Hypothesis

In aged gut microbiota, increased production of secondary bile acids (e.g., deoxycholic acid) activates the nuclear receptor FXR in enteric neurons, which transcriptionally upregulates Rubicon and stimulates mTORC1 via SREBP-1c–dependent lipid signaling, thereby suppressing autophagy as a protective response to limit neuronal excitability. Chronic FXR activation locks autophagy in a suppressed state, leading to accumulation of damaged proteins and organelles, contributing to enteric neuropathy and motility decline.

Testable Predictions

1. Pharmacological or genetic inhibition of FXR in aged mice will restore neuronal autophagy markers (LC3‑II, p62 degradation) and improve gut motility.
2. FXR activation in young enteric neurons (via agonist or bile acid supplementation) will recapitulate the aged phenotype: increased Rubicon, mTORC1 hyperactivity, reduced autophagosome formation, and heightened susceptibility to oxidative stress.
3. Depleting gut microbiota or bile acid synthesis (e.g., antibiotics, cholestyramine) in aged animals will lower FXR activity, decrease Rubicon expression, and rescue autophagy without altering mTORC1 upstream nutrients.
4. FXR will bind directly to the Rubicon promoter in enteric neurons; ChIP‑qPCR will show enriched FXR occupancy that increases with age.

Mechanistic Rationale

FXR is a bile acid‑activated receptor known to regulate lipid homeostasis and inflammation. In hepatocytes, FXR activation can increase mTORC1 signaling via SREBP-1c–mediated phospholipid synthesis that enriches lysosomal membranes, facilitating Rag GTPase activation. We propose a similar axis in enteric neurons: FXR drives SREBP-1c expression, boosting phosphatidylcholine and lysophosphatidic acid production, which are sensed by the lysosomal v‑ATPase‑Ragulator complex to activate mTORC1. Concurrently, FXR binds to enhancer regions of the Rubicon gene (supported by bioinformatic prediction of FXR response elements), increasing its transcription. Elevated Rubicon then sequesters BECN1, inhibiting VPS34 lipid kinase activity and blocking both autophagosome formation and fusion. This dual mechanism—mTORC1‑mediated ULK1 inhibition and Rubicon‑mediated VPS34 blockade—creates a robust, starvation‑insensitive suppression of autophagy that persists even when nutrients are low.

Experimental Approach

• Animal models: Aged (24‑month) C57BL/6 mice; FXR knockout (global or neuron‑specific Vil‑Cre;Fxr^fl/fl) and wild‑type littermates.
• Interventions: FXR antagonist (glycine‑β‑muricholic acid) or agonist (GW4064) administered orally; bile acid sequestrant (cholestyramine); microbiota depletion via broad‑spectrum antibiotics.
• Readouts: LC3‑II/I ratio, p62 levels by Western blot of isolated myenteric plexus; immunofluorescence for autophagosome markers; Rubicon mRNA and protein qPCR/Western; phospho‑S6K (mTORC1 activity); FXR ChIP‑qPCR on Rubicon promoter; gut transit time; colonic motility ex vivo.
• Expected outcomes: Inhibition of FXR or bile acid synthesis in aged mice will reduce phospho‑S6K, lower Rubicon, increase LC3‑II turnover, and improve motility to levels comparable to young controls. Conversely, FXR activation in young mice will mimic the aged suppression.

Falsifiability

If FXR manipulation does not alter Rubicon expression, mTORC1 activity, or autophagy markers in aged enteric neurons, or if bile acid depletion fails to rescue autophagy despite lowering FXR signaling, the hypothesis would be falsified. Additionally, if ChIP shows no FXR binding to the Rubicon promoter irrespective of age, the proposed transcriptional mechanism would be unsupported.

Implications

This work would reveal a microbiome‑host signaling axis that actively suppresses autophagy in the aging gut, offering a novel target (FXR or bile acid pathways) to mitigate enteric neurodegeneration and age‑related motility disorders. It also extends the concept of active autophagy suppression beyond cell‑intrinsic stressors to include extrinsic metabolic cues from the microbiota.

References

• mTORC1 hyperactivation suppresses ULK1 in aged tissues 1
• Rubicon upregulation blocks VPS34 in aged models 1
• TFEB cytoplasmic sequestration by mTORC1 2
• Senescent cells develop starvation‑insensitive mTORC1 3
• Rubicon knockdown restores autophagy and lifespan 4
• Young plasma reverses hepatic autophagy impairment 5
• Rapamycin reactivates ULK1 in senescent cells 6