Hypothesis

Transient expression of OSKM via mRNA in the myenteric plexus of aged mice will restore cholinergic enteric neuron numbers, reduce circulating phenylacetic acid, strengthen the blood‑brain barrier, and attenuate microglial neuroinflammation, thereby disrupting the self‑reinforcing gut‑brain axis that drives inflammaging.

Mechanistic Rationale

Age‑related loss of cholinergic myenteric neurons compromises gut motility and barrier integrity, permitting dysbiotic microbes to leak neurotoxic metabolites such as phenylacetic acid into the circulation. Phenylacetic acid directly induces endothelial senescence, which propagates systemic inflammation and further damages the gut epithelium. Recent work shows that brief, cyclic OSKM expression can reset epigenetic age without triggering tumorigenesis, primarily by reactivating chromatin remodeling pathways and enhancing mitochondrial function. We propose that OSKM rejuvenation in enteric neurons will:

1. Reactivate PGC‑1α‑driven mitochondrial biogenesis, improving ATP supply for neuronal maintenance and neurotransmitter synthesis.
2. Induce autophagy‑mediated clearance of damaged proteins and mitochondria, lowering the senescence‑associated secretory phenotype (SASP) in the enteric nervous system.
3. Upregulate tight‑junction proteins (claudin‑1, occludin) via improved mitochondrial ROS handling, reducing intestinal permeability.
4. Decrease microbial production of phenylacetic acid by restoring normal motility and secretory patterns, limiting substrate availability for phenylalanine‑degrading bacteria.
5. Lower circulating phenylacetic acid, thereby reducing endothelial senescence and downstream microglial activation.

Predictions & Experimental Design

1. Cellular rescue – Aged mice receiving lipid nanoparticle‑encapsulated OSKM mRNA via retrograde intestinal injection will show a ≥30% increase in ChAT‑positive myenteric neurons compared with saline controls after two weekly doses (measure by immunohistochemistry).
2. Metabolite shift – Plasma phenylacetic acid concentrations will drop by ≥25% within 7 days of the final OSKM dose (LC‑MS quantification).
3. Barrier integrity – FITC‑dextran gut permeability assay will reveal reduced flux (≤1.5‑fold of young baseline) and increased colonic claudin‑1 expression (Western blot).
4. Neuroinflammation – Brain sections will display decreased Iba‑1‑positive microglial morphology scores and lowered IL‑1β mRNA (qPCR) relative to aged controls.
5. Behavioral outcome – Treated mice will perform better in the novel object recognition test (discrimination index ↑20%), indicating improved cognition.

Controls

• Age‑matched mice receiving empty LNP.
• Young mice receiving OSKM to rule out over‑activation effects.
• Germ‑free mice receiving OSKM to test microbiota‑dependence of metabolite changes.

Potential Pitfalls & Mitigations

• Off‑target transfection: Use neuron‑specific promoters (e.g., ChAT) driving mRNA caps or incorporate miRNA‑target sites to restrict expression to cholinergic enteric neurons.
• Transient overexpression toxicity: Limit dosing to two cycles with 48‑hour intervals; monitor for weight loss or hyperplasia.
• Compensatory microbial shifts: Perform 16S rRNA sequencing pre‑ and post‑treatment to ensure phenylacetic acid reduction is not confounded by blooms of other neurotoxic taxa; if observed, adjust with targeted prebiotics.

If OSKM fails to improve any of the primary outcomes (neuron count, phenylacetic acid, barrier function, neuroinflammation, cognition), the hypothesis is falsified, suggesting that ENS rejuvenation alone is insufficient to break the gut‑brain inflammaging loop or that alternative mediators dominate the phenotype.