背景:自噬是一种对稳态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机器STX17与溶酶体融合,形成降解受损细胞器的自溶酶体。而自噬缺陷会增强溶酶体中的胆固醇积累和自噬通量受损,从而导致Niemann-PickC1型(NPC1)疾病。然而,由于存在有争议的报道,NPC1和自噬通量之间的精确互连仍然不清楚。
结果:本研究旨在比较三种自噬抑制剂药物的作用,包括氯喹,U18666A,和巴弗洛霉素A1,对胞内胆固醇转运和自噬通量的影响。氯喹,自噬通量抑制剂;U1866A,NPC1抑制剂,和巴弗洛霉素A,众所周知,一种促溶酶体剂通过不同的机制抑制自噬。在这里,我们表明,用U1866A和bafilomycinA治疗诱导溶酶体胆固醇积累,通过减少自噬体-溶酶体融合来防止自噬通量。我们还证明了胆固醇在溶酶体内的积累不影响溶酶体pH。尽管通过环糊精清除积累的胆固醇恢复了自噬体-溶酶体融合缺陷,只有当溶酶体酸化没有改变时,自噬通量恢复才有可能.此外,STX17转运至自噬体的失败在预防细胞内胆固醇转运抑制剂引起的自噬通量中起关键作用.
结论:我们的数据提供了一个新的见解,即自噬通量受损不一定导致溶酶体胆固醇积累,即使它阻止了自噬-溶酶体融合。视频摘要。
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. Video abstract.