NAD⁺ supplementation reduces mitochondrial adenine methylation (6mA) to limit heteroplasmy-driven inflammation in aging

Aging is marked by falling nuclear NAD⁺, which dampens SIRT1 activity and skews the NAD⁺/NADH ratio toward NADH (NAD⁺ decline reduces SIRT1 activity). This shift impairs sirtuin‑mediated deacetylation of histones and mitochondrial proteins, while simultaneously altering TCA‑cycle flux and lowering α‑ketoglutarate (α‑KG) relative to succinate. α‑KG is a required cofactor for the Fe(II)/α‑KG‑dependent demethylase ALKBH1, which removes N⁶‑methyladenine (6mA) from mitochondrial DNA. When NAD⁺ declines, the α‑KG/succinate ratio falls, ALKBH1 activity wanes, and 6mA accumulates on the mtDNA strand. Elevated 6mA interferes with TFAM binding and promotes error‑prone mtDNA replication, accelerating the rise of heteroplasmic single‑nucleotide variants after age 70. The resulting increase in cytosolic chromatin fragments from damaged mtDNA fuels cGAS/STING signaling unless cleared by mitophagy.

We hypothesize that restoring NAD⁺ with precursors such as nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN) re‑elevates the α‑KG/succinate ratio, reactivates ALKBH1‑mediated demethylation of mtDNA 6mA, and thereby improves replication fidelity. Lower 6mA should diminish heteroplasmy load, reduce mtDNA‑derived cytosolic DNA, and attenuate cGAS/STING‑dependent interferon production. Consequently, NAD⁺‑precursor treatment would decrease inflammatory senescence markers while enhancing mitophagy through FOXO3‑NIX/Parkin pathways (NAD⁺ precursors restore mitophagy via FOXO3‑NIX and Parkin).

To test this, we will treat senescent human mesenchymal stem cells (MSCs) from donors >70 years with 1 mM NR or NMN for 14 days. Parallel cultures receive vehicle. We will quantify mtDNA 6mA using dot‑blot immunoprecipitation followed by qPCR of mtDNA loci, assess heteroplasmy by single‑cell mtDNA sequencing, measure cGAS/STING activation via phospho‑TBK1 and IFN‑β ELISA, and evaluate mitophagy flux with mt‑Keima assay. We predict that NR/NMN‑treated cells will show a ≥30 % drop in mtDNA 6mA, a comparable reduction in heteroplasmic variant frequency, and a ≥40 % decrease in IFN‑β secretion relative to controls. If NAD⁺ precursors fail to alter 6mA levels or heteroplasmy despite raising total NAD⁺ (Treatment with NAD⁺ precursors increases intracellular NAD⁺ levels and rejuvenates senescent human mesenchymal stem cells), the hypothesis is falsified, indicating that NAD⁺‑dependent redox changes do not gate ALKBH1 activity in mitochondria.

A secondary aim examines sex‑specific effects. Preliminary data suggest female MSCs retain higher basal ALKBH1 expression; we will therefore compare male and female cultures to determine whether NAD⁺‑precursor efficacy differs by sex, which could inform timing of interventions before the heteroplasmy threshold is crossed.

Linking NAD⁺ metabolism to mitochondrial epigenetics provides a mechanistic bridge between metabolite depletion and mtDNA quality control, offering a clear, falsifiable route to refine anti‑aging strategies.