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

描述（由申请人提供）：DYT12是一种快速发作的帕金森性肌张力障碍（RDP）。DYT12患者有全身性肌张力障碍和帕金森症状。一般情况下，患者在轻度至重度应激源（如剧烈运动、创伤性脑损伤）诱发前无症状。应激后症状在几分钟到几天内出现，并且是永久性的。第一次发作通常发生在青春期晚期或成年早期。DYT12肌张力障碍已被确定为常染色体显性遗传。致病基因是ATP1A3，它编码Na？atp酶（13），仅在神经元中表达。虽然ATP1A3引起DYT12肌张力障碍的遗传学已被成功阐明，但其突变形式在引起DYT12肌张力障碍中的作用尚不清楚。此外，患者受影响的神经回路或突触连接尚未确定。最后，对于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对DYT12肌张力障碍病理生理学的充分理解，从而阻碍了对患者有效治疗策略的发展。我们研究的广泛而长期的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蛋白的功能丧失如何导致DYT12肌张力障碍。本应用程序的目的是表征Atp1a3突变小鼠来回答这些问题。我们假设，我们将能够区分行为渗透和非渗透的Atp1a3突变小鼠使用一种新的自愿轮跑范式。我们进一步假设，行为渗透突变小鼠会出现神经回路功能障碍，特别是在基底节区，多巴胺能调节，皮层和纹状体可能会改变突触传递和可塑性，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在引起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验证我们的假设：(1)验证压力诱导行为渗透突变小鼠的假设，定义为自愿跑轮活动减少。a.我们将对有压力的动物和无压力的动物进行差异性研究。b.行为不渗透的压力小鼠将接受另一个重复的压力源，并重新测试，看看它们是否变得具有行为渗透性。(2)为了验证行为渗透与非渗透突变小鼠会不同地表现出多巴胺能功能紊乱的假设，我们将通过高效液相色谱法测量组织多巴胺及其代谢产物的水平，通过放射配体结合试验和western blot分析测量纹状体多巴胺受体的水平，通过动物对多巴胺能激动剂和拮抗剂药理学管理的反应来测量多巴胺转运蛋白活性和多巴胺系统功能。(3)为了验证行为渗透突变小鼠与非行为渗透突变小鼠在运动控制、平衡和感觉知觉方面表现出的差异，我们将对小鼠进行开阔场实验、beam walking、rottarod运动协调和平衡测试、pole测试（主要是纹状体特异性运动缺陷）、tail flick和von Frey测试。上述Specific Aims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DYT12肌张力障碍。该结果将显著增加我们对DYT12肌张力障碍病理生理学的认识，最终有助于DYT12肌张力障碍患者治疗方法的开发。

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.