Pharmacological modulators of autophagy activate a parallel noncanonical pathway driving unconventional LC3 lipidation

Abstract

The modulation of canonical macroautophagy/autophagy for therapeutic benefit is an emerging strategy of medical and pharmaceutical interest. Many drugs act to inhibit autophagic flux by targeting lysosome function, while others were developed to activate the pathway. Here, we report the surprising finding that many therapeutically relevant autophagy modulators with lysosomotropic and ionophore properties, classified as inhibitors of canonical autophagy, are also capable of activating a parallel noncanonical autophagy pathway that drives MAP1LC3/LC3 lipidation on endolysosomal membranes. Further, we provide the first evidence supporting drug-induced noncanonical autophagy in vivo using the local anesthetic lidocaine and human skin biopsies. In addition, we find that several published inducers of autophagy and mitophagy are also potent activators of noncanonical autophagy. Together, our data raise important issues regarding the interpretation of LC3 lipidation data and the use of autophagy modulators, and highlight the need for a greater understanding of the functional consequences of noncanonical autophagy.

Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion

“…In line with these observations, we did not find a decrease in LysoTracker Red-positive puncta upon CQ treatment as previously shown [29,[55][56][57]. Our results and other recent studies [30,58,59], indicate that CQ does not substantially decrease lysosomal acidity, and the lysosomes retain their capacity to degrade delivered material.…”

“…Surprisingly, however, CQ led to a higher LDH sequestration compared to the control and BafA 1treated cells shortly upon addition (Figures 2(h) and 7(C)), suggesting that brief CQ treatments could stimulate autophagosome formation. Data that we obtained by analyzing the subcellular distribution of early autophagy protein markers such as ZFYVE1 and WIPI1 (data not shown), confirmed that CQ can stimulate an autophagic response shortly upon addition to cells as previously suggested [29,49]. Co-treatment with the PIK3C3 inhibitor SAR405 [18] completely abolished LDH sequestration, showing that the measured sequestration was indeed mediated by autophagy (Figure 7(C)).…”

“…Because there are discrepancies in the literature about whether or not CQ raises the lysosomal pH [29,30], and our data indicated that a concentration of 100 µM had no major effect ( Figure 1(C,D) and S1(B)), we assessed the lysosomal acidity in U2OS cells exposed to increasing concentrations of CQ using the LysoTracker Red dye (Figure S1(C)). Surprisingly, LysoTracker Red-positive puncta were present in cells treated with CQ at concentrations ranging from 25 to 200 µM (Figure S1(C)), but also at concentrations of 400 and 800 µM when cells started to display clear signs of stress (data not shown).…”

“…Because it has been shown that CQ promotes LC3 conjugation on endosomes [29,49] and we indeed observed that this compound severely affects and disorganizes components of the endo-lysosomal system (Figures 2(C,D), 3(A), S1(D), 2 (C-E)), we used SQSTM1 as an alternative autophagy marker protein to study the effects of CQ on the autophagic flux. We examined the subcellular distribution of SQSTM1 in U2OS cells also labeled for LAMP2, to determine whether the SQSTM1 puncta were accumulating either in the LAMP2decorated lysosomes (Figure 9(A), yellow arrows) or in the cytoplasm and autophagosomes, where they are not positive for LAMP2 ( Figure 9(A), red arrows).…”

Intrinsic lipid binding activity of ATG 16L1 supports efficient membrane anchoring and autophagy

“…Consistent with the LC3 lipidation results, p62 degradation was impaired in ATG16L1 LD -expressing cells, suggesting that autophagic flux was also inhibited ( Fig 5A). Given that noncanonical LC3 lipidation can occur on single membranes and requires the ATG5 complex but not additional upstream autophagy machinery, such as WIPI2 or the ULK1 complex, we further examined whether ATG16L1 LD could support LC3 lipidation during treatment with monensin, ammonium chloride (NH 4 Cl) or CCCP (100 lM), known to act as ionophores and/or lysosomotropic agents (Jacquin et al, 2017). Similar results were obtained during autophagy induced by glucose starvation, suggesting that the lipid binding domain of ATG16L1 is also required for autophagy induced in the absence of mTORC1 inhibition and ULK1-complex activation ( Fig 5C).…”