TRPML介导的Ca2+信号转导对糖原贮积病Ⅱ型（Pompe病）中溶酶体胞吐和肌细胞再生的调节机制研究

The rhGAA replacement therapy（ERT） has limited clinical applications for Glycogen Storage Disease Type II (Pompe Disease)，and has little effects on lysosome accumulation, excessive autophagy and progressive loss of muscle mass. Recently, a type of non-selective cation channel , TRPML, has been investigated and perceived as the ‘sensor’of lysosomal homeostasis, which can accelerate lysosome exocytosis and inhibit loss of muscle mass. Our initial findings have shown that the TRPML1 in Pompe’s skeletal muscle is up-regulated in the later stage of disease,and is in favor of lysosome exocytosis and muscle regeneration, although the mechanisms are unclear.We intend to use muscle specimen from Pompe patinets, and muscle from GAA KO mice, as well as myoblasts to investigate the mechanism of TRPML1/3 in regulating lysosomal exocytosis and retaining muscle mass under TRPML1/3 gene silencing or activated conditions. We also aim to explore the role of TFEB for lysosomal adaptation in regulating TRPML , as well as the role of AKT/FOXO1 and AKT/mTOR/p70S6K signaling pathway in inhibition of muscle wasting and improving proliferation. By investigating mechanisms of self-regulation of lysosome, exocytosis and muscle mass maintaining, the present study is expected to provide potential economic therapeutic targets for early treatment of Pompe disease.

Pompe病治疗中重组GAA酶替代疗法对骨骼肌症状改善并不理想，原因是它无法彻底缓解溶酶体堆积、自噬过量和肌容积的破坏。新近研究发现溶酶体膜上的非选择性阳离子通道TRPML，能够作为溶酶体“感受器”诱发钙离子流向胞浆，促进溶酶体的转运和胞吐。我们初步研究显示Pompe病患者肌肉组织内TRPML1上调时间较晚，其上调有利于溶酶体胞吐和肌细胞再生，但具体机制尚不清楚。本研究拟通过患者临床肌肉标本、GAA KO小鼠和体外成肌细胞培养三方面研究，探讨TRPML1/3的沉默和激活对骨骼肌内溶酶体的胞吐和肌容积维持的作用；证实TRPML1/3受到溶酶体适调中TFEB的调控，并通过激活下游AKT/FOXO1和AKT/mTOR/p70S6K通路分别抑制肌萎缩和促进再生。该研究可望通过深入探索溶酶体自身钙离子调节及其对胞吐和肌容积的调控机制，为Pompe病更为经济有效的早期治疗提供新的思路和靶点。

Pompe病治疗中重组GAA酶替代疗法对骨骼肌症状改善并不理想，原因是它无法彻底缓解溶酶体堆积、自噬过量和肌容积的破坏。我们在对44例晚发型Pompe病患者的临床随访和对18例患者的骨骼肌酶组织化学和免疫组织化学染色中发现，Stbd1在LOPD肌肉组织中的表达含量上调，尤其是疾病晚期，提示糖噬必然参与了Pompe病的病理改变，并且很可能加速了病情恶化。TRPML介导的自噬是也可能参与其中。Glycogenin作为糖原合成的引物，也是糖原分子中唯一的蛋白分子，其降解可能是糖原经溶酶体代谢的根源所在，探明这一问题或许能够找到阻止糖原过多经溶酶体代谢的过程，从而延缓发病；另一方面，结合其分布及相关蛋白的表达特点，推测糖噬作用的上调可能与自噬、糖原合成及能量调控机制相关，减少糖原储备（尤其在疾病早期），或能下调糖噬作用，从而减少糖原累积。不同病理进程中糖噬作用存在差异，早诊断、早治疗仍是决定患者病情进展和预后的关键。值得一提的是，以上成果被研究者在临床上用于指导患者低碳水化合物饮食，可能减少肌肉组织中糖原合成的原料和糖原储备，从而通过降低糖噬作用达到减少糖原累积的目的，是缓解Pompe病情的潜在治疗靶点。