Age-associated decline in ileal apical sodium‑dependent bile acid transporter (ASBT/SLC10A2) limits bile acid recycling and contributes to metabolic dysregulation, yet the upstream trigger remains unclear. We hypothesize that accumulating mitochondrial DNA (mtDNA) heteroplasmy in aged ileal enterocytes releases mtDNA fragments into the cytosol, where they activate the cGAS‑STING pathway and subsequently the NLRP3 inflammasome. This inflammasome signaling elevates IL-1β and type I interferon levels, which stimulate DNMT3A-mediated methylation of CpG islands in the SLC10A2 promoter, silencing transcription and reducing ASBT protein at the brush-border membrane. Consequently, bile acid reabsorption falls, altering the enterohepatic circulation and exacerbating systemic aging phenotypes.

Key predictions are: (1) mtDNA heteroplasmy load will positively correlate with NLRP3 inflammasome activation markers (ASC specks, cleaved caspase-1) and negatively with SLC10A2 mRNA and protein levels in ileal tissue across the lifespan; (2) pharmacological inhibition of NLRP3 or genetic ablation of STING will prevent age-dependent SLC10A2 promoter methylation and preserve ASBT function despite high mtDNA heteroplasmy; (3) targeted correction of pathogenic mtDNA mutations using a mitochondrially delivered DdCBE base editor will reduce heteroplasmy, dampen inflammasome signaling, restore SLC10A2 expression, and improve bile acid uptake in aged mice.

To test this, we will isolate ileal enterocytes from young (3 mo), middle-aged (12 mo), and old (24 mo) C57BL/6 mice, quantify mtDNA heteroplasmy by duplex sequencing, assess inflammasome activation via western blot for NLRP3, ASC, and cleaved caspase-1, measure SLC10A2 promoter methylation by bisulfite pyrosequencing, and evaluate ASBT activity using radiolabeled taurocholate uptake assays. Parallel groups will receive the NLRP3 inhibitor MCC950 or be crossed with Sting-/- mice. A separate cohort will be treated with an AAV9-mito-DdCBE construct designed to convert prevalent pathogenic mtDNA mutations (e.g., m.5024C>T) back to wild-type. Success of the hypothesis will be demonstrated if reducing mtDNA heteroplasmy or blocking inflammasome signaling rescues ASBT expression and bile acid reabsorption, whereas inflammasome activation or DNMT3A overexpression in young enterocytes reproduces the aged phenotype independent of mtDNA load. This framework directly links mitochondrial genome integrity to ileal transporter regulation, offering a mechanistic basis for the claim that mtDNA, not the nuclear genome, drives a key facet of intestinal aging.