Hypothesis

Aging cells shift from transient, soluble protein oligomers to stable, amyloid‑like deposits as a chaperone‑driven protective strategy. This conversion depends on the coordinated action of HSP70/HSP110 holdase activity and NAD⁺‑dependent SIRT1 signaling, which together promote the formation of inert aggregates that sequester toxic species and attenuate stress‑kinase pathways. When epigenetic reprogramming lowers NAD⁺ or reduces chaperone expression, the balance tips toward oligomer accumulation, accelerating epigenetic clock readouts. Thus, modulating the HSP70/HSP110‑SIRT1 axis can toggle aggregation between protective and pathological states.

Mechanistic Basis

1. Holdase chaperones bind exposed hydrophobic regions on misfolded proteins, preventing oligomerization and instead guiding them into β‑sheet rich fibrils that mature into IPOD/JUNQ‑like compartments.
2. SIRT1 deacetylates HSP70 and HSP110, enhancing their holdase capacity and linking aggregate formation to cellular NAD⁺ levels.
3. Elevated NAD⁺ (e.g., via NR supplementation) boosts SIRT1 activity, favoring aggregate maturation; conversely, NAD⁺ decline reduces holdase function, allowing oligomers to persist and seed pathology.
4. Protective aggregates sequester upstream activators of stress‑activated protein kinases (e.g., ASK1), dampening downstream p38/JNK signaling that otherwise drives inflammaging and epigenetic drift.
5. Epigenetic clocks such as DNAmGrimAge rise when autophagy genes (Atg5, LC3) become hypermethylated; restored chaperone‑mediated aggregation reduces the load on autophagy, permitting demethylation and clock reversal.

Predictions and Tests

• In aged mice, neuronal overexpression of HSP110 will increase insoluble, thioflavin‑S positive deposits without raising soluble oligomer levels, measured by filter trap and ELISA.
• This manipulation will lower p38/JNK phosphorylation and improve performance on rotarod and maze tasks.
• Co‑administration of a NAD⁺ booster (nicotinamide riboside) will amplify the HSP110 effect, whereas SIRT1 inhibition (EX‑527) will block deposit formation and restore oligomer toxicity.
• Chromatin profiling of sorted neurons will show reduced methylation at autophagy gene promoters correlating with deposit load.
• Flipping the experiment: knocking down HSP70 in young mice should increase soluble oligomers, elevate DNAmGrimAge, and shorten lifespan, confirming the protective role of the chaperone‑aggregate axis.

Potential Confounds

Aggregate burden may impair axonal transport; therefore, motility assays will be performed to ensure that increased deposits do not cause neurodegeneration independent of oligomer clearance. Additionally, off‑target effects of NAD⁺ boosters will be controlled with vehicle and NR‑only groups.