Pompe病是一种溶酶体贮积症,优先影响肌肉,它是由溶酶体中编码酸性α-葡萄糖苷酶的GAA突变和糖吞噬缺陷引起的。虽然Pompe病的最初病理是溶酶体中糖原的积累,溶酶体途径在糖原降解中的特殊作用尚不完全清楚。因此,我们研究了Pompe病中糖原积累的特征和糖吞噬障碍的机制。从患有庞普氏病的患者和小鼠模型的受影响部位获得骨骼肌样本。组织学分析,免疫印迹分析,免疫荧光测定,并利用溶酶体分离分析了糖原积累的特征。细胞培养,慢病毒感染,和CRISPR/Cas9方法用于研究糖积累的调节。我们证明了残留的糖原,通过暴露的糖原和更多的α-淀粉酶抗性与成熟糖原区分开来,在Pompe病的骨骼肌中积累。溶酶体分离显示野生型小鼠溶酶体中无糖原的糖原蛋白和Gaa-/-小鼠溶酶体中不同大小的糖原蛋白。我们的研究发现,溶酶体中糖原暴露的残留糖原的降解缺陷是庞贝病的基本病理机制。同时,在由细胞质糖原分解缺陷引起的其他糖原贮积病中,不存在糖原暴露的残留糖原。体外,残余糖原的产生是由细胞质糖原分解引起的。值得注意的是,糖原磷酸化酶的抑制导致Pompe病细胞模型中糖原暴露的残余糖原和糖原积累减少。因此,溶酶体水解途径在残余糖原降解为糖原的过程中起着至关重要的作用,与细胞质糖原分解同时发生。这些发现可能为Pompe病提供一种新的底物减少治疗策略。©2024英国和爱尔兰病理学会。
Pompe disease is a lysosomal storage disorder that preferentially affects muscles, and it is caused by GAA mutation coding acid alpha-glucosidase in lysosome and
glycophagy deficiency. While the initial pathology of Pompe disease is glycogen accumulation in lysosomes, the special role of the lysosomal pathway in glycogen degradation is not fully understood. Hence, we investigated the characteristics of accumulated glycogen and the mechanism underlying
glycophagy disturbance in Pompe disease. Skeletal muscle specimens were obtained from the affected sites of patients and mouse models with Pompe disease. Histological analysis, immunoblot analysis, immunofluorescence assay, and lysosome isolation were utilized to analyze the characteristics of accumulated glycogen. Cell culture, lentiviral infection, and the CRISPR/Cas9 approach were utilized to investigate the regulation of
glycophagy accumulation. We demonstrated residual glycogen, which was distinguishable from mature glycogen by exposed glycogenin and more α-amylase resistance, accumulated in the skeletal muscle of Pompe disease. Lysosome isolation revealed glycogen-free glycogenin in wild type mouse lysosomes and variously sized glycogenin in Gaa-/- mouse lysosomes. Our study identified that a defect in the degradation of glycogenin-exposed residual glycogen in lysosomes was the fundamental pathological mechanism of Pompe disease. Meanwhile, glycogenin-exposed residual glycogen was absent in other glycogen storage diseases caused by cytoplasmic glycogenolysis deficiencies. In vitro, the generation of residual glycogen resulted from cytoplasmic glycogenolysis. Notably, the inhibition of glycogen phosphorylase led to a reduction in glycogenin-exposed residual glycogen and
glycophagy accumulations in cellular models of Pompe disease. Therefore, the lysosomal hydrolysis pathway played a crucial role in the degradation of residual glycogen into glycogenin, which took place in tandem with cytoplasmic glycogenolysis. These findings may offer a novel substrate reduction therapeutic strategy for Pompe disease. © 2024 The Pathological Society of Great Britain and Ireland.