Hypothesis

Transient, inducible expression of Yamanaka factors (OSKM) specifically in the enteric nervous system (ENS) of aged mice will rejuvenate enteric neurons, restore normal gut motility and barrier function, thereby correcting age‑associated gut dysbiosis and reducing circulating microbial metabolites that drive neuroinflammation. If the gut‑to‑brain axis is dominant, rescuing the ENS should break the inflammaging cascade and improve cognitive performance without direct brain reprogramming.

Mechanistic Rationale

• Age‑related loss of enteric neurons and altered autonomic signaling precedes central pathology in Parkinson’s models [3].
• Enteroendocrine cells secrete hormones that modulate systemic aging in Drosophila [4]; similar vertebrate cells likely influence immune tone.
• Transient OSKM expression rejuvenates liver, spleen and blood phenotypes [5]; we posit that the same epigenetic reset applies to enteric glia and neurons, restoring neurotrophic factor release (e.g., GDNF) that supports microbiota‑friendly environments.
• A healthy ENS enhances mucosal immunoglobulin secretion and regulates epithelial tight junctions, limiting LPS translocation.

Testable Predictions

1. Cellular level – After doxycycline‑induced OSKM pulse (2 days/week for 4 weeks), aged mice will show increased HuC/D+ and Sox10+ cell counts in the myenteric plexus compared with controls.
2. Functional gut readouts – Colonic transit time will normalize, fecal lipocalin‑2 will drop, and intestinal permeability (FITC‑dextran assay) will decrease to youthful levels.
3. Microbiome shift – 16S rRNA sequencing will reveal increased Lactobacillus/Bifidobacterium ratios and decreased Bacteroides‑derived LPS genes.
4. Systemic inflammation – Serum phenylacetic acid, IL‑6, and TNF‑α will be reduced proportionally to microbiome changes.
5. Neuroimmune outcome – Microglial Iba1 activation in hippocampus and cortex will decline, and performance in the Morris water maze will improve to young adult levels.
6. Specificity – Delivering OSKM to hepatocytes or brain neurons under the same regimen will not reproduce the gut‑centric improvements, confirming the ENS as the critical node.

Experimental Design (brief)

• Use Rosa26‑LSL‑OSKM;Advillin‑CreERT2 mice for ENS‑specific, tamoxifen‑inducible OSKM expression.
• Cohorts: young (3 mo), aged untreated (24 mo), aged OSKM‑induced, aged vector control.
• Induce OSKM for 4 weeks, then wash‑out 4 weeks before analysis.
• Collect tissues for immunohistochemistry, motility assays, FITC‑dextran permeability, metabolomics, 16S sequencing, flow cytometry of immune cells, and behavioral testing.

Falsifiability

If OSKM activation in the ENS fails to improve any of the gut functional readouts (transit, permeability, microbiome composition) and does not reduce systemic inflammatory markers or neurogliosis, the hypothesis is falsified. Conversely, if improvements occur only when OSKM is expressed in non‑enteric tissues, the directional claim that gut‑to‑brain signaling is primary would be weakened.

Potential Impact

Demonstrating that neural rejuvenation can reshape the gut microbiome would invert the prevailing top‑down bias in longevity research, suggesting that enteric neurogenesis is a leverage point for breaking the inflammaging loop and delaying cognitive decline.