Developing a zebrafish model of Slc6a1/GAT1 hypofunction and an in vitro assay to identify novel treatments

项目总结/摘要 癫痫是一种高度流行的疾病，影响着世界上1%的人口，但我们对癫痫的理解是， 癫痫的分子机制仍然不完整。GABA再摄取功能缺陷 由GABA转运蛋白1（GAT 1）介导，与失神癫痫和杂合子癫痫有关。 编码GAT 1的基因SLC 6A 1突变导致肌阵挛性定向性癫痫，一种全身性癫痫发作 婴儿期发病的疾病。 关于GAT 1功能减退的病理生理学仍然存在一些问题，包括是否 疾病可以在斑马鱼中建模，因为它可以在啮齿动物中建模，以及这种疾病是否与原发性 GAT 1功能减退的组成性与继发性发育后果。从治疗学上讲， 问题是是否有可能确定GABA再摄取缺陷的疾病特异性治疗方法。 这些问题代表了知识上的重大差距，其答案可以告知时间和性质， 治疗MAE和其他涉及GAT 1功能减退的全身性癫痫综合征患者。 的 目前的提议将通过建立一种新的GAT 1功能减退的斑马鱼模型来缩小这些差距， 解决与疾病的发病机制有关的问题，并通过测试新的体外 GAT 1功能的荧光介导的基于细胞的测定可用于鉴定阳性调节剂， 治疗价值。拟议的研究将提供必要的见解的机制， 与slc 6a 1/GAT 1功能低下相关的癫痫综合征，并为未来的药物筛选建立平台， 体内测试以逆转病症的病理生理学。 正在考虑的提案将一个具有转化影响的创新研究项目结合起来， 在科学和职业发展方面的优秀导师，以及波士顿广泛的机构资源 儿童医院，马萨诸塞州总医院和哈佛医学院，这应该有助于 在奖励期结束时转变为独立的医生科学家。总而言之，该提案规定， 一个强大的独立研究计划的框架，平衡机械和翻译研究 癫痫的症状它与申请人的研究和临床兴趣很好地结合在一起，其职业目标是 成为一名独立的医生科学家，专注于高通量生物学和药物筛选， 了解癫痫的机制并确定相应的治疗方法。

PROJECT SUMMARY/ABSTRACT Epilepsy is a highly prevalent disorder affecting 1% of the world’s population but our understanding of the molecular mechanisms that underlie epilepsy is still incomplete. Deficiency in the function of GABA reuptake mediated by GABA transporter type 1 (GAT1), has been implicated in absence epilepsy and heterozygous mutations in the gene SLC6A1 which encode GAT1 cause myoclonic astatic epilepsy, a generalized seizure disorder with onset in infancy. Several questions remain regarding the pathophysiology of GAT1 hypofunction, including whether the disease can be modeled in zebrafish as it can in rodents and whether the disorder is related to primary constitutive versus secondary developmental consequences of GAT1 hypofunction. Therapeutically, a major question is whether it is possible to identify disease-specific treatments for GABA reuptake deficiency. These questions represent major gaps in knowledge whose answers could inform the timing and nature of treatment for patients with MAE and other generalized epilepsy syndromes involving GAT1 hypofunction. ​The current proposal will close these gaps by establishing a novel zebrafish model of GAT1 hypofunction to address questions related to the pathogenesis of the disorder and by testing whether a novel in vitro fluorescence-mediated cell-based assay of GAT1 function can be used to identify positive modulators with therapeutic value.​The proposed research will provide essential insights into the mechanisms of a generalized epilepsy syndrome related to slc6a1/GAT1 hypofunction and establish platforms for future drug screening and in vivo testing to reverse the pathophysiology of the disorder. The proposal under consideration combines an innovative research project with translational implications, excellent mentorship in science and career development, and extensive institutional resources at Boston Children’s Hospital, Massachusetts General Hospital, and Harvard Medical School, which should facilitate the transition into an independent physician-scientist by the end of the award period. In sum, the proposal provides a framework for a robust independent research program balancing mechanistic and translational investigations of epilepsy. It is well-integrated with the research and clinical interests of the applicant, whose career goal is to become an independent physician scientist with a focus on high-throughput biology and drug screening to understand the mechanisms of epilepsy and to identify corresponding treatments.