Disruption of ATG9A-dependent basal autophagy causes an accumulation of ubiquitin-rich condensates which act as a platform for inflammatory signaling.

Excessive inflammation underlies many human diseases, such as neurodegeneration, autoimmune disorders, cancer, and COVID-19. Thus, cellular mechanisms that control inflammation are of high therapeutic interest. Autophagy has been implicated in the suppression of inflammation, yet mechanistic links between autophagy and inflammation are not completely understood. Previous work demonstrated that loss of ATG9A, but not ATG5 or ATG7, increased inflammatory signaling through the STING-IRF3 cascade, suggesting that perhaps an autophagy-independent function of ATG9A regulates inflammation. Here, we show that loss of the essential basal autophagy regulators ATG9A and ATG101 sensitizes cells to dsDNA-induced IRF3 activation. Importantly, this effect was not observed with the loss of ATG5 or ATG7, suggesting that the canonical LC3-lipidation autophagy machinery is not required for suppression of IRF3 and inflammatory signaling. In an effort to understand the role of ATG9A and ATG101 in suppressing inflammatory signaling, we found that loss of ATG9A and ATG101, but not ATG5 or ATG7, caused an accumulation of ubiquitin-rich condensates. We also found that the accumulation of ubiquitin-rich condensates coincided with an overactivation of the ubiquitin-sensing, IRF3-targeted kinase TBK1. Importantly, we found that inhibiting the accumulation of ubiquitin-rich condensates via knock-out of p62/SQSTM1 abrogated the increased inflammatory signaling caused by loss of ATG9A. Together, our data suggest a model in which the loss of basal autophagy machinery causes an accumulation of ubiquitin-rich condensates, which, in turn, act as a platform for the aberrant activation of TBK1-mediated inflammatory signaling. We propose that these data have important implications for diseases of protein aggregation wherein similar ubiquitin-rich condensates form and aberrant inflammatory signaling plays a pathological role. REFERENCES: (1) Saitoh, T.; Fujita, N.; Hayashi, T.; Takahara, K.; Satoh, T.; Lee, H.; Matsunaga, K.; Kageyama, S.; Omori, H.; Noda, T.; Yamamoto, N.; Kawai, T.; Ishii, K.; Takeuchi, O.; Yoshimori, T.; Akira, S. Atg9a Controls DsDNA-Driven Dynamic Translocation of STING and the Innate Immune Response. PNAS 2009, 106 (49). https://doi.org/10.1073/pnas.0911267106. (2) Kannangara, A. R.; Poole, D. M.; McEwan, C. M.; Youngs, J. C.; Weerasekara, V. K.; Thornock, A. M.; Lazaro, M. T.; Balasooriya, E. R.; Oh, L. M.; Soderblom, E. J.; Lee, J. J.; Simmons, D. L.; Andersen, J. L. BioID Reveals an ATG9A Interaction with ATG13-ATG101 in the Degradation of P62/SQSTM1-ubiquitin Clusters. EMBO reports 2021, 22 (10). https://doi.org/10.15252/embr.202051136.