Hypothesis

Extended fasting triggers a two‑phase response in protein homeostasis: first, protective sequestration of misfolded proteins into IPOD compartments; second, lysosomal activation that solubilizes these aggregates. If lysosomal acidification and cathepsin activity lag behind autophagosome formation, the disaggregation of IPOD releases soluble oligomers that are more toxic than the original aggregates. Enhancing lysosomal function during the intermediate fasting window should shift the balance toward safe clearance and reduce oligomer‑mediated damage.

Background

• Cells sort misfolded proteins into JUNQ (soluble, ubiquitinated, degradation‑bound) and IPOD (insoluble, sequestered) compartments [1][2].
• Autophagy initiates ~18 h after food withdrawal as glycogen stores deplete [3].
• Calorie restriction or rapamycin lowers insoluble protein loads, showing aggregate clearance is pharmacologically tractable [4].
• In Alzheimer models, starvation‑induced autophagy increases autophagosome formation but fails to clear intracellular Aβ, leading to accumulation [5].

Mechanistic Insight

The proteasome‑autophagy axis and lysosomal degradation are not simultaneously engaged. Early fasting activates ULK1‑Beclin1 complexes, expanding the autophagosome pool, while lysosomal biogenesis via TFEB requires sustained mTORC1 inhibition and lysosomal acidification. A temporal mismatch creates a window where IPOD‑resident aggregates are accessed by disaggregases (Hsp110/Hsp70/Hsp40) but not yet processed by active cathepsins. The resulting fragments are soluble oligomers that expose hydrophobic surfaces, seeding further misfolding and impairing membrane integrity.

Testable Predictions

1. Oligomer surge – In neurons or yeast subjected to 12‑24 h fast, levels of SDS‑soluble, oligomer‑specific Aβ/tau (or Sup35) species will peak between 18‑22 h, coincident with LC3‑II accumulation but preceding LAMP1 lysosomal maturation.
2. Lysosomal rescue – Pharmacological elevation of lysosomal V‑ATPase activity (e.g., with MLN‑CF‑) or TFEB overexpression during the 18‑22 h window will attenuate oligomer accumulation and reduce cell death markers.
3. Disaggregase dependence – Knock‑down of Hsp110 or Hsp70 will blunt the oligomer surge despite ongoing autophagy, indicating that disaggregase‑mediated fragmentation is required for toxin release.
4. Functional readout – Synaptic vesicle recycling assays (FM dye uptake) will show acute impairment during the oligomer peak, rescued by lysosomal priming.

Experimental Approach

• Model systems: Primary hippocampal neurons from WT mice and a yeast strain expressing fluorescently tagged Sup35‑NM.
• Fasting regime: HBSS‑based nutrient withdrawal with time points at 0, 12, 18, 22, 28, 36 h.
• Readouts:
  • Filter trap and sucrose gradient sedimentation for insoluble aggregates.
  • Conformation‑specific antibodies (A11 for oligomers, OC for fibrils) quantified by dot blot and flow cytometry.
  • Live‑cell imaging of LC3‑RFP and LAMP1‑GFP to track autophagosome‑lysosome fusion.
  • Western blot for phosphorylated TFEB (nuclear translocation) and cathepsin D activity.
  • Viability assays (LDH release, caspase‑3 cleavage) and synaptic markers (synaptophysin, PSD‑95).
• Interventions:
  • MLN‑CF‑ (V‑ATPase activator) or torin1 (mTORC1 inhibitor) added at 12 h.
  • siRNA/shRNA against Hsp110, Hsp70, or TFEB.
  • Control: rapamycin alone (induces autophagy without fasting).

Potential Outcomes and Interpretation

• If oligomer levels rise specifically when LC3‑II accumulates but LAMP1 signal remains low, and lysosomal activation blunts this rise, the hypothesis is supported.
• If oligomer levels do not change with lysosomal modulation, or if disaggregase knockdown fails to affect oligomer levels, the hypothesis is falsified, suggesting alternative toxicity mechanisms.
• Demonstrating that enhancing lysosomal function converts a potentially harmful fasting interval into a net protective outcome would reframe therapeutic fasting regimens: timing lysosomal activators (e.g., TFEB agonists) to coincide with the autophagy‑lysosome uncoupling phase could maximize proteostatic benefit while minimizing proteotoxic risk.