GDNF-RELATED FACTORS AND RECEPTORS

GDNF 相关因子和受体

• 批准号: 6243414
• 负责人: LARS OLSON
• 金额: $ 12.28万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6243414
• 关键词: Parkinson's disease; basal ganglia; cell senescence; chick embryo; corpus striatum; disease /disorder model; dopamine; dopamine agonists; gene targeting; glia; growth factor receptors; immunocytochemistry; in situ hybridization; laboratory mouse; laboratory rabbit; nervous system transplantation; neurogenesis; neurotransmitter transport; neurotrophic factors; substantia nigra; tissue /cell culture; transfection; transforming growth factors

The objective of this project is an understanding of the neurotrophic support responsible for the development and maintenance of the nigrostriatal dopamine system. A central working hypothesis of Project 4 states that glial cell line-derived neurotrophic factor (GDNF) is the endogenous dopaminotrophic factor. This hypothesis leads to four postulates; 1) Exogenous GDNF must exert dopaminotrophic activity in vivo: This postulate is tested by a series of intraocular brain tissue grafting experiments. We will test the effect of neuron age as well as the effect of GDNF on target specificity and target=-elicited fiber growth in the intraocular model. 2a) GDNF must be expressed in the appropriate dopamine target areas: This postulate is tested by mapping the expression of GDNF mRNA by in situ hybridization and the presence of GDNF protein by immunohistochemistry. Treatment with neuroexcitatory drugs will test if GDNF expression in striatum is regulated. 2b) GDNF must be taken up by dopamine nerve terminals and transported retrogradely to their cell bodies of origin; The postulate is tested by transport studies using radiolabeled and unlabelled GDNF and autoradiographic and immunohistochemical detection methods, respectively. 3) Absence of GDNF must lead to a disturbed development and/or maintenance of the nigrostriatal dopamine system: This postulate is tested by a set of gene targeting experiments. We will generate "classical" knockouts of the GDNF gene. Several ways to handle the possibility that a "classical" GDNF knockout leads to early lethality, such as grafting mutated embryonic tissue to healthy animals, cross- breeding with animals overexpressing GDNF under a muscle-specific promoter (to rescue motor neurons) or studying heterozygous animals, which might have a milder phenotype, are also proposed. Using the Cre-loxP system, promoter-driven specific knockouts of the GDNF gene will also be generated. To cause a null-mutation of the GDNF gene in only neurons, only astrocytes or only striatum, respectively, we will use the nestin, thy-1 or neurofilament promoter (neurons), the GFAP promoter (astrocytes) or specific RARbeta or RXR gamma transcription factor promotors (striatum). 4) GDNF should have unique dopaminotrophic properties: This postulate is tested by studying the cellular distribution of another cloned TGFbeta superfamily member, OP-1. Additionally, the cloned OP01 receptor BMPR-type II will be mapped. Taken together, these experiments will provide new knowledge about the normal trophic support of the nigrostriatal dopamine system.

这个项目的目的是了解神经营养 负责开发和维护 黑质纹状体多巴胺系统 项目4的核心工作假设 指出胶质细胞源性神经营养因子（GDNF）是 内源性多巴胺营养因子 这一假设导致四个 假设：1）外源性GDNF必须发挥多巴胺营养活性， 体内：这一假设通过一系列眼内脑组织进行了测试。 嫁接实验 我们将测试神经元年龄的影响以及 GDNF对靶特异性和靶诱导的纤维生长的影响 眼内模型。 2a）GDNF必须以适当的 多巴胺靶区：这一假设是通过映射表达测试 GDNF mRNA的原位杂交和GDNF蛋白的存在 免疫组化 神经兴奋性药物治疗将测试是否 GDNF在纹状体中的表达受到调节。 2b）GDNF必须被以下物质摄取： 多巴胺神经末梢和运输逆行到他们的细胞体 通过使用放射性标记的 未标记GDNF及放射自显影和免疫组化检测 方法中选择一个. 3)GDNF的缺失一定会导致 黑质纹状体多巴胺系统的发展和/或维持：这 通过一组基因打靶实验验证了这一假设。 我们将 产生GDNF基因的“经典”敲除。 几种处理方式 “经典”GDNF敲除导致早期致死的可能性， 例如将突变的胚胎组织移植到健康的动物身上， 在肌肉特异性启动子下过表达GDNF的动物繁殖 (to拯救运动神经元）或研究杂合子动物，这可能 有一个温和的表型，也提出了。 使用Cre-loxP系统， 还将产生GDNF基因的启动子驱动的特异性敲除。 只在神经元和星形胶质细胞中引起GDNF基因的无效突变 或仅纹状体，分别，我们将使用巢蛋白，thy-1或 神经丝启动子（神经元）、GFAP启动子（星形胶质细胞）或 特异性RAR β或RXR γ转录因子启动子（纹状体）。 4)GDNF应该具有独特的多巴胺营养特性：这一假设是 通过研究另一种克隆的TGF β的细胞分布进行测试， 超家族成员OP-1 此外，克隆的OP 01受体BMPR型 二是要做地图。 综合起来，这些实验将提供新的 关于黑质纹状体多巴胺的正常营养支持的知识 系统