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

描述(由申请人提供)：智力低下(MR)是发育性大脑疾病的常见表现，目前几乎没有有效的治疗方法。这一建议是基于这样一种假设，即对神经元形态或可塑性缺陷的药物纠正将增强MR患者认知技能的获得。许多不同基因的突变可以引起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。这种保守提供了在临床试验之前使用遗传模型系统来探索新的治疗策略的机会。我们已经建立了一种原代细胞培养方法，用于鉴定具有MR基因突变的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(果蝇)蘑菇体，这是参与学习和记忆的最具特征的昆虫大脑区域，在类固醇激素依赖的发育可塑性高峰期采集。当野生型神经元在体外培养时，他们精心制作了一种具有高度定型形态的乔木。化验结果显示正常的性别二型性，以及由于单基因突变而产生的表型。在某些情况下，体外培养系统是高度敏感的，可以暴露出体内表现太过微妙而无法用传统显微镜检测到的缺陷。为了提高筛选的速度，我们正在开发NeuronMorphometrics，这是一套用于ImageJ的Java插件，可以执行半自动的数据分析。大约75%的人类MR基因在果蝇中有一个对应的基因，该基因在序列上足够相似，似乎可以在大脑中执行类似的功能。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基因。在平行研究中，我们将专注于“丝状”表型，这是一种由于细胞骨架蛋白遗传缺失而导致的体外突起生长的异常模式，并筛选出使这一表型正常化的化合物。总而言之，表型和矫正化学试剂的筛查应该为使用这种检测方法作为治疗MR疾病的药物发现和开发的垫脚石奠定基础。与公共卫生相关：我们的长期目标是用治疗大脑发育潜在异常的药物来治疗精神发育迟滞障碍。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.