Pathophysiology and Animal Model of Restless Legs Syndrome (RLS): Btbd9 Null Mic

描述（由申请人提供）：不宁腿综合征（RLS）是一种慢性睡眠运动障碍，其特征是腿部的不愉快感觉和无法控制的移动它们以缓解的冲动。过去的病理生理学研究将RLS与中枢多巴胺能系统和铁代谢紊乱相关联。家族和双生子研究强烈支持遗传因素对RLS的发病机制的贡献。最近在发现与RLS相关的候选基因方面取得了巨大进展。过去两年发表的三项独立研究都指出了BTBD 9在RLS中的作用。BTBD 9蛋白的功能尚不清楚。目前的动物模型包括6-羟基多巴胺损伤的啮齿动物、铁缺乏小鼠和多巴胺受体3敲除小鼠。RLS候选基因的鉴定为建立RLS的基因型模型铺平了道路，该模型将在阐明RLS的病理生理学和开发治疗方法方面更加相关。我们研究的广泛、长期目标是使用转基因小鼠来确定：1）BTBD 9蛋白在体内的功能作用，2）BTBD 9蛋白功能的改变如何导致RLS。本申请的目的是表征我们已经制造的Btbd 9（人BTBD 9基因的小鼠同源物）敲除小鼠，这将使我们能够回答这些问题。我们假设BTBD 9突变导致脑铁代谢和中枢多巴胺能系统的改变，特别是纹状体D2受体介导的间接途径，或间脑脊髓多巴胺能束（A11），或两者兼而有之。这反过来又会导致不愉快的感觉增强，昼夜节律功能障碍和强烈的运动冲动。我们计划用以下具体目标来检验我们的假设：1.为了检验BTBD 9突变破坏多巴胺能功能的假设，我们将测量动物在开放视野装置中对安非他明给药的反应，并检查已知投射到脊髓的下丘脑中A11神经元的形态和数量。还将在纹状体中测定组织多巴胺、其代谢物和多巴胺受体的水平。2.为了检验BTBD 9突变导致强烈的运动冲动和多动的假设，我们将在几天内监测动物在饲养笼中的活动。此外，突变小鼠将在带有自由进入转轮的笼子中进行测试，转轮被移植到计算机上。这些实验将揭示突变Btbd 9小鼠是否显示出运动活动的任何昼夜变化。3.为了检验BTBD 9突变影响铁代谢的假设，我们将测量Btbd 9突变小鼠的纹状体和黑质铁蛋白、转铁蛋白和总铁。4.为了检验BTBD 9突变影响感觉系统的假设，我们将使用甩尾和热板测定法测量疼痛阈值，并使用Von Frey纤维测量触觉。 上述特异性目的的成功完成将有助于我们确定BTBD 9蛋白在体内的功能以及BTBD 9突变如何导致RLS。这些结果将显著增加我们对RLS病理生理学的理解，这最终将有助于RLS患者治疗方法的发展。 公共卫生相关性：该研究项目的成功完成将有助于我们确定BTBD 9蛋白的功能以及BTBD 9基因突变如何导致不宁腿综合征。这些结果最终将有助于不宁腿综合征患者的治疗方法的发展。

DESCRIPTION (provided by applicant): Restless legs syndrome (RLS) is a chronic sleep motor disorder characterized by unpleasant sensations in the legs and an uncontrollable urge to move them for relief. Past pathophysiological studies have associated RLS to the disorder of the central dopaminergic system and iron metabolism. Family and twin studies strongly support a genetic contribution to the pathogenesis of RLS. Tremendous progress has been made recently of uncovering candidate genes linked to RLS. Three independent studies published in the last two years all pointed to the role of BTBD9 in RLS. The function of BTBD9 protein is not known. Current animal models include 6-hydroxydopamine-lesioned rodents, iron deficiency mice, and dopamine receptor 3 knockout mice. The identification of the RLS candidate genes paves the way for making genotypic model of RLS that will be more relevant in elucidating the pathophysiology of RLS and developing therapeutic treatments. The broad, long- term objective of our research is to use transgenic mice to determine: 1) the functional role of BTBD9 protein in vivo, 2) how the alteration of BTBD9 protein function could lead to RLS. The objective of this application is to characterize Btbd9 (mouse homolog of the human BTBD9 gene) knockout mice we have already made that will enable us to answer these questions. We hypothesize that BTBD9 mutations lead to alteration of brain iron metabolism and central dopaminergic system especially the striatal D2 receptor mediated indirect pathway, or diencephalic-spinal dopaminergic tracts (A11), or both. This in turn leads to enhanced unpleasant sensations, circadian dysfunction, and strong urge to move. We plan to test our hypothesis with the following Specific Aims: 1. To test the hypothesis that BTBD9 mutation disrupts dopaminergic function, we will measure animal's response to amphetamine administration in open field apparatus and examine the morphology and number of A11 neurons in hypothalamus that are known to project to spinal cord. Levels of tissue dopamine, their metabolites, and dopamine receptors will also be determined in striatum. 2. To test the hypothesis that BTBD9 mutation leads to the strong urge to move and hyperactivity, we will monitor animals' activity in home cages over several days. In addition, the mutant mice will be tested in cages with free access running wheels that are ported to a computer. These experiments will reveal whether mutant Btbd9 mice show any circadian variation of motor activity. 3. To test the hypothesis that BTBD9 mutation affects iron metabolism, we will measure striatal and nigral ferritin, transferrin and total iron in the Btbd9 mutant mice. 4. To test the hypothesis that BTBD9 mutation affects sensory system, we will measure the pain threshold using tail flick and hot plate assays, and sense of touch using Von Frey fibers. The successful completion of the above Specific Aims will help us to determine the function of BTBD9 protein in vivo and how the mutation of BTBD9 causes RLS. The results should significantly increase our understanding of the pathophysiology of RLS, which can ultimately aid the development of therapeutic treatments for RLS patients. PUBLIC HEALTH RELEVANCE: The successful completion of the proposed research project will help us to determine the function of BTBD9 protein and how the mutation of BTBD9 gene causes Restless Legs Syndrome. The results should ultimately aid the development of therapeutic treatments for Restless Legs Syndrome patients.