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。平均每个细胞都含有成百上千个母系遗传的线粒体，这些线粒体充满了它们自己的线粒体编码基因组。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。核和线粒体编码的OXPHOS基因突变已经确定，但很少有针对性的动物模型存在。OXPHOS基因缺陷与多种代谢性疾病密切相关，包括Leigh病等严重的儿童疾病。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。利氏病可能是由线粒体或核突变引起的，这种突变会在3个月到2岁之间导致毁灭性的肌肉和神经退化，并伴随一系列使人衰弱的病理。该项目侧重于开发基因工程小鼠模型，以表征特定OXPHOS基因的作用及其在OXPHOS通路和疾病进展中的参与。大多数Leigh病病例，无论是获得性还是散发性，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造成的。NADH脱氢酶-泛素- fes 4 （NDUFS4）基因的各种突变与线粒体电子传递和Leigh病中复合物I活性降低有关。NDUFS4蛋白的特异性磷酸化发生在camp依赖性激酶激活的响应中，并与复合体i的激活相关。条件Cre-LoxP NDUFS4杂合敲除小鼠胚胎干细胞将被创建以评估体外表型，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。将构建含有杂合NDUFS4点突变的第二种胚胎干细胞系。这将有助于创建纯合点突变小鼠模型，该模型将解决功能NDUFS4蛋白缺失导致Leigh病的具体机制。除了复合体I活性、细胞呼吸和线粒体功能的具体测量外，表型将通过大体和组织病理学分析来确定。2种NDUFS4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，该项目代表了开发治疗策略和针对衰弱线粒体疾病的针对性干预的第一步。

DESCRIPTION (provided by applicant): Mitochondria provides 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 2 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.