Hypothesis

Chronic hexosamine biosynthetic pathway (HBP) flux promotes age‑associated protein aggregation by O‑GlcNAcylating low‑complexity domains of RNA‑binding proteins, thereby lowering their saturation concentration for liquid‑liquid phase separation and stabilizing pathological aggregates.

Mechanistic Rationale

O‑GlcNAc addition introduces a bulky, polar N‑acetylglucosamine moiety onto serine/threonine residues within intrinsically disordered regions. This modification disrupts transient π‑π and cation‑π contacts that drive reversible phase separation, effectively decreasing the critical concentration required for droplet formation. In aged tissues, sustained HBP activity—driven by elevated GFAT1 flux—produces chronic O‑GlcNAcylation of proteins such as TDP‑43, FUS, and hnRNPA1, shifting their equilibrium toward insoluble, amyloid‑prone states. This mechanism operates independently of extracellular glucose because HBP flux is regulated by nutrient‑derived fructose‑6‑phosphate and glutamine levels, not by glycemia.

Testable Predictions

1. Reducing GFAT1 activity in aged mice will decrease O‑GlcNAcylation of TDP‑43 in heart and brain, reduce its cytoplasmic aggregation, and improve contractile or cognitive performance without altering blood glucose.

2. In vitro, recombinant TDP‑43 modified with O‑GlcNAc at specific low‑complexity sites will exhibit a lower critical concentration for droplet formation and accelerated conversion to amyloid fibrils compared with unmodified protein.

3. Pharmacological O‑GlcNAc removal (using OGA activators) will rescue phase‑separation behavior and aggregation propensity of TDP‑43 in lysates from aged diabetic hearts, even when glucose remains high.

4. Tissue‑specific thresholds exist: cardiac myocytes tolerate a higher O‑GlcNAc load on metabolic enzymes before dysfunction, whereas neurons exhibit aggregation at lower modification levels due to distinct proteostasis networks.

Experimental Approach

• Generate cardiomyocyte‑ and neuron‑specific GFAT1 knockout mice aged 18‑24 months; assess O‑GlcNAc levels on TDP‑43 by immunoprecipitation‑Western blot, aggregate load by filter‑trap assay, and functional readouts (echocardiography, memory tests).
• Use CRISPR knock‑in of O‑GlcNAc‑site mutants (Ser→Ala) in TDP‑43 to confirm causality.
• Perform recombinant protein phase‑separation assays with purified OGT/OGA to modulate modification status, measuring turbidity and ThT fluorescence over time.
• Apply GFAT inhibitor DON or OGA activator Thiamet‑G to aged diabetic rodent models and monitor aggregation versus glucose tolerance.

Falsifiability

If GFAT1 inhibition fails to reduce TDP‑43 O‑GlcNAcylation or aggregation in aged tissues, or if O‑GlcNAc modification of TDP‑43 does not alter its phase‑separation behavior in vitro, the hypothesis would be refuted. Likewise, demonstrating that glucose normalization alone rescues aggregation without affecting HBP flux would challenge the claim of glucose‑independent mechanism.

Potential Impact

Establishing a direct link between HBP‑driven O‑GlcNAcylation and phase‑separation pathology would reveal a nutrient‑sensing axis that governs aging‑related proteostasis collapse, suggesting that targeting GFAT1 or OGA could mitigate multiple age‑related diseases beyond diabetes.