Rapid-onset Dystonia Parkinsonism (DYT12 Dystonia): Pathophysiology & Atp1a3 Mice

描述（由申请人提供）：DYT12是一种快速发作的肌张力障碍伴帕金森综合征（RDP）。DYT12患者具有全身性肌张力障碍和帕金森症状。一般来说，患者在受到轻度至重度应激源（例如剧烈运动、创伤性脑损伤）的刺激之前不会出现症状。症状在压力后的几分钟到几天内发展，并且是永久性的。在DYT12中，首次发作常见于青春期后期或成年早期。DYT12肌张力障碍的遗传已被确定为常染色体显性遗传。致病基因是ATP 1A3，它编码Na？ATP酶（13），其仅在神经元中表达。 虽然ATP 1A3导致DYT12肌张力障碍的遗传学已被成功阐明，但其突变形式在导致DYT12肌张力障碍中的作用尚不清楚。此外，患者中受影响的神经回路或突触连接未被识别。最后，DYT12没有有效的治疗方法，因为没有办法确定压力源之前症状的程度。这些未知因素阻碍了充分理解DYT12肌张力障碍的病理生理学的努力，从而阻碍了为患者开发有效的治疗策略。 我们研究的广泛、长期目标是使用转基因小鼠来确定：1）13蛋白在体内的功能作用，以及2）13蛋白功能的丧失如何导致DYT12肌张力障碍。本申请的目的是表征Atp1a3突变小鼠以回答这些问题。 我们假设，我们将能够区分行为的渗透性和非渗透性Atp1a3突变小鼠使用一种新的自愿轮运行范例。我们进一步假设，行为外显突变小鼠将有神经回路功能障碍，特别是在基底神经节，多巴胺能调制，并在皮层和纹状体可能改变了突触传递和可塑性，最终影响运动控制和姿势。这项研究的基本原理是，一旦确定了Atp1a3在导致大脑运动控制功能障碍中的作用，就可以开发出纠正DYT12的可能干预措施。 我们计划通过以下具体目的来检验我们的假设：（1）检验应激诱导行为渗透突变小鼠的假设，即自主轮跑活动减少。 a.我们将在自愿的车轮运行中对应激动物和非应激动物进行差异化研究。 B.应激的行为非渗透小鼠将经历另一个重复的应激源，并重新测试，看看它们是否成为行为渗透。 (2)为了检验行为渗透突变小鼠与非渗透突变小鼠将不同地表现出多巴胺能功能被破坏的假设，我们将通过HPLC测量组织多巴胺及其代谢物的水平，通过放射性配体结合测定和蛋白质印迹分析测量纹状体多巴胺受体的水平，多巴胺转运蛋白活性，和多巴胺系统功能通过动物对在旷场装置中多巴胺能激动剂和拮抗剂的药理学给药的反应，（3）为了验证行为渗透突变小鼠与非渗透突变小鼠将不同地表现出运动控制、平衡和感觉知觉的破坏的假设，我们将在旷场装置、横梁行走、旋转棒中测试小鼠的运动协调和平衡，杆测试主要用于纹状体特异性运动缺陷，甩尾和von Frey用于感觉测试。 上述特定目的的成功完成将有助于我们确定Atp1a3在体内的功能以及Atp1a3的突变形式如何导致DYT 12肌张力障碍。这些结果将显著增加我们对DYT12肌张力障碍的病理生理学的理解，这将最终有助于DYT12肌张力障碍患者治疗方法的发展。 公共卫生相关性：仅在美国就有超过50万人患有肌张力障碍。该项目旨在开发和分析DYT 12肌张力障碍的遗传小鼠模型，以了解蛋白质，Na？可能导致这种衰弱性疾病。拟议研究的结果应显着增加对DYT12肌张力障碍的病理生理学的理解，这最终有助于DYT12肌张力障碍患者和其他肌张力障碍患者的治疗方法的开发。

DESCRIPTION (provided by applicant): DYT12 is a rapid-onset dystonia with parkinsonism (RDP). DYT12 patients have both generalized dystonia and parkinsonian symptoms. Generally patients exhibit no symptoms until precipitated by a mild to severe stressor (e.g. strenuous exercise, traumatic brain injury). Symptoms develop within minutes to days post- stressor and are permanent. In DYT12 the first attack is common in late adolescence or early adulthood. The inheritance of DYT12 dystonia has been determined to be autosomal dominant. The causative gene is ATP1A3, which encodes the 13 subunit of the Na?ATPase (13), which is only expressed in neurons. While the genetics of ATP1A3 causing DYT12 dystonia has been successfully elucidated, the role of its mutated forms in causing DYT12 dystonia is unknown. Furthermore, neural circuits or synaptic connections affected in the patients are not identified. Finally, there is no effective therapeutics for DYT12 since there is no way to determine the extent of symptoms prior to the stressor. These unknowns hamper efforts to adequately understand the pathophysiology of DYT12 dystonia, thus preventing the development of effective therapeutic strategies for patients. The broad, long-term objective of our research is to use transgenic mice to determine: 1) the functional role of 13 protein in vivo, and 2) how the loss of function of 13 leads to DYT12 dystonia. The objective of this application is to characterize Atp1a3 mutant mice to answer these questions. We hypothesize that we will be able to distinguish from behaviorally penetrant and non-penetrant Atp1a3 mutant mice using a novel voluntary wheel running paradigm. We further hypothesize that behaviorally penetrant mutant mice will have neural circuitry dysfunction especially in the basal ganglia, dopaminergic modulation, and in the cortex and striatum may have altered synaptic transmission and plasticity, ultimately affecting motor control and posture. The rationale for the proposed research is that once the roles of Atp1a3 in causing dysfunction of movement control in the brain are determined, possible interventions to correct DYT12 can be developed. We plan to test our hypothesis with the following Specific Aims: (1) To test the hypothesis that stress induces behaviorally penetrant mutant mice as defined as decreased activity in voluntary wheel running. a. We will differentially examine stressed animals versus non-stressed animals in voluntary wheel running. b. Stressed behaviorally non-penetrant mice will undergo another repetitive stressor and retested to see if they become behaviorally penetrant. (2) To test the hypothesis that the behaviorally penetrant versus non-penetrant mutant mice will differentially exhibit disrupted dopaminergic function, we will measure levels of tissue dopamine and its metabolites by HPLC, striatal dopamine receptors by radioligand binding assays and western blot analysis, dopamine transporter activity, and dopamine system function via animals' response to pharmacological administration of dopaminergic agonists and antagonists in the open field apparatus, (3) To test the hypothesis that the behaviorally penetrant versus non-penetrant mutant mice will differentially exhibit disrupted motor control, balance, and sensory perception, we will test mice in open field apparatus, beam-walking, rotarod for motor coordination and balance, pole test mainly for striatum-specific motor deficits, tail flick and von Frey for sensory tests. The successful completion of the above Specific Aims will help us to determine the function of Atp1a3 in vivo and how the mutant form of Atp1a3 causes DYT12 dystonia. The results should significantly increase our understanding of the pathophysiology of DYT12 dystonia, which will ultimately aid the development of therapeutic treatments for DYT12 dystonia patients. PUBLIC HEALTH RELEVANCE: Dystonia affects more than half of a million people in US alone. The project is aimed at developing and analyzing a genetic mouse model of DYT12 dystonia to understand how malfunction of a protein, 13 subunit of the Na?ATPase, could lead to this debilitating disorder. The results from the proposed research should significantly increase the understanding of the pathophysiology of DYT12 dystonia, which can ultimately aid the development of therapeutic treatments for DYT12 dystonia patients and other dystonia patients.