Mitochondrial NAD ⁺ -mediated mitophagy alleviates type I interferon response to the cytosolic mitochondrial DNA

Mitochondrial nicotinamide adenine dinucleotide (NAD⁺) plays a central role in energy metabolism, yet its roles and mechanisms in mitophagy and innate immunity remain poorly understood. In this study, we identify mitochondrial NAD⁺ depletion that causes mitophagy dysfunction and inflammation. We find that depletion of mitochondrial NAD⁺ owing to deficiency of the mitochondrial NAD⁺ transporter SLC25A51 impairs BNIP3-mediated mitophagy. Loss of mitochondrial NAD⁺ inhibits SIRT3-mediated deacetylation of FOXO3, leading to transcriptional downregulation of <i>BNIP3</i> and subsequent disruption of MAP1LC3B/LC3B recruitment. Notably, mitochondrial NAD⁺ depletion promotes mitochondrial DNA (mtDNA) release from mitochondria to the cytosol upon oxidative stress, thereby exacerbating the type I interferon response to free cytosolic mtDNA via activation of the CGAS-STING1 signaling pathway. Our findings reveal a novel mechanistic link among mitochondrial NAD⁺, mitophagy, and mtDNA-induced inflammation by genetic manipulation of cell lines, highlighting mitochondrial NAD⁺ as a potential therapeutic target for mitigating sterile inflammation triggered by free cytosolic mtDNA. Thus, the study provides new insights into the crosstalk among mitochondrial homeostasis, inflammation, and innate immunity.<b>Abbreviations</b>: Baf A1: bafilomycin A₁; BNIP3: BCL2 interacting protein 3; CCCP: carbonyl cyanide m-chlorophenyl-hydrazone; CCL5: C-C motif chemokine ligand 5; CGAS: cyclic GMP-AMP synthase; COX4/COX-IV: cytochrome c oxidase subunit 4; CXCL10: C-X-C motif chemokine ligand 10; D-LOOP: displacement loop; EBSS: Earle's balanced salt solution; ELISA: enzyme-linked immunosorbent assay; FIS1: fission, mitochondrial 1; FOXO3: forkhead box O3; IFN: interferon; IFNB/IFNβ: interferon beta; IRF3: interferon regulatory factor 3; KO: knockout; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; mtDNA: mitochondrial DNA; NAD: nicotinamide adenine dinucleotide; MT-ND1: mitochondrially encoded NADH dehydrogenase 1; RT-PCR: real-time polymerase chain reaction; SIRT3: sirtuin 3; SLC25A51: solute carrier family 25 member 51; SoNar: sensor of NAD⁺ and NADH redox; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC2: voltage dependent anion channel 2.