Hypothesis

We propose that cytosolic protein aggregates in senescent T cells act as NAD+-dependent scaffolds that bind mitochondrial ROS and keep NLRP3 inflammasomes in an inactive state. When NAD+ falls and SIRT3 deacetylase activity drops, aggregates lose their protective acetylation state, expose inflammasome‑activating surfaces, and trigger caspase‑1 activation. Thus, aggregation is not merely a by‑product of proteostatic failure but a regulated switch that can be protective or pathogenic depending on the redox‑metabolic context.

Mechanistic Rationale

• Aggregates sequester misfolded proteins and damaged mitochondria, limiting their ability to activate NLRP3 (see Protein aggregates serve as adaptive cytosolic “sinks”).
• SIRT3, a mitochondrial NAD+-dependent deacetylase, acetylates lysine residues on aggregate‑associated proteins, reducing their affinity for ASC specks (hypothetical based on SIRT3’s role in ROS control).
• Declining NAD+ in aging T cells (driven by CD38 overexpression) reduces SIRT3 activity, leading to hyper‑acetylated aggregates that now nucleate NLRP3 (link to CD38 overexpression in aging T cells depletes NAD+).
• Consequently, the same aggregates that once sequestered danger signals become platforms for inflammasome assembly, creating a feedback loop that drives inflammaging.

Testable Predictions

1. In young T cells, artificially induced aggregates (e.g., via transient proteasome inhibition) will reduce NLRP3‑dependent IL‑1β release after LPS/ATP stimulation, an effect that disappears when SIRT3 is knocked down.
2. Boosting NAD+ with NR or inhibiting CD38 will preserve the anti‑inflammatory state of aggregates in aged T cells, measurable by reduced ASC speck formation and lower caspase‑1 activity.
3. Acetyl‑mimic mutants of a representative aggregate‑bound protein (e.g., HSP70) will increase NLRP3 activation, whereas deacetyl‑mimic mutants will suppress it, even in the presence of excess aggregates.
4. Imaging of aggregate–NLRP3 colocalization will show a shift from peripheral sequestration in young cells to perinuclear speck association in aged cells, reversible by NAD+ supplementation.

Experimental Approach

• Isolate naïve and memory CD8+ T cells from young (3 mo) and old (24 mo) mice.
• Treat subsets with proteasome inhibitor MG132 (2 h) to induce aggregates, with or without SIRT3 siRNA, NR (1 g/kg) or CD38 antibody.
• Measure aggregate formation (filter‑trap assay), NAD+ levels (enzymatic assay), SIRT3 activity (fluorometric deacetylase assay), NLRP3 activation (ASC speck imaging, caspase‑1 FLICA, IL‑1β ELISA).
• Perform rescue experiments expressing acetyl‑mimic (K→Q) and deacetyl‑mimic (K→R) HSP70 constructs.
• Statistical analysis: two‑way ANOVA with post‑hoc Tukey; n≥5 per group; significance set at p<0.05.

If predictions hold, the data will support aggregation as a redox‑regulated checkpoint that can be tipped toward inflammation by NAD+ loss, suggesting that preserving SIRT3 activity or NAD+ levels maintains aggregates in a harmless, sequestering state.