Mitochondrial NAD+ Fuels Cellular Cleanup That Blocks Inflammatory DNA Signals

Chronic, low-grade inflammation driven by cytosolic mitochondrial DNA (mtDNA) is increasingly recognized as a hallmark of aging and numerous age-related diseases. Understanding what keeps mtDNA safely inside mitochondria — and what happens when that containment fails — is therefore a high-priority question in longevity biology.

This study from Wuhan University focused on SLC25A51, the primary transporter responsible for importing NAD+ into the mitochondrial matrix. Using genetic knockout and overexpression models in human cell lines, the team systematically mapped the consequences of mitochondrial NAD+ depletion. They employed the SoNar fluorescent biosensor to confirm compartment-specific NAD+ changes and used oxidative stress (H2O2) as a physiologically relevant trigger for mtDNA release.

The central mechanistic finding is a linear pathway: mitochondrial NAD+ → SIRT3 deacetylase activity → FOXO3 transcription factor activation → BNIP3 expression → MAP1LC3B (LC3B) recruitment → mitophagy completion. When SLC25A51 is knocked out, intramitochondrial NAD+ falls, SIRT3 cannot deacetylate FOXO3, BNIP3 transcription drops, and LC3B fails to localize to damaged mitochondria. The result is an accumulation of dysfunctional mitochondria that, under oxidative stress, rupture and release mtDNA into the cytosol.

Once free in the cytoplasm, mtDNA is detected by the innate immune sensor CGAS, which produces cGAMP and activates STING1, TBK1, and IRF3, culminating in elevated type I interferon (IFNB/IFNβ), CCL5, and CXCL10 production. The study demonstrates that SLC25A51 knockout cells exhibit significantly greater mtDNA cytosolic accumulation and a stronger interferon signature after oxidative stress compared to wild-type controls. Importantly, rescuing mitochondrial NAD+ by restoring SLC25A51 expression or supplementing NAD+ precursors reversed the mitophagy block and attenuated the interferon response, validating the causal chain.

These findings establish mitochondrial NAD+ as a molecular gatekeeper at the intersection of mitochondrial quality control and innate immune activation. The work is particularly relevant to aging biology because mitochondrial NAD+ levels decline with age, mitophagy efficiency decreases with age, and CGAS-STING-driven inflammation is increasingly implicated in inflammaging, neurodegenerative diseases, and autoimmune conditions. Boosting mitochondrial NAD+ through NMN, NR, or other strategies may therefore simultaneously improve mitophagy and dampen sterile inflammation.