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，其特征是腿部不愉快的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联系起来。家庭和双胞胎研究强烈支持基因对RLS发病机制的贡献。近年来，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BTBD9在RLS中的作用。BTBD9蛋白的功能尚不清楚。目前的动物模型包括6-羟多巴胺损伤的啮齿动物、缺铁小鼠和多巴胺受体3敲除小鼠。RLS候选基因的鉴定为建立RLS的基因型模型奠定了基础，对阐明RLS的病理生理和制定治疗方法具有重要意义。我们研究的长远目标是利用转基因小鼠来确定：1)BTBD9蛋白在体内的功能作用，2)BTBD9蛋白功能的改变如何导致RLS。本应用程序的目的是表征我们已经制造的Btbd9（人类Btbd9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BTBD9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），或两者兼有。这反过来又会导致不愉快的感觉增强，昼夜节律紊乱，以及强烈的运动欲望。我们计划以以下具体目标来检验我们的假设：为了验证BTBD9突变破坏多巴胺能功能的假设，我们将在开放场装置中测量动物对安非他明的反应，并检查下丘脑中已知投射到脊髓的A11神经元的形态和数量。纹状体中组织多巴胺及其代谢物和多巴胺受体的水平也将被确定。2. 为了验证BTBD9突变导致强烈的移动冲动和多动的假设，我们将在几天内监测动物在家庭笼子中的活动。此外，突变小鼠将在笼子里进行测试，笼子里有可自由进入的跑步轮，这些轮子被移植到电脑上。这些实验将揭示突变Btbd9小鼠是否表现出任何运动活动的昼夜变化。3. 为了验证BTBD9突变影响铁代谢的假设，我们将测量BTBD9突变小鼠的纹状体和黑质铁蛋白、转铁蛋白和总铁。4. 为了验证BTBD9突变影响感觉系统的假设，我们将使用甩尾法和热板法测量疼痛阈值，并使用Von Frey纤维测量触觉。上述Specific Aims的成功完成将有助于我们确定BTBD9蛋白在体内的功能以及BTBD9突变如何导致RLS。该结果将显著增加我们对RLS病理生理的理解，最终有助于RLS患者治疗方法的发展。

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.