Study on generation, source and effect of lysosomal superoxide with novel small-molecule tools

Abstract

Lysosomes, rather than simple recycle bins, now emerge as comprehensive organelles that integrate diverse signal to control multiple aspects of cell physiology (e.g., secretion, plasma membrane repairing, cell death and nutrient sensing). Superoxide (O2•−) is the primary and most upstream reactive oxygen species (ROS), which participates in physiology and pathology. Apart from mitochondria, recent findings about NADPH Oxidase 2 (NOX2) distribution and labile iron pool in lysosomes make lysosomes another potential source for endogenous ROS. However, shortage of reliable sensors that can precisely detect lysosome-localized ROS hinders the investigation of lysosomal oxidative stress. To address those issues, we developed lysosome-targeting turn-on fluorescent sensors, HKSOX-2 (Lyso) and HKSOX-2L, which are selective and sensitive to O2•−. With HKSOX-2 (Lyso), we have established two efficient and reproducible approaches (i.e., PMA challenge and autophagic stimuli) for inducing lysosomal O2•−. Unfortunately, the inappropriate subcellular distribution of HKSOX-2 (Lyso) in breast cancer cells limited its further application. We then modified the directing moiety of HKSOX-2 (Lyso) to obtain the improved lysosomal O2•− indicator, HKSOX-2L. A specific lysosome-directing ROS scavenger Lyso-NAC was also designed, based on known antioxidant N-acetylcysteine (NAC). Taking advantages of these novel molecules, we successfully proved that O2•− can be in situ generated inside lysosomes under PMA stimulation. With HKSOX-2L, we further demonstrated the involvement of lysosomal O2•− in diverse cellular models, including nutrient deprivation, anticancer drug (Dp44mT) treatment and neuronal injury, and captured significant elevation of fluorescence on multiple platforms. In particular, we studied the upstream source of lysosomal O2•− upon PMA-induced inflammation. NOX2 was identified to be responsible for lysosomal O2•− generation via pharmacological intervention and gene silencing. The distribution of NOX2 in lysosomes was then validated through lysosome enrichment and subsequent immunobloting. Moreover, the activation of lysosome-resident NOX2 was confirmed by examining serine phosphorylation of p47phox. In summary, these data strongly suggest that NOX2 does localize in lysosomes for in situ O2•− generation, raising the possibility that lysosomes have their independent oxidant sensing and modulating systems. The essential roles of lysosomes in autophagy put these organelles at the crossroads of multiple cellular processes. Global ROS elevation is well documented during autophagy, but lysosomal ROS is largely unclear. HKSOX-2L allowed us to probe the kinetics of lysosomal O2•− during starvation-induced autophagy. NOX2 was also validated to be the mediator for lysosomal O2•− production. Interestingly, we discovered that Lyso-NAC upregulated basal autophagy under starvation condition. In addition, we found that cathepsin L (CTSL) as a target for lysosomal O2•− during autophagy. Since lysosomal O2•− downregulates CTSL activity directly (oxidative modification) and indirectly (lysosome alkalinization), the removal of lysosomal O2•− by Lyso-NAC facilitates CTSL activation, induces autophagy enhancement and eventually promotes cell survival upon metabolic stress. To our knowledge, this is the first report addressing the underlying mechanism of autophagy under lysosomal oxidative stress. HKSOX-2L and Lyso-NAC are promising tools for investigation on the function of local O2•− in more lysosome-related biological contexts.