CADDMR: a Cell-based Assay for Drug Discovery for Mental Retardation Disorders

描述（由申请人提供）：精神发育迟滞（MR）是一种常见的发展性脑部疾病，目前没有有效的治疗方法。这一建议是基于一种假设，即对形态学或可塑性的神经元缺陷进行药物纠正将增强MR患者认知技能的习得。许多不同基因的突变可导致MR，许多MR基因是相互关联的遗传途径的成员，这些遗传途径在动物物种中非常保守。这种保护提供了一个机会，利用遗传模型系统，探索新的治疗策略之前，临床试验。我们开发了一种原代细胞培养试验，用于鉴定具有核磁共振基因突变的大脑神经元的异常形态或可塑性，以及鉴定使缺陷正常化的化合物。这些神经元来自果蝇（Drosophila melanogaster）的蘑菇体，这是昆虫大脑中与学习和记忆有关的最具特征的区域，在类固醇激素依赖的发育可塑性高峰期收获。当野生型神经元在体外培养时，它们精心制作了具有高度定型形态的乔木。该分析揭示了正常的两性二态性，以及由于单基因突变的表型。在某些情况下，体外培养系统是高度敏感的，可以揭示体内表现过于微妙而无法通过常规显微镜检测到的缺陷。为了提高筛选速度，我们正在开发NeuronMorphometrics，这是一组用于ImageJ的Java插件，用于执行半自动数据分析。大约75%的人类MR基因在果蝇中有对应的序列，它们在大脑中有相似的功能。我们将筛选果蝇MR基因的一个子集，我们认为这些基因是揭示体外神经元形态和可塑性缺陷的主要候选者。在平行研究中，我们将重点关注“丝状体”表型，这是一种由于细胞骨架蛋白遗传缺失而导致的体外神经突生长的异常模式，并筛选使这种表型正常化的化合物。总之，表型和纠正性化学试剂的筛选应该为使用该分析作为MR疾病药物发现和开发的垫脚石奠定基础。公共卫生相关性：我们的长期目标是用药物治疗智力迟钝障碍，修复潜在的大脑发育异常。拟议中的实验是朝着这一目标迈出的重要一步。

DESCRIPTION (provided by applicant): Mental retardation (MR), for which few effective treatments are available, is a common manifestation of developmental brain disorders. This proposal is based on the hypothesis that pharmacological correction of neuronal defects of morphology or plasticity will enhance the acquisition of cognitive skills in MR patients. Mutations of many different genes can cause MR, and many MR genes are members of interconnected genetic pathways that are very highly conserved among animal species. Such conservation provides an opportunity to use genetic model systems to explore novel therapeutic strategies prior to clinical testing. We have developed a primary cell culture assay for identification of abnormal morphology or plasticity of brain neurons with MR-gene mutations, as well as for identification of compounds that normalize the defects. The neurons are from fruit fly (Drosophila melanogaster) mushroom bodies, the best-characterized insect brain region involved in learning and memory, harvested during a stage of peak steroid-hormone-dependent developmental plasticity. When wild-type neurons are cultured in vitro they elaborate an arbor with a highly stereotyped morphology. The assay has revealed normal sexual dimorphism, as well as phenotypes due to single-gene mutations. In some cases, the in vitro culture system is highly sensitive, unmasking defects whose in vivo manifestations are too subtle to be detected by conventional microscopy. To enhance the speed of screening, we are developing NeuronMorphometrics, a set of Java plug-ins for ImageJ that perform semi-automated data analysis. About 75% of human MR genes have a counterpart in Drosophila that is similar enough by sequence to plausibly perform similar functions in the brain. We will screen a subset of Drosophila MR genes which we believe are prime candidates for revealing neuronal morphology and plasticity defects in vitro. In parallel studies, we will focus on the "filagree" phenotype, an abnormal mode of neurite outgrowth in vitro due to genetic loss of a cytoskeletal protein, and screen for compounds that normalize this phenotype. Together, the screens for phenotypes and for corrective chemical agents should set the stage for use of this assay as a stepping stone for drug discovery and development for MR disorders. Public health relevance: Our long-term goal is to treat mental retardation disorders with drugs that fix the underlying abnormalities of brain development. The proposed experiments are an important step toward that goal.