Hypothesis

We hypothesize that intermittent rapamycin dosing creates a pulsatile mTORC1 inhibition that preserves mTORC2‑AKT‑SGK1 signaling on lysosomes, thereby sustaining lysosomal acidification and enhancing autophagic clearance of damaged macromolecules. This mechanism explains why pulses of rapamycin extend lifespan without the metabolic drawbacks seen with continuous inhibition.

Mechanistic Rationale

mTORC2 phosphorylates AKT and SGK1, which in turn phosphorylate the V‑ATPase subunits that drive lysosomal proton pumping. Maintaining lysosomal acidity is essential for autophagosome‑lysosome fusion and proteolysis. Continuous rapamycin suppresses mTORC2 in tissues with high FKBP12 expression, lowering AKT/SGK1 activity and compromising lysosomal function. Intermittent dosing allows mTORC2 reactivation during drug‑free intervals, restoring V‑ATPase activity and lysosomal pH. The resulting cycles of mTORC1 suppression (inducing autophagy initiation) followed by mTORC2‑mediated lysosomal reacidification (completing autophagic flux) create a “two‑step” clearance process that is more effective than sustained mTORC1 inhibition alone.

Novel Prediction

If this model is correct, then:

• Lysosomal pH measured in vivo will be more acidic (lower pH) under intermittent rapamycin than under continuous dosing or control.
• Autophagic flux, assessed by LC3‑II turnover with lysosomal blockade, will show higher peak activity during the drug‑free window of an intermittent schedule.
• Markers of inflammaging (serum IL‑6, TNF‑α) will be reduced to a greater extent with intermittent regimens, correlating with preserved mTORC2‑AKT signaling in macrophages and microglia.
• Genetic disruption of SGK1 in lysosome‑rich cells will abolish the lifespan benefit of intermittent rapamycin despite normal mTORC1 inhibition.

Experimental Test

1. Treat cohorts of male mice with either continuous rapamycin (2 mg/kg/day) or intermittent rapamycin (6 mg/kg once weekly) for 6 months.
2. Use LysoSensor‑Blue DND‑160 imaging of live tissue slices to quantify lysosomal pH in liver, brain microglia, and peritoneal macrophages.
3. Measure autophagic flux by administering chloroquine and tracking LC3‑II accumulation via Western blot.
4. Collect blood for cytokine ELISA and monitor survival.
5. In a parallel arm, delete SGK1 specifically in LysM‑positive myeloid cells and repeat the intermittent regimen to test necessity.

Additionally, we will measure mitochondrial membrane potential (Δψm) in the same tissues using TMRM fluorescence to test whether preserved lysosomal acidification correlates with maintained mitochondrial health, addressing the civilizational versus survival trade‑off at the organelle level.

An outcome where intermittent dosing yields lower lysosomal pH, higher autophagic flux, reduced inflammaging, and extended lifespan—while SGK1 loss abrogates these effects—would support the hypothesis. Conversely, if lysosomal acidification and autophagic flux are unchanged between regimens, the model would be falsified.