted by sterotaxy in precise and functional areas of the brain without causing damage to the surrounding tissue. Moreover, the release profile of the GDNF-loaded microspheres showed a sustained release over 56 days of biologically active GDNF at clinically relevant doses. The present study shows that the implantation of GDNF-loaded microspheres at a distance to the site of FVM cells in the 6-hydroxydopamine-lesioned rat model of PD improves dopaminergic graft survival and function. Furthermore, the unloaded and the GDNF-loaded microspheres, when they are mixed with FVM cells, may provide a mechanical support and a 3D environment inducing differentiation and increased function of dopaminergic neurons. Taken together, these results show that GDNF microspheres represent an efficient delivery system for cell transplantation studies.     

Pharmacokinetics and bioactivity of glial cell line-derived factor (GDNF) and neurturin (NTN) infused into the rat brain

Convection-enhanced delivery (CED) of GDNF and NTN was employed to determine the tissue clearance of these factors from the rat striatum and the response of the dopaminergic system to a single infusion. Two doses of GDNF (15 and 3 μg) and NTN (10 μg and 2 μg) were infused into the rat striatum. Animals were euthanized 3, 7, 14, 21, and 28 days post-infusion. Brains were processed for ELISA, HPLC, and immunohistochemistry (IHC). Both doses of the infused GDNF resulted in a sharp increase in striatal GDNF levels followed by a rapid decrease between day 3 and 7. Interestingly, IHC revealed GDNF in the septum and the base of the brain 14 days after GDNF administration. Dopamine (DA) turnover was significantly increased in a dose-dependent manner for more than 7 days after a single GDNF infusion. NTN persisted in the brain for at least two weeks longer than GDNF. It also had more persistent effects on DA turnover, probably due to its precipitation in the brain at neutral pH after infusion. Our data suggest that daily or continuous dosing may not be necessary for delivering growth factors into the CNS.     

Targeting the glial-derived neurotrophic factor and related molecules for controlling normal and pathologic pain

Introduction: Glial-derived neurotrophic factor (GDNF) and its family of ligands (GFLs) have several functions in the nervous system. As a survival factor for dopaminergic neurons, GDNF was used in clinical trials for Parkinson’s disease. GFLs and their receptors are also potential targets for new pain-controlling drugs. Although molecules with analgesic activities in rodents mostly failed to be effective in translational studies, this potential should not be underestimated.

Areas covered: The circuitry, molecular, and cellular mechanisms by which GFLs control nociception and their intervention in inflammatory and neuropathic pain are considered first. The problems related to effective GDNF delivery to the brain and the possibility to target the GFL receptor complex rather than its ligands are then discussed, also considering the use of non-peptidyl agonists.

Expert opinion: In nociceptive pathways, an ideal drug should either: i) target the release of endogenous GFLs from large dense-cored vesicles (LGVs) by acting, for example, onto the phosphatidylinositol-3-phosphate [PtdIns₍₃₎P] pool, which is sensitive to Ca²⁺ modulation, or ii) target the GFL receptor complex. Besides XIB403, a tiol molecule that enhances GFRα family receptor signaling, existing drugs such as retinoic acid and amitriptyline should be considered for effective targeting of GDNF, at least in neuropathic pain. The approach of pain modeling in experimental animals is discussed.     

The RET receptor tyrosine kinase: activation, signalling and significance in neural development and disease

The RET receptor tyrosine kinase was first identified in a screen for human oncogenes and has subsequently been linked to several human syndromes: Hirschprung's disease, multiple endocrine neoplasia types 2A and 2B and familial thyroid carcinoma. Interestingly, all of the tissues affected by mutations in RET are derived from the neural crest during development. RET transduces a signal following activation by ligands of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophins which currently comprises GDNF, neuturin (NTN), artemin (ART) and persephin (PSP). To activate RET they form a tripartite complex with RET and a member of a family of four extracellular, GPI-linked alpha receptors (GFR alpha 1-4). Specificity is achieved by each GFR alpha binding only one member of the GDNF family with high affinity. Current evidence indicates that signal transduction by RET activates several second messenger systems including the PLC gamma, Ras, JNK and inositol phosphate pathways. Targeted mutagenesis in transgenic mice has shown that Ret, GFR alpha 1 and GDNF are required for multiple developmental events including development of the enteric nervous system (ENS) affected in Hirschsprung's disease. We describe experiments in chick neural crest cells which provide evidence for the normal function of RET and the basis of the defect in Hirschsprung's disease.     

GDNF family receptor complexes are emerging drug targets

Glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), which consist of GDNF, neurturin, artemin and persephin, regulate the development and maintenance of the nervous system. GDNF protects and repairs dopamine-containing neurons, which degenerate in Parkinson's disease, and motoneurons, which die in amyotrophic lateral sclerosis. GDNF and neurturin have shown promise in clinical trials of Parkinson's disease, and artemin is currently undergoing clinical trials for chronic pain treatment. However, the delivery of GFLs into the brain through invasive approaches such as neurosurgery, viral vectors or by the use of encapsulated cells is associated with multiple obstacles. The development of small molecules that specifically activate GFL receptors and that can be applied systemically would overcome most of these problems. The unique nature of the GFL receptors, recent progress in elucidation of the 3D structures of GFLs and GFL-receptor complexes and the use of high-throughput screening have resulted in the development of the first small molecules that mimic the effects of the different GFLs.     

Glial-derived neurotrophic factor regulates enteric mast cells and ameliorates dextran sulfate sodium-induced experimental colitis

Background & AimsAlthough interactions between enteric glial cells (EGCs) and enteric mast cells have been demonstrated to play an important role in the pathogenesis of inflammatory bowel disease (IBD), the exact mechanisms by which EGCs regulate enteric mast cells are still unknown. The aims of this study were to investigate whether glial-derived neurotrophic factor (GDNF), which has been confirmed to be produced mostly by EGCs, might regulate enteric mast cells and ameliorate dextran sulfate sodium (DSS)-induced experimental colitis.MethodsRecombinant adenoviral vectors encoding GDNF (Ad-GDNF) were administered intracolonically in experimental colitis induced by DSS. The disease activity index and histological score were measured. The expression of tumour necrosis factor-α (TNF-α), interleukin-6 and myeloperoxidase (MPO) activity were measured by ELISA assay. The expression of trypsin and β-hexosaminidase were evaluated. GDNF specific receptor (GFR-α1/RET) was detected. The calcium reflux was tested by microplate reader. The expression p-JNK was analyzed by western blot assay.ResultsGDNF resulted in a significant inhibition of the activation of enteric mast cells by down-regulating JNK signal pathway, lessening intracellular calcium influx, and then reducing the degranulation as well as the expression of pro-inflammatory cytokines via combing with its receptor (GFR-α1/RET) in mast cells, and these inhibitory effects were abrogated by treatment with neutralizing antibody against GDNF. Moreover, the administration of GDNF led to an amelioration of experimental colitis.ConclusionsGDNF are able to regulate enteric mast cells and ameliorate experimental colitis. GDNF might be an important mediator of the cross-talk between EGCs and enteric mast cells, and GDNF might be a useful therapeutic drug for IBD.     

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.     

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.     

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.