Metabolite Profiling of a Novel Mitochondrial Kinase in Neuromuscular Disease

描述(申请人提供)：这项建议的目标是利用代谢组学分析来了解神经肌肉疾病的机制，包括一种新的小鼠Nadkd1突变，Nadkd1是一种定位于线粒体的未鉴定的NAD Kinase家族成员，并开发信息学工具来分析从实验室小鼠获得的代谢组学数据。使用基于质谱学的低分子代谢物分析(代谢组学)有可能根据直接受疾病过程影响的代谢物的变化来确定疾病机制。当未知功能的新基因发生突变时，这种公正、广泛的方法尤其有价值。Nadkd1的突变就是这种情况，它会导致小鼠的神经肌肉退化，在大约8周大的时候明显出现神经肌肉疾病的症状，在大约5个月大的时候死亡。在这些小鼠中，神经肌肉接头似乎逐渐失去神经，导致类似于远端脊肌萎缩的运动功能丧失。NADKD1蛋白属于小分子蛋白激酶NAD Kinase家族，定位于线粒体，但其底物和功能尚不清楚。考虑到推测的小分子激酶活性和线粒体的定位，代谢物图谱将通过鉴定积累的底物和还原的磷酸化产物来确定该蛋白质的底物和生化途径。我们有一个来自Charcot-Marie-Tooth 2D周围神经病小鼠模型的初步代谢组学数据集，将用于比较。然而，这项初步研究也强调了代谢组学数据的分析和解释中的一些挑战。因此，我们提出了两个具体目标。首先，我们将进行额外的代谢分析，将Nadkd1突变小鼠与其他相关模型和适当的对照进行比较。这些比较将包括1)发病前和2)发病后Nadkd1突变小鼠，以及3)Pla2g6突变小鼠，这是一种婴儿神经轴索营养不良(INAD)模型，也会导致失神经和外周轴突退化。每个比较研究都有其自身的优点，总的来说，我们将能够将Nadkd1相关的图谱与其他遗传神经肌肉模型进行比较，以确定哪些变化是在Nadkd1突变小鼠中专门发现的，因此可能与基因功能和疾病机制直接相关。其次，我们将开发一个基于网络的生物信息学资源，专门为分析为实验室小鼠生成的代谢组学数据而设计。这个被称为老鼠代谢分析平台(MMAP)的资源将促进代谢组学数据与其他基因组数据的集成，并利用现有的关于小鼠基因和蛋白质的生化途径和生物学注释的知识库中的信息。为这一探索性R21开发的信息学工具将普遍适用于未来人类疾病小鼠模型的代谢组学研究。

DESCRIPTION (provided by applicant): The goals of this proposal are to use metabolomics analysis to understand the mechanisms of neuromuscular diseases including a novel mouse mutation in Nadkd1, an uncharacterized NAD Kinase family member that localizes to mitochondria, and to develop informatics tools for the analysis of metabolomics data obtained from the laboratory mouse. The use of mass spectroscopy-based analysis of low molecular weight metabolites (metabolomics) has the potential to identify disease mechanisms based on changes in metabolites that are directly impacted by the disease process. Such an unbiased, broad-scope approach is particularly valuable when novel genes with unknown functions are mutated. This is the case for mutations in Nadkd1, which cause neuromuscular degeneration in mice, with overt onset of neuromuscular disease symptoms at approximately eight weeks of age and death by approximately 5 months of age. Neuromuscular junctions appear to progressively denervate in these mice, leading to loss of motor function resembling a distal spinal muscular atrophy. The NADKD1 protein is in the NAD Kinase family of small molecule kinases, and localizes to mitochondria, but the substrate and function of NDAKD1 is unknown. Given the putative small molecule kinase activity and the mitochondrial localization, metabolite profiling will be used to determine the substrate and biochemical pathway for this protein by identifying accumulated substrate and reduced phosphorylated product. We have a preliminary metabolomics data set from a mouse model of Charcot-Marie-Tooth 2D peripheral neuropathy that will be used for comparison. However, this preliminary study also highlighted a number of challenges in the analysis and interpretation of metabolomics data. We are therefore proposing two specific aims. First, we will perform additional metabolomic analyses to compare the Nadkd1 mutant mice to other relevant models and appropriate controls. These comparisons will include 1) pre- and 2) post-onset Nadkd1 mutant mice, and 3) Pla2g6 mutant mice, a model of Infantile Neuroaxonal Dystrophy (INAD), which also results in denervation and peripheral axon degeneration. Each comparative study has merit on its own, and in aggregate, we will be able to compare Nadkd1-associated profiles to other genetic neuromuscular models to determine which changes are specifically found in the Nadkd1 mutant mouse and therefore may be directly related to the gene function and disease mechanism. Second, we will develop a web-based bioinformatics resource specifically designed for the analysis of data from metabolomics data generated for the laboratory mouse. This resource, called the Mouse Metabolic Analysis Platform (mMAP) will facilitate the integration of metabolomics data with other genomics data and with information from existing knowledgebases of biochemical pathways and biological annotations for mouse genes and proteins. The informatics tools developed for this exploratory R21 will be generally applicable to future metabolomics studies of mouse models of human disease.