Takikita Shoichi, Myerowitz Rachel, Zaal Kristien, Raben Nina, Plotz Paul H
Arthritis and Rheumatism Branch, National Institutes of Arthritis and Musculoskeletal and Skin Diseases, NIH, Building 50 Room 1345, 50 South Drive, Bethesda, MD 20892, USA.
Mol Genet Metab. 2009 Apr;96(4):208-17. doi: 10.1016/j.ymgme.2008.12.012. Epub 2009 Jan 22.
Lysosomes filled with glycogen are a major pathologic feature of Pompe disease, a fatal myopathy and cardiomyopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme, acid alpha-glucosidase (GAA). To facilitate studies germane to this genetic disorder, we developed two in vitro Pompe models: myotubes derived from cultured primary myoblasts isolated from Pompe (GAA KO) mice, and myotubes derived from primary myoblasts of the same genotype that had been transduced with cyclin-dependent kinase 4 (CDK4). This latter model is endowed with extended proliferative capacity. Both models showed extremely large alkalinized, glycogen-filled lysosomes as well as impaired trafficking to lysosomes. Although both Pompe tissue culture models were derived from fast muscles and were fast myosin positive, they strongly resemble slow fibers in terms of their pathologic phenotype and their response to therapy with recombinant human GAA (rhGAA). Autophagic buildup, a hallmark of Pompe disease in fast muscle fibers, was absent, but basal autophagy was functional. To evaluate substrate deprivation as a strategy to prevent the accumulation of lysosomal glycogen, we knocked down Atg7, a gene essential for autophagosome formation, via siRNA, but we observed no effect on the extent of glycogen accumulation, thus confirming our recent observation in autophagy-deficient Pompe mice [N. Raben, V. Hill, L. Shea, S. Takikita, R. Baum, N. Mizushima, E. Ralston, P. Plotz, Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease, Hum. Mol. Genet. 17 (2008) 3897-3908] that macroautophagy is not the major route of glycogen transport to lysosomes. The in vitro Pompe models should be useful in addressing fundamental questions regarding the pathway of glycogen to the lysosomes and testing panels of small molecules that could affect glycogen biosynthesis or speed delivery of the replacement enzyme to affected lysosomes.
充满糖原的溶酶体是庞贝病的主要病理特征，庞贝病是一种致命的肌病和心肌病，由溶酶体糖原降解酶酸性α-葡萄糖苷酶（GAA）缺乏引起。为了促进与这种遗传疾病相关的研究，我们建立了两种体外庞贝病模型：源自从庞贝病（GAA基因敲除）小鼠分离的原代成肌细胞培养得到的肌管，以及用细胞周期蛋白依赖性激酶4（CDK4）转导的相同基因型原代成肌细胞培养得到的肌管。后一种模型具有延长的增殖能力。两种模型均显示出极大的碱化、充满糖原的溶酶体以及溶酶体运输受损。尽管两种庞贝病组织培养模型均源自快肌且快肌球蛋白呈阳性，但就其病理表型及其对重组人GAA（rhGAA）治疗的反应而言，它们与慢肌纤维极为相似。快肌纤维中作为庞贝病标志的自噬积累不存在，但基础自噬功能正常。为了评估底物剥夺作为防止溶酶体糖原积累的策略，我们通过小干扰RNA敲低了自噬体形成所必需的基因Atg7，但我们未观察到对糖原积累程度有任何影响，从而证实了我们最近在自噬缺陷型庞贝病小鼠中的观察结果 [N. 拉本、V. 希尔、L. 谢伊、S. 泷木启太、R. 鲍姆、N. 水岛、E. 拉尔斯顿、P. 普洛茨，骨骼肌自噬抑制揭示泛素化蛋白的积累及其在庞贝病肌肉损伤中的潜在作用，《人类分子遗传学》17（2008年）3897 - 3908]，即巨自噬不是糖原运输到溶酶体的主要途径。体外庞贝病模型应有助于解决有关糖原到溶酶体途径的基本问题，并测试可能影响糖原生物合成或加快替代酶向受影响溶酶体递送的小分子药物。
