Hypothesis

Aging gut microbiota shifts increase luminal D‑lactate, which translocates to the brain and activates the hydroxycarboxylic acid receptor 1 (HCAR1) on astrocytes, lowering intracellular cAMP and protein kinase A (PKA) activity. Reduced PKA diminishes TFEB Ser142 phosphorylation, promoting its cytosolic retention and suppressing transcription of autophagy genes (ATG5, ATG7, BECN1) across tissues. This mechanism links microbial metabolism to the gut‑brain‑immune axis described by et al. and provides a testable node where modulating D‑lactate levels or HCAR1 signaling restores TFEB‑driven autophagy.

Mechanistic Rationale

• It's known that Lactobacillus and Bifidobacterium strains produce D‑lactate as a fermentation byproduct; their relative abundance rises with age‑associated dysbiosis (2).
• Age‑related intestinal permeability permits D‑lactate entry into circulation; plasma D‑lactate correlates with cognitive decline in humans (https://doi.org/10.1016/j.cellmet.2020.02.005).
• HCAR1 is Gi‑coupled; activation reduces cAMP, decreasing PKA‑mediated TFEB phosphorylation at Ser142, a key step for nuclear translocation (1).
• Cytosolic TFEB fails to drive CLEAR network transcription, lowering ATG5/ATG7/BECN1 mRNA in neurons, hepatocytes, and muscle, mirroring the tissue‑autonomous decline reported across multiple organs (1).
• Reduced autophagy in astrocytes exacerbates neuroinflammation, further increasing gut permeability—a vicious loop that aligns with the observed cholinergic anti‑inflammatory deficit (3).

Testable Predictions

1. Correlation: In aged mice, plasma D‑lactate levels will inversely correlate with nuclear TFEB abundance in astrocytes and with ATG5/ATG7 expression in brain and peripheral tissues.
2. Causality: Oral administration of a D‑lactate‑degrading probiotic (e.g., Lactobacillus plantarum engineered to express D‑lactate dehydrogenase) will lower plasma D‑lactate, increase astrocytic TFEB nuclear localization, and rescue autophagy gene expression.
3. Pharmacological block: HCAR1 antagonism in aged animals will mimic the effects of D‑lactate reduction, restoring TFEB activity even when plasma D‑lactate remains high.
4. Genetic validation: Astrocyte‑specific HCAR1 knockout mice will resist age‑dependent autophagy decline despite exhibiting gut dysbiosis and elevated plasma D‑lactate.
5. Human relevance: Older adults with high fecal D‑lactate will show reduced TFEB target gene expression in peripheral blood mononuclear cells; supplementation with D‑lactate‑scavenging prebiotics will normalize these markers.

Experimental Design (brief)

• Cohort: Young (3 mo) and aged (24 mo) C57BL/6 mice, plus HCAR1 floxed lines crossed with Aldh1l1‑CreERT2 for astrocyte‑specific deletion.
• Interventions: (a) D‑lactate‑degrading probiotic gavage daily for 8 weeks; (b) HCAR1 antagonist (MK‑1026) via osmotic pump; (c) vehicle controls.
• Readouts: Plasma D‑lactate (LC‑MS), intestinal permeability (FITC‑dextran), cortical astrocyte TFEB immunofluorescence (nuclear/cytoplasmic ratio), qPCR for ATG5, ATG7, BECN1 in brain, liver, blood; autophagic flux (LC3‑II/I with bafilomycin A1); behavioral assays (Morris water maze).
• Analysis: Two‑way ANOVA (age × treatment) with post‑hoc Tukey; correlation plasma D‑lactate vs TFEB nuclear index (Pearson). Significance set at p < 0.05.

If predictions hold, the hypothesis establishes a microbial metabolite–receptor axis that directly links gut aging to transcriptional autophagy failure, offering a precise intervention point distinct from broad anti‑inflammatory approaches.