Mouse Modeling of Leigh Disease and Complex I Assembly

描述（由申请人提供）：线粒体通过氧化磷酸化（OXPHOS）为细胞提供能量。平均每个细胞包含数百到数千个母系遗传的线粒体，其中充满了它们自己的染色体编码基因组。线粒体和核编码的OXPHOS基因的相互作用在能量产生中至关重要。已经鉴定了核编码的OXPHOS基因和脑编码的OXPHOS基因中的突变，但是存在很少的靶向动物模型。OXPHOS基因内的缺陷与各种代谢疾病密切相关，包括严重的儿童疾病，包括Leigh病。Leigh病是一种以运动控制丧失和局部坏死病变为特征的神经退行性线粒体疾病。Leigh病可能由线粒体或核突变引起-导致3个月至2岁之间的破坏性肌肉和神经变性沿着一系列使人衰弱的病理。该项目的重点是开发基因工程小鼠模型，以表征特定OXPHOS基因的作用及其参与OXPHOS途径和疾病进展。大多数Leigh病病例，无论是获得性的还是散发性的，都是由于参与氧化磷酸化的特定电子传递蛋白的产生或功能不足所致。NADH脱氢酶-泛醌-FeS 4（NDUFS 4）基因中的各种突变与线粒体电子传递中复合物I活性降低和Leigh病相关。NDUFS 4蛋白的特异性磷酸化响应cAMP依赖性激酶活化而发生，并与复合物I的活化相关。将创建条件性Cre-LoxP NDUFS 4杂合敲除小鼠胚胎干细胞以评价体外表型，并随后建立纯合敲除小鼠模型以检查体内表型和组织特异性。将构建第二个胚胎干细胞系，使其含有杂合NDUFS 4点突变。这将有助于创建纯合子点突变小鼠模型，该模型将解决功能性NDUFS 4蛋白丧失可导致Leigh病的特定机制。除了复合物I活性、细胞呼吸和线粒体功能的特定测量外，还将通过大体和组织病理学分析来鉴定表型。两种NDUFS 4小鼠模型将允许检查导致Leigh病的机制。这些模型将进一步加深我们对线粒体功能和复合体I组装的理解。因此，该项目代表了为使人衰弱的线粒体疾病开发治疗策略和有针对性的干预措施的第一步。

DESCRIPTION (provided by applicant): Mitochondria provide energy for cells via oxidative phosphorylation (OXPHOS). The average cell contains hundreds to thousands of maternally-inherited mitochondria replete with their own mitochondrially-encoded genome. The interactions of mitochondrial and nuclear-encoded OXPHOS genes are critical in energy generation. Mutations in both nuclear- and mitochondrially-encoded OXPHOS genes have been identified but few targeted animal models exist. Defects within the OXPHOS genes are intimately associated with various metabolic diseases including severe childhood disorders including Leigh disease. Leigh disease is a neurodegenerative mitochondrial disorder characterized by a motor control loss and localized necrotic lesions. Leigh Disease can result from either mitochondrial or nuclear mutations - that cause devastating muscle and neurological degeneration between 3 months and 2 years of age along with a cascade of debilitating pathologies. This project focuses on developing genetically-engineered mouse models to characterize the role of specific OXPHOS genes and their involvement in OXPHOS pathways and disease progression. The majority of Leigh disease cases, whether acquired or sporadic, result from an insufficiency in production or function of particular electron transport proteins involved in oxidative phosphorylation. Various mutations in the NADH dehydrogenase-ubiquinone-FeS 4 (NDUFS4) gene have been associated with decreased Complex I activity in mitochondrial electron transport and Leigh disease. Specific phosphorylation of the NDUFS4 protein occurs in response to cAMP-dependent kinase activation and correlates with activation of Complex I. Conditional Cre-LoxP NDUFS4 heterozygous knockout mouse embryonic stem cells will be created to evaluate in vitro phenotype and to subsequently establish a homozygous knockout mouse model to examine in vivo phenotype and tissue specificity. A second embryonic stem cell line will be constructed to contain a heterozygous NDUFS4 point mutation. This will facilitate the creation of a homozygous point mutant mouse model that will address the specific mechanisms by which loss of a functional NDUFS4 protein can lead to Leigh disease. Phenotype will be identified by gross and histopathological analyses, in addition to specific measures of Complex I activity, cellular respiration and mitochondrial function. The two NDUFS4 mouse models will allow examination of mechanisms leading to Leigh disease. These models will further our understanding of mitochondrial function and Complex I assembly. As such, this project represents a first step toward developing therapeutic strategies and targeted interventions for debilitating mitochondrial disorders.