Genetic Control of Phospholipid Biosynthesis and Muscular Dystrophy

描述（由申请人提供）：该项目的长期目标是确定细胞器膜脂生物合成和维持的失调如何导致先天性肌营养不良（CMD），我们如何成功地设计治疗这种疾病的策略，以及这种疾病机制如何影响其他类型的肌营养不良。我们首先在头尾肌萎缩症（rmd）突变小鼠中发现了PC稳态对骨骼肌维持的关键作用，随后在胆碱激酶β (CHKB)基因功能突变缺失的CMD患者中发现了PC稳态。CHKB是在肯尼迪途径中催化胆碱磷酸化为磷酸胆碱的两种哺乳动物酶之一。CHKB活性的丧失导致骨骼肌PC水平显著降低和进行性肌肉营养不良表型，包括核膜畸形和线粒体明显增大（大线粒体），并伴有呼吸功能降低。我们假设膜PC含量的改变直接影响骨骼肌细胞器（细胞核和线粒体）的功能特性，并且恢复膜PC水平的策略将在治疗上有益。在目标1中，我们将定义调节线粒体和核功能障碍的机制。我们建议：a)确定PC缺陷是否在ER/线粒体接触点破坏线粒体裂变；b)使用高分辨率FPALM显微镜确定线粒体裂变/融合的调节机制，以测试线粒体膜曲率变化的实时动态；c)确定核膜变化是否与LMNA emry - dreifuss MD中所见的核膜变化在功能上相关。我们将测试治疗策略，a)确定chk - α (CHKA)是否可以用转基因方法替代CHKB缺陷，b)测试线粒体裂变蛋白的过表达或线粒体融合蛋白的敲除是否可以减轻大线粒体疾病的表型，c)测试PC或中间代谢物是否可以治疗性地恢复磷脂稳态。

DESCRIPTION (provided by applicant): The long-term goals of this project are to determine how the dysregulation of organelle membrane lipid biosynthesis and maintenance leads to a congenital muscular dystrophy (CMD), how we can successfully design strategies to treat this disorder, and how this disease mechanism can inform upon other types of muscular dystrophy. We first identified PC homeostasis as critical for skeletal muscle maintenance in the rostrocaudal muscular dystrophy (rmd) mutant mouse, and later in human CMD patients with loss of function mutations in the choline kinase beta (CHKB) gene. CHKB is one of two mammalian enzymes catalyzing the phosphorylation of choline to phosphocholine in the Kennedy pathway. Loss of CHKB activity results in significantly reduced skeletal muscle PC levels and a progressive muscular dystrophy phenotype with nuclear membrane dysmorphology and distinctly enlarged mitochondria (megamitochondria) with reduced respiratory function. We hypothesize that alterations in membrane PC content directly affect the functional properties of skeletal muscle organelles (nuclei and mitochondria) and that a strategy to restore membrane PC levels will be therapeutically beneficial. In aim 1, we will define the mechanisms regulating mitochondrial and nuclear dysfunction. We propose to a) determine if PC deficiency disrupts mitochondrial fission at points of ER/mitochondrial contact, b) define mechanisms regulating mitochondrial fission/fusion using high-resolution FPALM microscopy to test the real-time dynamics of mitochondrial membrane curvature changes, and c) determine if nuclear membrane changes are functionally related to those seen in LMNA Emery-Dreifuss MD. In aim 2, we will test therapeutic strategies by a) determining if CHK-alpha (CHKA) can substitute for CHKB deficiency using a transgenic approach, b) testing if overexpression of mitochondrial fission proteins, or knockout of mitochondrial fusion proteins can alleviate the megamitochondrial disease phenotype, and c) testing if PC or an intermediate metabolite can be administered therapeutically to restore phospholipid homeostasis.