Hypothesis

Coordinated tissue-wide expansions and contractions observed during aging are triggered by discrete, oscillatory bursts of proteasome activity that reset chromatin accessibility and nucleolar phase‑state, thereby imposing stage‑specific transcriptional programs.

Mechanistic Basis

Recent single‑cell trajectory work shows nuclear proteasome concentration decline predicts yeast lifespan before mitochondrial dysfunction [1] and that mouse organs undergo simultaneous expansion‑contraction waves at specific life stages [2]. We propose that proteasome activity does not merely decline monotonically; instead, it exhibits pulsed troughs and peaks synchronized across cells. Each trough reduces degradation of specific ubiquitinated transcription factors and nucleolar proteins, allowing their transient accumulation. This accumulation drives liquid‑liquid phase separation of chromatin regulators, opening or closing accessibility domains in a coordinated fashion. The resulting transcriptional shift produces the observed cellular expansion (e.g., proliferative or secretory states) or contraction (e.g., senescent or quiescent states). Because these pulses are entrained by metabolic cues (NAD⁺/NADH ratio, ATP levels), they occur uniformly across tissues, explaining the simultaneous organ‑wide patterns.

Testable Predictions

1. Pulse Detection – Live‑cell reporters of proteasome activity (e.g., Ub‑G76V‑GFP) will reveal rhythmic fluctuations with a period matching the interval between expansion‑contraction waves in aged mouse tissues.
2. Causal Link – Pharmacologically boosting proteasome activity during predicted troughs (using low‑dose IU1 or overexpressing PA28γ) will suppress the ensuing expansion/contraction shift and restore youthful bifurcating trajectories in hair follicle stem cells [5].
3. Transcriptomic Signature – RNA‑velocity vectors will point away from aged states toward younger branches only when proteasome activity is experimentally elevated at the trough phase, measurable as increased Kendall’s τ between pseudotime and chronological age [3].
4. Lifespan Impact – Mice receiving timed proteasome‑enhancing interventions will show delayed onset of molecular hallmarks (mitochondrial ROS, DNA methylation age) and extended median lifespan compared to continuous treatment or controls.

Experimental Design

• Monitoring: Generate Ub‑G76V‑GFP knock‑in mice; perform intravital two‑photon imaging of liver, muscle, and skin across the lifespan to quantify fluorescence oscillations. Correlate peaks/troughs with scRNA‑seq expansion/contraction markers identified in [2].
• Intervention: Administer IU1 (0.5 mg/kg) or vehicle via osmotic pump synchronized to detected trough windows (determined by real‑time reporter feedback). Control groups receive continuous IU1 or vehicle.
• Readouts: At 3, 6, 12 months, collect tissues for scRNA‑seq + RNA velocity, ATAC‑seq for chromatin accessibility, and proteomics for ubiquitinated substrates. Compute pseudotime with psupertime [3] and compare trajectory shapes to youthful controls.
• Outcome Metrics: Shift in Kendall’s τ, proportion of cells occupying youthful bifurcating branches, mitochondrial membrane potential (TMRE), and survival curves.

Potential Outcomes and Falsifiability

If proteasome pulses drive stage‑specific transitions, we expect:

• Detectable, synchronous oscillations in proteasome reporter signal preceding expansion/contraction waves.
• Timed enhancement to reduce expansion/contraction amplitude, increase youthful trajectory occupancy, and raise Kendall’s τ toward 0.9.
• Lifespan extension only in the timed‑treatment group, not in continuous or vehicle groups. Failure to observe rhythmic proteasome activity, or lack of effect from timed enhancement despite adequate target engagement, would falsify the core claim that proteasome pulses are the upstream trigger of coordinated aging transitions.

Significance

This hypothesis links molecular proteostasis dynamics to tissue‑scale morphometric changes, offering a mechanistic explanation for why aging appears stage‑based rather than linear. It also suggests a precise therapeutic window: interventions aimed at boosting proteasome function at predicted troughs could redirect cells away from pathogenic trajectories toward regenerative, youthful states.