Modeling PKAN disease through neuron-specific degradation of coenzyme A

 描述(申请人提供)：泛酸激酶相关神经退行性变(PKAN)是一种持续进行性的神经疾病，也是神经退行性变伴脑铁蓄积(NBIA)障碍中最常见的综合征。PKAN患者过早死亡的风险更高，临床范围广泛，与成人相比，受影响的儿童表现出更快的疾病进展和更严重的运动受累。目前对PKAN没有有效的治疗方法，缺乏这种疾病的小鼠模型是开发治愈方法的主要障碍。PKAN是由PANK2基因突变引起的，PANK2基因是合成辅酶A(CoA)所必需的基因，辅酶A是一种对脂质合成和能量代谢至关重要的通用辅因子。PANK2的大部分突变降低或取消了该酶的活性，导致假设CoA减少可能是PKAN患者神经退行性变的根本原因；然而，目前尚无小鼠模型可用于研究神经元CoA水平与神经退行性变之间的关系，导致PKAN的分子机制仍不清楚。以前建立PKAN小鼠模型的尝试集中在通过遗传(单独或联合敲除每一只小鼠Pank)和/或饮食操作来减少全身CoA的合成或可获得性。这些方法培育出的小鼠要么没有神经退化的迹象，要么因为寿命极短(18天)和/或全身新陈代谢严重受损而无法用于开发PKAN疗法。最近，我们通过使用一种基于选择性降解小鼠神经元辅酶A的新方法，在建立具有临床意义的PKAN小鼠模型方面取得了重大进展。为了实现这一点，我们使用腺相关病毒(AAV)和神经元特异性启动子的组合来在大脑和脊髓的神经元中传递和过度表达CoA降解酶Nudt7cyt。与表达绿色荧光蛋白(GFP)的对照小鼠相比，表达Nudt7cyt的小鼠的大脑CoA和运动协调性显著降低；然而，通过AAV方法获得的高度可变的转基因表达使得CoA和运动协调性降低之间的联系难以表征。这项建议的目的是通过产生神经元特异性Nudt7cyt表达的转基因小鼠来改进这一策略。这些小鼠将通过行为、运动技能和组织分析相结合的方式进行评估。结果将与不同大脑区域的CoA测量结果相关联。这种建立PKAN小鼠模型的新方法将提供一个模型系统来识别在PKAN疾病中被破坏的CoA依赖的通路，并提供一个测试潜在治疗方法的平台。

 DESCRIPTION (provided by applicant): Pantothenate kinase-associated neurodegeneration (PKAN) is a relentlessly progressive neurological disorder and the most common syndrome among the neurodegeneration with brain iron accumulation (NBIA) disorders. PKAN patients are at a higher risk for a premature death and exhibit a broad clinical spectrum, with affected children exhibiting a more rapid disease progression and a more severe motor involvement compared to adults. There is no effective treatment for PKAN and the lack of a mouse model of the disease is a major obstacle to the development of a cure. PKAN is caused by mutations in PANK2, a gene that is required for the synthesis of coenzyme A (CoA), a universal cofactor essential for lipid synthesis and energy metabolism. Most of the mutations in PANK2 reduce or abolish the activity of the enzyme, leading to the hypothesis that reduced CoA might be the underlying cause of the neurodegeneration in PKAN patients; however, no mouse model of the disease is currently available to investigate the connection between neuronal CoA levels and neurodegeneration, and the molecular mechanisms leading to PKAN remain unknown. Previous attempts to generate a PKAN mouse model have focused on reducing whole-body CoA synthesis or availability by genetic (knocking out every mouse Pank alone or in combination) and/or dietary manipulations. These approaches have produced mice that either do not show signs of neurodegeneration or cannot be used to develop PKAN therapeutics because of their extremely short life-span (18 days) and/or their significantly impaired whole-body metabolism. We recently made significant progress towards the development of a clinically relevant PKAN mouse model by using a novel approach based on the selective degradation of CoA in mouse neurons. To achieve this, we used the combination of an adeno-associated virus (AAV) and a neuron-specific promoter to deliver and over-express a CoA-degrading enzyme, Nudt7cyt, in neurons throughout the brain and spinal cord. Mice expressing Nudt7cyt exhibited a significant reduction in brain CoA and motor coordination compared to the control mice expressing green fluorescent protein (GFP); however, the highly variable transgene expression obtained with the AAV approach makes the connection between CoA and reduced motor coordination difficult to characterize. The objective of this proposal is to improve this strategy by generating transgenic mice with neuron-specific Nudt7cyt expression. These mice will be evaluated with a combination of behavior, motor skill and histological analyses. The results will be correlated with CoA measurements in different brain regions. This novel approach to the generation of a PKAN mouse model will provide a model system to identify CoA-dependent pathways that are disrupted in PKAN disease and a platform to test potential treatments.