Autophagy is an intracellular degradation process crucial for homeostasis. During autophagy, a double-membrane autophagosome fuses with lysosome through SNARE machinery STX17 to form autolysosome for degradation of damaged organelle. Whereas defective autophagy enhances cholesterol accumulation in the lysosome and impaired autophagic flux that results Niemann-Pick type C1 (NPC1) disease. However, exact interconnection between NPC1 and autophagic flux remain obscure due to the existence of controversial reports. This study aimed at a comparison of the effects of three autophagic inhibitor drugs, including chloroquine, U18666A, and bafilomycin A1, on the intracellular cholesterol transport and autophagy flux. Chloroquine, an autophagic flux inhibitor; U1866A, a NPC1 inhibitor, and bafilomycin A, a lysosomotropic agent are well known to inhibit autophagy by different mechanism. Here we showed that treatment with U1866A and bafilomycin A induces lysosomal cholesterol accumulation that prevented autophagic flux by decreasing autophagosome–lysosome fusion. We also demonstrated that accumulation of cholesterol within the lysosome did not affect lysosomal pH. Although the clearance of accumulated cholesterol by cyclodextrin restored the defective autophagosome–lysosome fusion, the autophagy flux restoration was possible only when lysosomal acidification was not altered. In addition, a failure of STX17 trafficking to autophagosomes plays a key role in prevention of autophagy flux caused by intracellular cholesterol transport inhibitors. Our data provide a new insight that the impaired autophagy flux does not necessarily result in lysosomal cholesterol accumulation even though it prevents autophagosome–lysosome fusion.

中文翻译：

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细胞内胆固醇转运抑制通过减少自噬体-溶酶体融合来损害自噬通量

自噬是一种对体内平衡至关重要的细胞内降解过程。在自噬过程中，双膜自噬体通过 SNARE 机制 STX17 与溶酶体融合，形成自噬溶酶体，用于降解受损细胞器。而有缺陷的自噬会增强溶酶体中的胆固醇积累和自噬通量受损，从而导致尼曼-匹克 C1 型 (NPC1) 病。然而，由于有争议的报道的存在，NPC1 和自噬通量之间的确切相互联系仍然不清楚。本研究旨在比较三种自噬抑制剂药物（包括氯喹、U18666A 和巴弗洛霉素 A1）对细胞内胆固醇转运和自噬通量的影响。氯喹，一种自噬通量抑制剂；U1866A，一种 NPC1 抑制剂，和巴弗洛霉素 A，众所周知，溶酶体促性剂通过不同机制抑制自噬。在这里，我们发现用 U1866A 和巴弗洛霉素 A 治疗可诱导溶酶体胆固醇积累，从而通过减少自噬体-溶酶体融合来阻止自噬通量。我们还证明，胆固醇在溶酶体中的积累不会影响溶酶体的 pH 值。尽管环糊精清除累积的胆固醇可以恢复有缺陷的自噬体-溶酶体融合，但只有在溶酶体酸化没有改变的情况下才有可能恢复自噬通量。此外，STX17 无法转运至自噬体在防止细胞内胆固醇转运抑制剂引起的自噬通量方面起着关键作用。