Hypothesis

Aggregates function as intracellular sinks that sequester not only misfolded proteins but also endogenous nucleic‑acid ligands that would otherwise activate the cGAS‑STING and NLRP3 inflammasome pathways Protein aggregates act as protective sinks. When proteostasis collapses, the cell diverts dangerous species into highly ordered, thermodynamically stable deposits Specialized backup systems sequester aggregates in specific tissues. This sequestration reduces the pool of cytosolic DNA/RNA that can trigger innate immune sensors, thereby limiting SASP amplification Aggregation prevents chaperones from being sequestered in futile cycles. Disaggregating these deposits without restoring proteostasis releases both protein toxins and nucleic‑acid danger signals, provoking a surge of inflammasome activity and exacerbating inflammaging Skin‑derived ApoE forms aggregates with bacterial toxins to aid clearance. Senolytics break the cycle by eliminating senescent cells that are the primary source of cytosolic nucleic‑acid release (e.g., mitochondrial DNA), lowering the inflammasome trigger load and allowing the protective aggregate compartmentalization to persist without causing secondary inflammation Proteostasis collapse is an early molecular event in aging that triggers cellular senescence; Senescent cells induced by proteotoxic stress release SASP factors; Disrupting aggregates without resolving underlying proteostasis collapse releases toxic misfolded proteins and amplifies harm.

Testable Predictions

1. In models of proteotoxic stress (e.g., express mutant Huntington exon‑1 or tau‑P301L), chemical disaggregation (using Hsp104 overexpression or small‑molecule dissolvers) will increase cytosolic dsDNA levels and caspase‑1 activation compared with stressed controls, even when aggregate load is reduced.
2. Co‑treatment with a senolytic (dasatinib + quercetin) will blunt the disaggregation‑induced rise in inflammasome markers, despite persistent aggregates, because senolytics reduce mitochondrial DNA release from senescent cells.
3. Genetic ablation of cGAS or NLRP3 in neurons will protect against disaggregation‑induced toxicity, rescuing synaptic function without altering aggregate burden.
4. Longitudinal imaging of aggregate‑containing cells will show a negative correlation between aggregate volume and cytosolic mtDNA specks; senolytic treatment will shift this relationship toward lower mtDNA specks at comparable aggregate levels.

Experimental Approach

• Cell models: Primary mouse cortical neurons transfected with aggregation‑prone constructs; iPSC‑derived microglia.
• Interventions: (a) Aggregation inducer (e.g., arsenite); (b) Disaggregator (Hsp104‑AA or small molecule AR‑12); (c) Senolytic (D+Q).
• Readouts: Filter‑trap assay for aggregates; PicoGreen for cytosolic DNA; Western blot for cleaved caspase‑1, IL‑1β; ELISA for SASP cytokines; Seahorse for mitochondrial ROS; live‑cell imaging of mtDNA‑GFP specks.
• Controls: Non‑targeting scrambles, vehicle, and cGAS/NLRP3 knock‑down via siRNA.

Potential Outcomes and Interpretation

If disaggregation raises cytosolic nucleic‑acid sensors and inflammasome activity, and senolytics suppress this rise, the hypothesis gains support: aggregates act as a protective buffer against nucleic‑acid‑driven inflammation. Failure to observe increased inflammasome signaling upon disaggregation would falsify the core claim that aggregates sequester immune‑activating ligands.

Broader Implications

This reframes anti‑aggregate therapies: rather than dissolving deposits, strategies should aim to stabilize the sequestered state while removing the senescent‑cell source of inflammasome ligands. Combinatorial regimens pairing proteostasis enhancers with senolytics may preserve the proteome’s last‑ditch order‑making effort while curbing the inflammatory feedback loop that drives neurodegeneration.