Background

Enterochromaffin (EC) cells are the gut’s main source of serotonin, responding to microbial metabolites via receptors such as FFAR2/3 for short‑chain fatty acids, GPBAR1 for bile acids, and GPR142 for amino acids [1]. Microbial metabolites like isovalerate depolarize EC cells electrophysiologically, while aromatic amino acids promote 5‑HT secretion [2]. In inflammatory conditions, EC cells upregulate TPH1 and chromogranin A, shifting from sensation to secretion [3]. However, nothing is known about how normal aging alters these processes.

Hypothesis

We propose that aging induces mitochondrial dysfunction in EC cells, decreasing ATP‑dependent SERCA activity and blunting the calcium oscillations needed for vesicular 5‑HT release. Consequently, TPH1 transcription and enzyme activity remain intact, but stimulus‑evoked serotonin secretion declines with age. Simultaneously, senescent EC cells acquire a secretory‑associated phenotype (SASP) marked by elevated chromogranin A release and low‑grade inflammation, further disrupting normal serotonergic signaling.

Predictions

1. In aged mice (≥24 mo), duodenal EC cells will show unchanged TPH1 mRNA and protein levels compared with young mice (≥3 mo), but ex vivo 5‑HT release in response to SCFA (e.g., isovalerate) or aromatic amino acids will be reduced by ≥40 %.
2. Mitochondrial markers (e.g., reduced ATP, increased ROS, lowered membrane potential) will be elevated specifically in EC isolates from aged animals.
3. Pharmacological rescue of mitochondrial function (e.g., with SS‑31 peptide or NAD⁺ booster) will restore calcium transients and 5‑HT secretion in aged EC cells to youthful levels.
4. Senescence markers (p16^INK4a, SASP cytokines IL‑6, CXCL1) will be increased in EC cells from aged tissue, correlating with higher baseline chromogranin A secretion but lower evoked 5‑HT.

Experimental Approach

• Isolate EC cells from young and aged mice using fluorescent‑activated cell sorting based on chromogranin A immunoreactivity.
• Measure TPH1 expression by qPCR and Western blot; assess basal and stimulated 5‑HT release via HPLC.
• Evaluate mitochondrial health with Seahorse OCR/ATP production, MitoSOX ROS, and TMRE membrane potential.
• Use calcium imaging (Fluo‑4 AM) to compare oscillation amplitudes after FFAR2/3 activation.
• Treat aged EC isolates with SS‑31 (10 nM) or NAD⁺ precursor (NR, 1 mM) for 24 h and repeat secretion assays.
• Assess senescence via p16 immunostaining and SASP cytokine ELISA.

If predictions hold, this work would identify mitochondrial EC cell dysfunction as a mechanistic link between aging gut microbiota changes, reduced serotonergic tone, and age‑related dysmotility, offering a target for interventions.