PROJECT SUMMARY: Mitochondria are the evolutionary product of the endosymbiosis between the ancestral eukaryotic cell and an obligate aerobe bacterium, which brought new functionalities to eukaryotic cells. During their evolution, mitochondria transferred more than 99% of their known genetic material to the nucleus – with the exception of a small, multi-copy genome referred to as mtDNA. In Homo sapiens, the 16.6 Kbp of mtDNA is circular and encodes for 13 members of the oxidative phosphorylation chain (OXPHOS) and other structural RNAs. Beyond their canonical role in the generation of ATP through OXPHOS, the mitochondria are also critical stakeholders in several cellular processes from metabolite fluxes and calcium signaling to cell death and aging. Recently, mitochondrial stress has been implicated in the aberrant activation of innate immunity, mediated by the release of organellar components, such as mitochondrial nucleic acids, recognized by cytosolic sensors as foreign and potentially dangerous. Aberrant immunity has deep implications in human health and is a driver of human diseases, such as neuroinflammation and autoimmunity. Mitochondrial immunity has been studied both in vitro and in vivo, but the complete repertoire of its triggers and effectors has yet to be characterized. I recently uncovered a previously unknown source of stress conducive to aberrant immunity: stress to the mtDNA in the form of double-stranded breaks (mtDSBs). The presence of this stressor is relayed to the cytosolic compartment via mitochondrial herniation, a recently described form of Bax/Bak mediated organelle permeabilization, that exposes mitochondrial contents to the cytosol. After mtDSBs, mitochondrial RNA – rather than the recipient of the stress, mtDNA – initiated the innate immunity cascade by activating the sensor RIG-I. Our proposed research plan builds on this past work to ask essential questions: (1) which sources of mtDNA stress are conducive to aberrant immunity and what is the impact of dysfunctional mitochondrial transcription or translation; (2) how is mitochondrial herniation regulated and how does it differ from other forms of mitochondrial permeabilization; and (3) what are the distinctive features of mitochondrial RNA activation of RIG-I and where do they originate? Our goal is to understand how mitochondria integrate and translate stress signals, particularly in the context of innate immunity. My lab will probe different sources of mtDNA stress and interrogate the mechanisms, effectors, and mitochondrial moieties engaging the cytosolic sensors of immunity. RIG-I is a key protein for the defense against viruses, but its aberrant activation is involved in both autoimmune conditions and the beneficial anti-tumor responses elicited by cancer treatments. By building upon our recent findings and prior experience, as well as by establishing collaborations and seeking the assistance of senior investigators with advanced expertise in both mitochondrial...