The fusion of the autophagosome to the lysosome to form the autolysosome is mediated by several different classes of proteins, including Rab GTPases, SNAREs (such as STX17), UVRAG and UVRAG’s binding-partner VPS16, which is a component of the endosome–autophagosome tethering complex HOPS.1 Which specific proteins are utilised depends the type of autophagy and cell type. In Nature Cell Biology, De Leo et al.2 recently published that the cargo of the autophagosome may actually regulate fusion to the lysosome, and therefore regulate autophagic flux (the rate of autophagic degradation). Specifically, they demonstrate that TLR9 is activated during starvation and drives autolysosome formation (using the RFP-GFP-LC3 reporter) and by extension autophagic flux (as also measured by LC3B-II and p62 turnover assays) (Figure 1). TLR9 is a toll-like receptor found on the endoplasmic reticulum and endosomes that has an important role in innate immunity. TLR9 is activated by unmethylated CpG dinucleotides found in both bacterial and mitochondrial (mt)DNA. Indeed, De Leo et al.2 show that eliminating mtDNA inhibits translocation of several proteins on the lysosome during starvation, whereas induction of mitophagy increases this effect. One protein that translocated to lysosomes was OCRL, a protein mutated in Lowe syndrome (Figure 1a). Indeed, they go on to demonstrate that depletion of OCRL causes an increase in autophagosomes (presumably due to an inhibition of autophagic flux) and that activation of it’s binding-partner MCOLN1 may rescue this defect in OCRL-depleted and Lowe syndrome patient samples. Some papers resolve old problems, whereas others raise lots of interesting follow-up questions. I believe this paper falls in the latter category, and how mtDNA might regulate autophagic flux by activating TLR9 will be the focus of this News & Views piece.
For instance, if whole mitochondria are engulfed and the mitochondrial inner and outer membranes protect the mtDNA, how does the mtDNA become available to bind TLR9? If TLR9 instigates autolysosome formation, as De Leo et al. suggest, then the mitochondria will not yet be in the presence of lysosomal hydrolases to degrade them (Figure 1a). Indeed, the lysosomal deoxyribonuclease (DNase) II may need to process CpG-containing DNA before it can be a suitable ligand for TLR9.3, 4 On the other hand, it has previously been reported by Otsu and colleagues5 that mtDNA from damaged mitochondria activates TLR9 from the autolysosome when mtDNA is unable to be properly degraded, such as when the DNase II is genetically ablated. In the experimental conditions of De Leo et al.2, DNAse II is expected to be active, but when it was inhibited, OCRL translocation to lysosomes was reduced. It would be interesting to formally demonstrate if OCRL can localise to autolysosomes as well as lysosomes, for instance with co-localisation assays between OCRL and LC3/LAMPs under starvation conditions. A model that fits data from both the De Leo and Otsu papers is that autolysosomes (made independently of mtDNA-driven TLR9 activation) expose mtDNA by degrading the mitochondrial membrane, which is then recognised by TLR9 in the autolysosome.2, 5 This would then cause a positive feedback loop via TLR9-dependent to increase autophagic flux, but would mean that TLR9 is not strictly required for autolysosome formation under these circumstances (Figure 1b). Another possibility is that TLR9 is brought to the cargo via endosome–autophagosome fusion (Figure 1a). It is conceivable that mitochondria could at least partially be degraded to expose mtDNA to the receptor at this stage.
