Genetic Metabolic Myopathies--phosphofructokinase/acid Maltase Deficiency

我们先前报告的治疗庞贝氏症小鼠（酸性α-葡萄糖苷酶敲除菌株）的经验和酶替代疗法（ERT）人体试验的经验表明，该药物在清除储存在心肌细胞中的糖原方面非常有效，而对骨骼肌细胞的效果较差。庞贝氏症中骨骼肌对重组酶反应的失败已成为我们关注的主要焦点，并使我们进入了细胞生物学的活跃新领域，这些领域本身就为我们的活动增添了极大的兴趣。在庞贝氏症快速纤维中，存在大面积的自噬积累，其可占据接近纤维直径的一半。庞贝氏症肌纤维中大量的自噬积聚不仅是由于自噬货物向溶酶体的低效递送，而且也是由于自噬的诱导。我们已经表明，这种诱导可能是细胞试图通过激活糖原合成酶激酶（GSK）（一种最近受到重视的自噬调节剂）来下调糖原合成的意外结果。我们还表明GSK上调C2 C12肌细胞系和原代庞贝氏症成肌细胞中的自噬。 自噬包涵体破坏肌纤维的微管网络和收缩装置。 此外，自噬积累对治疗酶的溶酶体递送造成问题，所述治疗酶通过甘露糖-6-磷酸受体介导的内吞作用运输至其目的地。 由于自噬和内吞途径沿着沿着在几个步骤上会聚，因此药物最终到达自噬区域并不奇怪。 因此，去除自噬积聚似乎是改善治疗的合理方法。 为此，我们已经取得了庞贝氏症小鼠品系，其中一个关键的自噬基因，Atg 5或Atg 7，是失活的骨骼肌特异性。 自噬基因的组织特异性失活是有道理的，因为它表明，在小鼠全身自噬抑制导致出生后早期致死。 正如预期的那样，自噬在两种（Atg 5和Atg 7）自噬缺陷型庞贝氏症小鼠的肌肉中被抑制，如通过不存在LC 3 II（自噬囊泡（自噬体）的高度特异性标志物）所示。 溶酶体的扩张，庞贝氏症的标志，持续存在，但自噬积累是不存在的。自噬缺陷型庞贝氏症小鼠的ERT导致糖原水平急剧降低，接近野生型水平。肌肉活检的PAS染色以及分离的单纤维的自噬体和溶酶体标记物的免疫染色也证明了糖原清除。这种结果在自噬未被篡改的庞贝氏症小鼠中从未见过。值得注意的是，ERT加自噬抑制将Pompe小鼠转化为肌肉特异性自噬缺陷野生型小鼠;这些小鼠的健康状况（寿命、活动性和单纤维收缩性）远远好于Pompe小鼠的健康状况。在肌肉特异性自噬缺陷的野生型小鼠中，已经报道了功能障碍的线粒体的积累、轻度萎缩和年龄依赖性的力下降，但是这些异常似乎是为庞贝氏症的病理逆转付出的合理代价。 为了促进自噬抑制剂的开发，我们开发了一种庞贝氏症小鼠模型，其中自噬体用LC 3标记。这些小鼠非常适合体内测试自噬抑制剂。我们还使用这些绿色庞培小鼠产生永生化成肌细胞系;这些细胞系被用于调节体外自噬。 在另一种使庞贝氏症骨骼快肌纤维对治疗有反应的方法中，我们使用了肌肉中主要调节蛋白PGC 1 α的过表达，其将快肌纤维的一些特征改变为慢肌纤维的特征。 为了将抗治疗纤维转化为适合治疗的纤维，我们产生了转基因Pompe小鼠，其在肌肉中表达PGC 1 α。我们已经证明，PGC 1 α表达确实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。虽然我们试图通过PGC-1的转基因表达来改善治疗失败，但我们认为纤维类型转换作为一种增加对ERT反应性的方法值得进一步探索。PGC-1实验的失败是由于这种蛋白质对能量代谢的多个方面的影响，包括糖原代谢。因此，我们寻找一种不同的方法来转换纤维类型。 我们已经制造了转基因庞贝氏症小鼠，其在MCK启动子的控制下表达肌球蛋白编码的miR-499。MCK-miR-499：WT小鼠中快速肌纤维重编程为较慢肌纤维类型是通过收缩特性而非代谢特性的变化实现的。 我们目前正在分析这些老鼠。 在一项研究中，我们观察到在ERT治疗前后庞贝氏症婴儿的单根肌纤维，我们注意到治疗前的主要病理是存在巨大的充满糖原的溶酶体，但自噬的积累，这在较轻的成人形式的疾病中非常突出，可以忽略不计。然而，在接受ERT治疗的婴儿中，随着充满糖原的溶酶体收缩，自噬的积累变得可见。这些数据表明婴儿和成人庞贝氏症的发病机制可能存在差异，可能对治疗设计产生影响。 我们还发现，肌纤维中的自噬积累在治疗多年后仍然存在，这可能是对治疗反应不满意的一个原因。我们现在正在进行一项长期的研究和大量的样本，以评估ERT治疗患者中自噬积累的命运。

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.