Hypothesis

AMPK activation under metabolic stress does not merely bulk‑remove damaged proteins; it directly promotes the formation of ordered, amyloid‑like assemblies that act as a final, reversible depot for irreversibly misfolded species. Dissolving these aggregates without restoring degradation capacity releases toxic oligomers, worsening proteotoxic stress.

Mechanistic Basis

AMPK phosphorylates low‑complexity domains of aggregation‑prone proteins (e.g., TDP‑43, α‑synuclein) at serine residues flanking prion‑like motifs. This modification shifts the phase‑behavior of these proteins from liquid droplets to hydrogel‑like condensates that nucleate β‑sheet‑rich amyloids. Concurrently, AMPK‑dependent ULK1 activation (Ser317/Ser777) initiates a selective autophagy flux that matures these condensates into insoluble, thioflavin‑positive deposits analogous to yeast IPODs. The process is reversible: Hsp104/AAA‑ ATPase disaggregases can solubilize the amyloids when autophagic flux is sufficient, allowing refolding or degradation. If disaggregation precedes adequate clearance, soluble oligomers re‑accumulate, causing synaptic dysfunction.

Testable Predictions

1. In neurons, pharmacological AMPK activation (AICAR, metformin) will increase the proportion of SDS‑resistant, thioflavin‑positive aggregates of TDP‑43 while decreasing soluble oligomeric TDP‑43 measured by FRET‑based sensors.
2. Genetic inhibition of AMPK in the same model will reduce amyloid‑like deposits but cause a proportional rise in soluble oligomers and accelerate neurite fragmentation.
3. Enhancing disaggregase activity (Hsp104 overexpression) alongside AMPK activation will prevent oligomer rebound and improve survival, whereas disaggregase overexpression alone will increase oligomer load and toxicity.

Potential Experiments

• Primary cortical neurons transfected with TDP‑43‑mCherry. Treat with AICAR (0.5‑2 mM) for 24 h, then filter‑trap assay and thioflavin‑S staining to quantify amyloid‑like species; parallel ELISA for oligomers. Include AMPKα1/α2 KO cultures as controls.
• Live‑cell imaging of Hsp104‑GFP aggregation dynamics after acute glucose starvation, with and without AMPK inhibitor (Compound C). Measure fluorescence recovery after photobleaching (FRAP) to assess material state transitions from liquid to solid.
• Fly model expressing human TDP‑43 in motor neurons. Cross with UAS‑AMPKα constitutively active line and UAS‑Hsp104 line. Assess lifespan, climbing ability, and biochemical fractions (soluble, SDS‑insoluble) by western blot.
• Rescue test: administer autophagy inducer (rapamycin) after AMPK‑driven aggregation; predict that blocking autophagy will trap toxic oligomers despite amyloid formation.

These experiments directly test whether AMPK‑mediated amyloidogenesis is a regulated, protective sequestration mechanism rather than a passive endpoint of failure. Confirmation would reshape therapeutic strategies: enhancing aggregation capacity or stabilizing the amyloid depot may be beneficial, whereas indiscriminate disaggregation could exacerbate disease.