Chronic mtDNA‑cGAS‑STING signaling locks autophagy suppression in aging via Rubicon upregulation and mTORC1 hyperactivity

Chronic cytosolic mtDNA activates the cGAS‑STING pathway in aged cells, driving IRF3‑dependent transcription of the autophagy inhibitor Rubicon (RUBCN) and sustaining mTORC1 activity through PKR‑mediated eIF2α phosphorylation. This creates a self‑reinforcing loop that suppresses autophagy initiation and lysosomal biogenesis, even when nutrient signals would normally induce it. The loop represents a short‑term adaptive response to limit mtDNA‑triggered inflammation but becomes maladaptive, allowing damaged molecules to accumulate. Restoring autophagy by breaking the cGAS‑STING‑Rubicon axis should re‑engage TFEB‑driven lysosomal gene expression and improve cellular homeostasis. ### Mechanistic Basis - mtDNA released from dysfunctional mitochondria accumulates in the cytosol of senescent and aged tissues [1]. - Cytosolic mtDNA binds cGAS, producing 2’3’‑cGAMP that activates STING, leading to TBK1‑IRF3 signaling [6]. - IRF3 directly promotes transcription of RUBCN, as shown by IRF3‑binding motifs in the Rubicon promoter (novel prediction). - Simultaneously, STING‑activated PKR phosphorylates eIF2α, which enhances mTORC1 signaling by reducing AMPK activity and increasing Raptor stability [2]. - Elevated mTORC1 keeps TFEB phosphorylated and cytoplasmic, blocking lysosomal gene transcription [3][4]. - Rubicon binds the BECN1‑VPS34 complex, inhibiting autophagosome nucleation [1]. - Together, these mechanisms create a bistable switch that maintains low autophagy despite upstream starvation signals. ### Testable Predictions 1. In aged mouse liver and muscle, IRF3 chromatin occupancy at the Rubicon locus will be higher than in young tissues. 2. Pharmacological inhibition of cGAS or STING will reduce Rubicon protein levels, decrease p‑S6K (mTORC1 read‑out), and increase nuclear TFEB. 3. Genetic knockout of Sting in aged mice will rescue autophagic flux (measured by LC3‑II turnover with bafilomycin) without exacerbating systemic inflammation. 4. Supplementation with NAD+ boosters will not restore autophagy unless the cGAS‑STING‑Rubicon axis is concurrently inhibited, indicating that the suppression is upstream of metabolic sensing. ### Experimental Approach - Isolate primary hepatocytes from young (3 mo) and aged (24 mo) mice; perform IRF3 ChIP‑qPCR for the Rubicon promoter. - Treat aged hepatocytes with the cGAS inhibitor RU.521 or STING antagonist C‑176; measure Rubicon by western blot, p‑S6K, TFEB localization (immunofluorescence), and LC3‑II flux. - Generate liver‑specific Sting‑KO aged mice; assess autophagy markers, p62 accumulation, and histological signs of damage. - Combine NAD+ precursor (NR) with cGAS inhibition to test for additive effects on TFEB target gene expression (Lamp1, Ctsb). If predictions hold, the data will support a model where chronic mtDNA‑cGAS‑STING signaling actively enforces autophagy suppression in aging, shifting the view from passive failure to an active, inflammatory‑driven blockade.