Genetic Metabolic Myopathies--phosphofructokinase/acid Maltase Deficiency

我们之前报道的治疗Pompe小鼠(一种酸性α葡萄糖苷酶基因敲除菌株)的经验以及在人类身上进行的酶替代疗法(ERT)试验的经验表明，该药物在清除储存在心肌细胞中的糖原方面非常有效，但对骨骼肌细胞的效果较差。Pompe病中骨骼肌对重组酶反应的失败已经成为我们关注的一个主要焦点，并引导我们进入活跃的细胞生物学新领域，这些领域本身就增加了我们的活动兴趣。在庞培快速纤维中，有很大面积的自噬积累，可能占据纤维直径的近一半。庞培肌纤维中大量的自噬堆积不仅是由于自噬货物向溶酶体的低效输送，也是自噬的诱导。我们已经证明，这种诱导可能是细胞试图通过激活糖原合成酶激酶(GSK)来下调糖原合成的意外结果，GSK是最近认识到的一种自噬调节因子。我们还表明，GSK上调了C2C12肌肉细胞系和原代Pompe成肌细胞的自噬。 自噬内含物破坏微管网络和肌肉纤维的收缩装置。此外，自噬的堆积对治疗酶的溶酶体输送提出了问题，该酶通过甘露糖-6-磷酸受体介导的内吞作用运输到目的地。由于自噬和内吞途径在过程中分几步汇聚，因此药物最终进入自噬区域也就不足为奇了。因此，去除自噬积聚似乎是提高治疗效果的合理途径。为此，我们制作了庞贝小鼠品系，在这些品系中，关键的自噬基因ATG5或ATG7专门在骨骼肌中失活。自噬基因的组织特异性失活是合理的，因为研究表明，抑制小鼠全身的自噬会导致出生后早期死亡。正如预期的那样，在自噬缺陷的庞贝小鼠(ATG5和ATG7)中，肌肉中的自噬都受到了抑制，这表现在没有LC3II，这是自噬小泡(自噬小体)的高度特异性标记物。庞培病的一个特征--溶酶体的扩张持续存在，但没有自噬积累。自噬缺陷的庞贝小鼠的ERT导致糖原水平急剧下降，接近野生型水平。通过肌肉活检的PAS染色和分离的单个纤维的自噬和溶酶体标记的免疫染色，也证明了糖原清除。这一结果在自噬没有被篡改的庞贝小鼠身上从未见过。值得注意的是，ERT加上对自噬的抑制可以将庞贝小鼠转变为肌肉特异性自噬缺陷的野生型小鼠；这些小鼠的健康(寿命、活动度和单纤维收缩能力)远远好于庞培小鼠的健康。据报道，在肌肉特异性自噬缺陷野生型小鼠中，存在功能障碍的线粒体积累、轻度萎缩和年龄相关性力量下降，但这些异常似乎是庞培病病理逆转的合理代价。 为了促进自噬抑制剂的开发，我们开发了一种Pompe小鼠模型，在该模型中，自噬小体被标记为LC3。这些小鼠非常适合在体内测试自噬抑制剂。我们还用这些绿色的庞贝小鼠来产生永生化的成肌细胞系；这些细胞系正在被用来调节体外的自噬。 在另一种呈现庞贝骨骼快肌纤维对治疗反应的方法中，我们使用了主要调节蛋白PGC1pha在肌肉中的过度表达，它将快肌纤维的一些特征改变为慢肌纤维的特征。为了将耐治疗的纤维转变为可治疗的纤维，我们培育了转基因庞培小鼠，这种小鼠在肌肉中表达PGC1α。我们已经证明，事实上，PGC1pha的表达导致了从快纤维到慢纤维的成功转换，但它们对治疗的反应仍然很差。虽然我们通过转基因表达PGC-1来改善治疗的尝试失败了，但我们认为纤维类型转换作为一种提高ERT反应性的方法值得进一步探索。PGC-1实验的失败是因为这种蛋白质对能量代谢的多个方面产生了影响，包括糖原代谢。因此，我们寻找了一种不同的方法来转换纤维类型。我们获得了在MCK启动子控制下表达肌球蛋白编码miR-499的转基因Pompe小鼠。在MCK-miR-499：WT小鼠中，快肌纤维重新编程为慢肌纤维类型是通过收缩而不是代谢特性的改变实现的。我们目前正在分析这些老鼠。 在一项对庞培病婴儿ERT前后的单一肌肉纤维的研究中，我们注意到治疗前的主要病理是巨大的、充满糖原的溶酶体的存在，但自噬的堆积在较轻微的成人形式中是如此显著，可以忽略不计。然而，在接受ERT的婴儿中，随着充满糖原的溶酶体缩小，自噬积聚变得可见。这些数据表明，在婴儿和成人中，庞贝病的发病机制可能存在差异，这可能对治疗设计有影响。我们还表明，在多年的治疗后，肌肉纤维中的自噬积聚仍然存在，这可能是治疗反应不满意的一个原因。我们现在正在进行一项长期研究和大量样本，以评估接受ERT治疗的患者自噬积聚的命运。

Our previously reported experience of treating Pompe mice (an acid alpha glucosidase knockout strain) and the experience in trials in humans with enzyme replacement therapy (ERT), demonstrated that the drug is highly effective in clearing glycogen stored in cardiac muscle cells and poorly effective for skeletal muscle cells. This failure of skeletal muscle response to recombinant enzyme in Pompe disease has become a major focus of our attention and has led us into active new areas of cell biology that in themselves add great interest to our activities. In Pompe fast fibers there are large areas of autophagic accumulation, which may occupy close to half the diameter of a fiber. Massive autophagic buildup in Pompe myofibers results not only from inefficient delivery of autophagic cargo to the lysosomes but also from the induction of autophagy. We have shown that this induction may be an unintended consequence of a cellular attempt to down-regulate glycogen synthesis by activating glycogen synthase kinase (GSK), a recently appreciated regulator of autophagy. We have also shown that GSK up-regulates autophagy in C2C12 muscle cell line and in primary Pompe myoblasts. The autophagic inclusions disrupt the microtubular network and the contractile apparatus of muscle fibers. Furthermore, autophagic buildup poses a problem for the lysosomal delivery of the therapeutic enzyme which traffics to its destination via mannose-6-phosphate receptor mediated endocytosis. Since autophagic and endocytic pathways converge at several steps along the way it is not surprising that the drug ends up the autophagic area. Therefore, the removal of autophagic buildup seemed a reasonable approach to improve the therapy. To this end we have made Pompe mouse strains in which a critical autophagic gene, Atg5 or Atg7, is inactivated specifically in skeletal muscle. The tissue-specific inactivation of autophagic genes is warranted, because it was shown that suppression of autophagy in the whole body in