PARP1‑SIRT1 Axis Controls TFEB Acetylation and Tissue‑Specific Autophagy Gene Silencing in Aging

Hypothesis

Aging raises nuclear PARP1 activity, consuming NAD+ and lowering SIRT1 deacetylase capacity. Reduced SIRT1 leads to hyperacetylation of TFEB on lysine residues outside the known K274/K279 sites (e.g., K292). This modification promotes TFEB binding to the HDAC3-containing NuRD complex, which is then recruited to CLEAR motifs in the promoters of ATG5, ATG7, and ATG12. The NuRD complex compacts chromatin, blocking transcription despite TFEB nuclear presence. This mechanism explains why ATG transcription falls in tissues with high PARP1 demand (brain, liver) while other tissues retain autophagy.

Testable Predictions

1. In old vs young mouse liver and brain, PARP1 activity will be elevated, SIRT1 activity reduced, and TFEB K292 acetylation increased (detected by acetyl-lysine immunoprecipitation followed by western blot).
2. Chromatin immunoprecipitation will show increased HDAC3 and NuRD subunit (MTA2) occupancy at ATG5/7/12 promoters in aged tissue, correlating with TFEB occupancy.
3. Pharmacological inhibition of PARP1 (e.g., with olaparib) or NAD+ supplementation (NR) in aged mice will lower TFEB K292 acetylation, reduce HDAC3/NuRD promoter binding, restore ATG5/7/12 mRNA levels, and improve autophagic flux (LC3-II turnover) without triggering autosis markers (e.g., focal LC3 aggregates, plasma membrane rupture).
4. Tissue-specific PARP1 knockdown via AAV-shRNA will rescue ATG transcription only in high-PARP1 tissues (brain, liver) but not in low-PARP1 tissues (skeletal muscle), confirming the mechanistic selectivity.

Falsification

If PARP1 inhibition fails to alter TFEB K292 acetylation or HDAC3 promoter recruitment, or if ATG gene expression does not rise despite restored TFEB nuclear localization, the hypothesis is refuted. Likewise, if SIRT1 overexpression does not reduce TFEB acetylation or rescue ATG transcription, the SIRT1-dependent deacetylation step is not essential.

Relevance to Existing Work

This builds on the established GCN5-mediated TFEB acetylation (2) and mTORC1 phosphorylation (1) by introducing a NAD+-sensitive SIRT1 checkpoint that gates TFEB’s interaction with repressive complexes. It also accounts for tissue-specific autophagy decline observed across models (1) without invoking global transcriptional shutdown.

Experimental Approach

• Measure PARP1 activity (ELISA) and NAD+ levels in young (3 mo) vs old (24 mo) mouse tissues.
• Assess SIRT1 activity using a fluorogenic deacetylase assay.
• Immunoprecipitate TFEB and probe for acetyl-K292 with site-specific antibody.
• Perform ChIP-qPCR for TFEB, HDAC3, MTA2 at ATG5/7/12 promoters.
• Treat aged mice with olaparib (50 mg/kg/day) or NR (400 mg/kg/day) for 4 weeks, then evaluate LC3-II/p62 turnover by western blot and autosis via electron microscopy.
• Use AAV-shPARP1 to knock down PARP1 selectively in hepatocytes or neurons and compare ATG mRNA rescue.

Outcome will clarify whether targeting the PARP1-SIRT1-TFEB node can safely rejuvenate autophagy in aging.