NAD+-Dependent Sirtuin Activation Promotes Ordered Proteostatic Deposits as a Protective Strategy in Aging Mammalian Cells

Hypothesis

Elevating intracellular NAD+ levels enhances the formation of ordered, Hsp42‑like protein deposits that act as a protective, phase‑separated reservoir for misfolded proteins in aging mammalian cells.

Mechanistic Rationale

In yeast, Hsp42 drives the asymmetric sequestration of aggregated proteins into a single, stable deposit that retains chaperone accessibility and stress resistance 1. This process depends on liquid‑liquid phase separation, which is tuned by post‑translational modifications such as phosphorylation and arginine methylation 2 3. NAD+‑dependent sirtuins (especially SIRT2) deacetylate lysine residues on small heat‑shock proteins (e.g., HspB5/αB‑crystallin), promoting their oligomerization into amyloid‑like structures that retain dynamic exchange. We propose that NAD+ boost mimics the yeast Hsp42 pathway by activating SIRT2, thereby shifting the equilibrium from disordered aggregates to ordered deposits that safely sequester toxic species.

Testable Predictions

• NAD+ supplementation (NR or NMN) increases the fraction of cells containing Thioflavin‑T‑positive, FRAP‑slow deposits compared with untreated aged controls.
• SIRT2 inhibition or knock‑down abolishes the NAD+‑induced increase in ordered deposit formation without changing total aggregate load.
• Cells with elevated NAD+ show faster clearance of an acute proteotoxic stressor (e.g., heat‑induced luciferase aggregation) than controls, but this advantage disappears when SIRT2 is blocked.
• Genetic mimic of SIRT2‑mediated deacetylation (acetyl‑lysine to arginine mutants of HspB5) recapitulates the protective deposit phenotype even in NAD+‑deficient cells.

Experimental Approach

1. Culture primary mouse fibroblasts or iPSC‑derived neurons from old donors; treat with NR (1 g kg⁻¹ day⁻¹) or vehicle for 7 days.
2. Quantify deposit number and order using immunofluorescence for HspB5, Thioflavin‑T staining, and fluorescence recovery after photobleaching (FRAP) to assess mobility.
3. Measure total insoluble protein burden via SDS‑resistant fractionation.
4. Apply a heat shock (42 °C, 30 min) followed by a luciferase refolding assay to gauge proteostatic capacity.
5. Repeat conditions with SIRT2 inhibitor (AGK2) or SIRT2 siRNA.
6. Include rescue experiments expressing acetylation‑mimetic (K→Q) and acetylation‑defective (K→R) HspB5 mutants.

Falsifiability

If NAD+ elevation doesn't increase the proportion of ordered, immobile deposits, or if the protective effect on stress recovery persists despite SIRT2 loss, the hypothesis is falsified. Likewise, if ordered deposits form but fail to confer improved stress resistance, the proposed mechanistic link between deposit quality and proteostasis would be refuted.