Hypothesis

Extracellular acetate fuels nuclear ACSS2 to replenish nuclear acetyl‑CoA, which directly acetylates H3K27 at promoters of TFEB‑regulated autophagy genes, thereby reversing age‑associated epigenetic silencing and extending healthspan.

Mechanistic Rationale

• Nuclear acetyl‑CoA sustains HAT activity (e.g., CBP/PCAF) that acetylates histone lysones, a permissive mark for transcription of lysosomal biogenesis genes.
• ACSS2 uniquely converts acetate to acetyl‑CoA inside the nucleus; its chromatin tethering couples metabolite availability to histone acetylation.
• Age‑related NAD+ decline reduces SIRT1‑mediated deacetylation of cytoplasmic ACSS2, favoring its nuclear import, but falling acetate levels limit substrate supply, uncoupling the NAD+/SIRT1 axis from acetyl‑CoA production.
• Restoring acetate bypasses mitochondrial citrate export deficits and ACLY dependence, providing a substrate‑driven route to nuclear acetyl‑CoA that does not require functional TCA flux.

Testable Predictions

1. Acetate supplementation in aged mice (≥20 mo) will increase nuclear acetyl‑CoA levels by ~1.5‑fold within 48 h, measurable by subcellular fractionation and LC‑MS.
2. This rise will correlate with elevated H3K27ac at promoters of Tfeb, Lamp1, and Cathepsin genes (ChIP‑qPCR), without altering global H3K9ac.
3. Enhanced histone acetylation will drive TFEB nuclear translocation, boosting lysosomal activity (LysoSensor assay) and autophagic flux (LC3‑II/I ratio, p62 degradation) in brain and muscle.
4. Functional outcomes include improved grip strength, increased spontaneous locomotion, and delayed onset of age‑related cognitive deficits in the Morris water maze.
5. Genetic ablation of nuclear ACSS2 (CRISPR‑mediated NLS mutation) will abolish acetate‑induced H3K27ac gains and downstream autophagy improvements, confirming enzyme specificity.
6. Pharmacological inhibition of ACLY (SB‑204990) will not attenuate acetate’s effects, indicating ACLY independence when acetate is abundant.
7. In aged human peripheral blood mononuclear cells, acetate treatment will raise nuclear acetyl‑CoA and H3K27ac at TFEB targets, rescuing autophagic capacity measured by mCherry‑GFP‑LC3 reporter.

Falsifiability

If acetate supplementation fails to increase nuclear acetyl‑CoA or does not produce the predicted histone acetylation and autophagy enhancements despite confirmed nuclear ACSS2 presence, the hypothesis is refuted. Conversely, observing the outlined molecular and physiological changes supports the mechanistic link between extracellular acetate, nuclear acetyl‑CoA homeostasis, and epigenetic rejuvenation during aging.

References

• Nuclear ACSS2 chromatin association and autophagy regulation [https://pmc.ncbi.nlm.nih.gov/articles/PMC8068152/]
• NAD+‑SIRT1 control of ACSS2 localization [https://elifesciences.org/articles/47866]
• Age‑related acetyl‑CoA decline in brain [https://pmc.ncbi.nlm.nih.gov/articles/PMC12402629/]
• Nutrient‑dependent acetyl-CoA source switching in exhausted T cells [https://www.science.org/doi/10.1126/science.adj3020]

Experimental Outline

• Cohorts: young (3 mo), aged untreated, aged + acetate (1 % w/v in drinking water), aged + acetate + nuclear ACSS2 NLS mutant.
• Readouts: subcellular acetyl‑CoA (LC‑MS), ChIP‑seq for H3K27ac, RNA‑seq for lysosomal genes, functional assays (grip strength, rotarod, cognition).
• Timeline: 8‑week treatment, assessments at baseline, 4 weeks, and endpoint.

This framework directly tests whether modulating a single extracellular metabolite can rewire nuclear acetyl‑CoA pools to reset epigenetic drivers of aging.