mice leads to early post-natal lethality. As expected, autophagy was suppressed in muscles in both (Atg5 and Atg7) autophagy-deficient Pompe mice as shown by the absence of LC3II, a highly specific marker for autophagic vesicles (autophagosomes). Expansion of lysosomes, a hallmark of Pompe disease, persisted, but autophagic accumulation was absent. ERT in autophagy-deficient Pompe mice resulted in a dramatic reduction in the glycogen level approaching wild type levels. Glycogen clearance was also demonstrated by PAS staining of muscle biopsies and by immunostaining of isolated single fibers for autophagosomal and lysosomal markers. This outcome was never seen in Pompe mice in which autophagy was not tampered with. Of note, ERT plus suppression of autophagy converts Pompe mice into muscle-specific autophagy-deficient wild type mice; the health (longevity, mobility, and single fiber contractility) of these mice is far better than the health of Pompe mice. The accumulation of dysfunctional mitochondria, mild atrophy and age-dependent decrease in force have been reported in muscle-specific autophagy-deficient wild type mice, but these abnormalities seem to be a reasonable price to pay for the reversal of pathology in Pompe disease. To facilitate the development of autophagy inhibitors we have developed a Pompe mouse model, in which autophagosomes are labeled with LC3. These mice are ideally suited for in vivo testing autophagy inhibitors. We have also used these green Pompe mice for the generation of immortalized myoblast cell lines; these cell lines are being used to modulate autophagy in vitro. In yet another approach to rendering Pompe skeletal fast muscle fibers responsive to therapy, we have used the over-expression in muscle of the major regulatory protein, PGC1alpha, which changes some of the characteristics of fast muscle fibers into those of slow muscle fibers. To turn the therapy-resistant fibers into fibers amenable to therapy, we generated transgenic Pompe mice, which express PGC1 alpha in muscle. We have shown that, indeed, PGC1alpha expression resulted in a successful switch from fast to slow fibers, but they still responded poorly to therapy. Although our attempt to improve therapy by transgenic expression of PGC-1 failed, we thought that fiber-type conversion as a way to increase responsiveness to ERT is worth exploring further. The failure of PGC-1 experiments resulted from the effect of this protein on multiple aspects of energy metabolism, including glycogen metabolism. Therefore we looked for a different way to convert fiber types. We have made transgenic Pompe mice, which express myosin-encoded miR-499 under the control of the MCK promoter. The reprogramming of fast myofibers to a slower myofiber type in MCK-miR-499:WT mice is achieved by a change in the contractile rather than the metabolic properties. We are currently analyzing these mice. In a study of single muscle fibers from infants with Pompe disease obtained before and after ERT, we have noted that the dominant pathology before therapy is the presence of huge, glycogen-filled lysosome but the autophagic buildup, which is so prominent in the milder adult form of the disease, is negligible. However, in infants on ERT, as the glycogen-filled lysosomes are shrunk, autophagic buildup becomes visible. The data point to the possibility of differences in the pathogenesis of Pompe disease in infants and adults, with possible implications for the design of therapy. We have also shown that autophagic buildup in muscle fibers persists after years on therapy, and it may be a reason for unsatisfactory response to therapy. We are now conducting a long-term study and a larger number of samples to evaluate the fate of autophagic buildup in ERT-treated patients.