In aged cells, lipid peroxidation-derived electrophiles such as 4‑HNE and MDA do not merely inhibit autophagy enzymes; they drive the formation of reversible, hydrogel‑like cytoplasmic aggregates that sequester core autophagy proteins (ATG5, ATG7, BECN1, LC3‑II) and autophagy‑initiating kinases (ULK1 complex). This sequestration acts as a tunable ‘brake’ that dampens autophagic flux to prevent excessive self‑digestion when proteostatic capacity is compromised, representing an active, adaptive suppression rather than passive failure.

Mechanistic Basis

1. Adduct‑induced phase separation – 4‑HNE and MDA form Michael adducts on lysine residues of ATG proteins, increasing their hydrophobicity and promoting low‑complexity domain interactions akin to stress‑granule formation. These modifications lower the saturation concentration for phase separation, leading to micron‑sized assemblies that retain enzymatic activity but restrict substrate access.
2. Regulation by redox‑sensitive chaperones – Hsp70 and Hsp110 bind adducted ATG proteins, modulating aggregate size and dynamics. In young cells, chaperone activity keeps aggregates transient; with age, chaperone capacity declines, stabilizing the assemblies.
3. Feedback to mitochondrial ROS – By limiting mitophagy, damaged mitochondria accumulate, elevating ROS and perpetuating lipid peroxidation, thus reinforcing aggregate formation—a self‑limiting loop that prevents catastrophic autophagy activation.
4. Reversibility – Hydrazine‑based scavengers (e.g., hydralazine) or reductants (e.g., N‑acetylcysteine) can reverse adducts, dissolving aggregates and restoring flux without inducing apoptosis, distinguishing this brake from irreversible damage.

Testable Predictions

• P1: In aged tissues, a detectable fraction of ATG5/ATG7/BECN1 will co‑localize with markers of cytoplasmic hydrogels (e.g., FUS, TIA‑1) in a 4‑HNE‑dependent manner.
• P2: Genetic reduction of Hsp70 ATPase activity will increase aggregate size and further suppress autophagy, whereas overexpression will disperse aggregates and rescue flux.
• P3: Acute treatment with a cell‑permeable 4‑HNE scavenger will rapidly decrease aggregate formation, increase LC3‑II turnover, and reduce mitochondrial ROS within 6 h.
• P4: Forced expression of aggregation‑resistant ATG5 (lysine‑to‑arginine mutants at major 4‑HNE sites) will maintain autophagy flux in old cells despite high lipid peroxidation.
• P5: Inhibition of autophagy (e.g., ATG5 KO) in young cells will phenocopy the aged aggregate profile, indicating that autophagy suppression drives, rather than follows, aggregate accumulation.

Experimental Approach

• Imaging: Proximity ligation assay (PLA) combined with immunofluorescence for 4‑HNE and ATG proteins; super‑resolution microscopy to assess aggregate morphology.
• Biochemical: Sequential detergent fractionation to quantify soluble vs. insoluble ATG pools; mass spectrometry to map adduct sites.
• Functional: Flux assays using mCherry‑GFP‑LC3 and mitochondrial‑Keima; ROS measurement with MitoSOX.
• Genetic: CRISPR‑knockin of aggregation‑resistant ATG alleles in mice; chaperone overexpression via AAV.
• Pharmacological: Dose‑response curves for hydralazine, NAC, and newly designed HNE‑traps.

Potential Outcomes and Interpretation

• If P1‑P4 are confirmed, the data support a reversible, adduct‑driven hydrogel mechanism that actively tunes autophagy, redefining age‑related decline as a adaptively regulated state.
• If aggregates form but scavenging fails to restore flux, alternative irreversible damage (e.g., protease cleavage) dominates, falsifying the hydrogel brake hypothesis.
• If chaperone manipulation does not affect aggregate size or autophagy, the proposed Hsp70/Hsp110 regulatory layer is incorrect, prompting exploration of other regulators (e.g., small HSPs, ubiquitin‑dependent segregation).

This hypothesis links lipid peroxidation chemistry to biomolecular phase separation, offers a concrete, falsifiable framework, and suggests therapeutic strategies that target adduct reversal or aggregate dynamism to rejuvenate autophagic capacity in aging.