Hypothesis

In aged, normoglycemic tissues, elevated hexosamine biosynthetic pathway (HBP) flux drives O-GlcNAcylation of the transcription factor TFEB, which blocks its nuclear import and lysosomal gene expression, thereby suppressing autophagosome‑lysosome fusion and flux. This mechanism explains autophagy stall as an active metabolic suppression, not just passive wear‑and‑tear.

Mechanistic rationale

• HBP flux rises with age due to increased glutamine uptake and altered glycolytic shunting, producing more UDP‑GlNAc even when blood glucose is normal.
• O-GlcNAc transferase (OGT) modifies TFEB on serine/threonine residues that overlap with phosphorylation sites required for its interaction with importins and 14‑3‑3 release.
• O-GlcNAcylated TFEB exhibits higher affinity for cytoplasmic 14‑3‑3 proteins, sequestering it away from the nucleus, reducing transcription of lysosomal genes (e.g., LAMP1, CATD) and autophagosome‑lysosome tethering factors.
• Reduced lysosomal biogenesis limits the availability of functional lysosomes, causing autophagosomes to accumulate despite intact upstream initiation—mirroring the SNAP29‑dependent block described in diabetic cardiomyocytes[1].
• The effect is nutrient‑sensitive and evolutionarily conserved, as O-GlcNAc cycling modulates autophagy in C. elegans independent of hyperglycemia[2], and OGT loss alleviates proteotoxicity via enhanced autophagy[3].
• Concurrently, lowered O-GlcNAc diminishes mTORC1 activity[4], creating a feedback loop where O-GlcNAc both directly blocks TFEB and indirectly sustains mTORC1‑mediated autophagy inhibition.

Testable predictions

1. Aged mouse liver and brain will show increased O-GlcNAc‑TFEB levels and decreased nuclear TFEB under euglycemic conditions compared with young controls.
2. Genetic or pharmacological reduction of O-GlcNAc (OGT knock‑down, OGA over‑expression, or GFAT inhibition) in aged tissues will restore TFEB nuclear localisation, boost lysosomal gene expression, and increase autophagic flux (measured by LC3‑II turnover and p62 degradation).
3. Mutating the O-GlcNAc acceptor sites on TFEB to alanine will prevent age‑dependent autophagy suppression, whereas mimicking O-GlcNAc (Asp/Glu) will reproduce the block even in young cells.
4. The autophagic defect will be rescued by lysosomal overexpression (e.g., LAMP1) despite persistent O‑GlcNAc‑TFEB, indicating that the block operates upstream of lysosome availability.

Experimental approach

• Cohorts: Young (3 mo) and aged (24 mo) C57BL/6 mice maintained on standard chow; fasting glucose confirmed normal.
• Readouts: Western blot for O-GlcNAc‑TFEB (using O‑GlcNAc antibody after TFEB IP), subcellular fractionation for nuclear vs cytoplasmic TFEB, qPCR for lysosomal genes, LC3‑II/p62 flux with bafilomycin A1, electron microscopy for autophagosome‑lysosome contacts.
• Interventions: AAV‑shOGT or AAV‑OGA in liver; CRISPR knock‑in of TFEB‑S/A mutants; GFAT inhibitor (azaserine) administered via drinking water.
• Controls: Scrambled shRNA, vehicle, and diabetic (high‑fat‑high‑sugar) mice to contrast hyperglycemia‑dependent effects.

Potential outcomes and falsification

• If aged tissues show no rise in O-GlcNAc‑TFEB or nuclear TFEB remains unchanged, the hypothesis is falsified.
• If OGT inhibition fails to increase autophagic flux despite lowering global O-GlcNAc, alternative suppression mechanisms dominate.
• If TFEB mutants resistant to O-GlcNAc still exhibit autophagy decline, the block likely acts elsewhere (e.g., on SNAP29 or other fusion proteins).

Implications

Demonstrating that a nutrient‑driven O‑GlcNAc switch actively disables the lysosomal transcriptional program reframes autophagy decline in aging as a reversible metabolic state. It suggests that targeting HBP flux or O‑GlcNAc cycling could restore clearance capacity without requiring glycemic control, offering a geroprotective strategy distinct from mTOR inhibition alone.