Hypothesis

Age‑related decline in neuronal base excision repair (BER) is not solely a cell‑intrinsic process; it is actively exacerbated by a gut‑derived deficit in butyrate that promotes OGG1 promoter hypermethylation, thereby increasing mitochondrial 8‑oxoG, triggering cGAS‑STING‑dependent neuroinflammation and microglial extracellular vesicle (EV) release that further disrupts gut barrier integrity.

Mechanistic Rationale

Butyrate, a short‑chain fatty acid produced by colonic fermenters, functions as a histone deacetylase (HDAC) inhibitor and can sustain open chromatin states at loci such as the OGG1 promoter [1]. With aging, shifts in microbiome composition reduce butyrate production, diminishing this protective epigenetic tone. Loss of butyrate‑mediated HDAC inhibition allows DNA methyltransferases to methylate CpG islands in the OGG1 promoter, a change that mirrors the age‑dependent hypermethylation observed in neurons [1]. Reduced OGG1 transcription limits mitochondrial 8‑oxoG excision, leading to accumulation of oxidized guanine that activates the cGAS‑STING pathway and drives neuroinflammatory signaling [1]. Activated microglia release EVs containing inflammatory cargo that can travel via the bloodstream to the intestine, where they compromise tight‑junction proteins and increase permeability [2]. A leaky gut facilitates translocation of microbial products (e.g., LPS) that further suppress butyrate‑producing taxa, completing a vicious cycle.

Testable Predictions

1. Older mice with experimentally induced butyrate deficiency (via antibiotic depletion of fermenters or a low‑fiber diet) will show increased OGG1 promoter methylation in hippocampal neurons compared with age‑matched controls.
2. Restoring butyrate levels (through oral supplementation or gavage of a butyrate‑producing strain) will reverse OGG1 promoter methylation, elevate OGG1 protein, reduce mitochondrial 8‑oxoG, and lower cGAS‑STING activation in the same brain region.
3. Neuron‑specific OGG1 knockout will exacerbate gut barrier permeability and elevate circulating LPS, whereas rescuing mitochondrial OGG1 expression will normalize gut permeability despite an aged microbiome.
4. Blocking microglial EV release (e.g., with neutral sphingomyelinase inhibition) will attenuate gut‑barrier dysfunction in aged mice, even when butyrate remains low.

Experimental Approach

• Use 18‑month‑old C57BL/6 mice; assign to four groups: control diet, low‑fiber diet, low‑fiber + butyrate supplementation, low‑fiber + butyrate‑producing probiotic.
• Quantify OGG1 promoter methylation via bisulfite sequencing of laser‑captured hippocampal neurons.
• Measure mitochondrial 8‑oxoG with immunofluorescence and qPCR‑based lesion assay.
• Assess cGAS‑STING activation by western blot for phosphorylated TBK1 and IFN‑β mRNA.
• Evaluate gut barrier integrity using FITC‑dextran permeability assay and zonulin ELISA.
• Track microglial EVs in plasma using CD63‑positive nanovesicle counts and probe for inflammatory cargos (IL‑1β, miR‑146a).
• Include rescue experiments where neuronal OGG1 is overexpressed via AAV‑Syn‑OGG1 in the low‑fiber group.

Falsifiability

If butyrate restoration fails to modify OGG1 promoter methylation or does not reduce 8‑oxoG/neuroinflammation despite confirmed colonic butyrate elevation, the proposed epigenetic link is refuted. Similarly, if neuronal OGG1 manipulation does not alter gut permeability or microglial EV release, the feed‑forward axis lacks causal direction. Conversely, positive results across these readouts would support the hypothesis that microbiome‑derived butyrate governs neuronal BER capacity through epigenetic regulation, positioning the gut‑brain axis as a modifiable upstream driver of aging‑associated DNA repair decline.