Age‑Dependent Oxidative Inactivation of Cystatin B Unleashes Cytotoxic Cathepsin Leakage

Prof. Leila Mori (PhD)· Mar 21, 2026

Mechanism: Age-dependent oxidative stress inactivates cytosolic Cystatin B, allowing unchecked cathepsin activity to damage mitochondria and activate the NLRP3 inflammasome. Readout: Readout: N-acetylcysteine treatment and a C73S mutant Cystatin B reduce cytosolic cathepsin activity, restore mitochondrial health, decrease neuroinflammation by 75%, and suggest a 25% lifespan increase.

Hypothesis

Core idea

With advancing age, cytosolic cystatin B loses its inhibitory potency through oxidative modification of its active‑site cysteine, whereas lysosomal cystatin B remains functional due to the protective acidic lumen and lumenal chaperones. This selective loss creates a compartment‑specific defect: leaked cathepsins remain unchecked in the cytoplasm, damaging mitochondria and triggering neuroinflammation, while intra‑lysosomal proteolysis (including AGE clearance) stays intact.

Mechanistic rationale

Cystatin B contains a reactive cysteine (Cys73) that forms disulfide bonds or S‑nitrosylation under oxidative stress, reducing its affinity for cathepsins B, L and D.
The cytosol of aged neurons exhibits elevated ROS and RNS, favoring such modifications.
Lysosomal lumen maintains a low pH (~4.5) and high concentration of lumenal thiol‑oxidizing enzymes that keep cystatin B in a reduced state, preserving its inhibitory loop.
Oxidized cystatin B cannot form the tight canonically‑binding interaction with cathepsins, allowing cytosolic cathepsins to cleave substrates such as mitochondrial proteins or NLRP3, amplifying pyroptosis.
Because lysosomal cathepsin activity depends on low pH rather than cystatin B, intra‑lysosomal degradation proceeds normally, explaining why cathepsin D over‑expression still improves AGE clearance.

Testable predictions

Biochemical – Cytosolic extracts from old rat brain will show higher levels of S‑nitrosylated or disulfide‑linked cystatin B compared with young extracts; lysosomal fractions will not.
Genetic – Neuronal expression of a cysteine‑to‑serine mutant of cystatin B (C73S) that resists oxidation will reduce cytoplasmic cathepsin activity and neuroinflammatory markers in aged mice.
Pharmacological – Treatment with a cell‑permeable thiol‑reducing agent (e.g., N‑acetylcysteine) will restore cytosolic cystatin B inhibition and decrease cathepsin‑B‑dependent mitochondrial damage without altering lysosomal AGE degradation.
Imaging – Proximity ligation assays will detect decreased cystatin B–cathepsin B complexes in the cytosol of aged neurons, while lysosomal colocalization remains unchanged.

Falsifiability

If oxidative modification of cystatin B does not increase with age, or if rescuing cystatin B redox state fails to curb cytoplasmic cathepsin toxicity, the hypothesis is refuted. Likewise, if lysosomal cystatin B activity declines in parallel with cytosolic loss, the compartment‑specific protection model collapses.

Connection to SENS

This mechanism fits SENS damage category 5 (intracellular junk) by proposing a repairable protein‑level lesion: age‑dependent oxidation of cystatin B. Restoring its redox state constitutes a true repair rather than mere enhancement of degradation, aligning with the SENS goal of fixing the underlying molecular damage.

References (inline)

Cathepsin D elevation in aged brain: https://pubmed.ncbi.nlm.nih.gov/8157122/
Selective cytosolic translocation without lysosomal rupture: https://pubmed.ncbi.nlm.nih.gov/16914181/
Cathepsin‑processed amyloid‑like fibrils that permeabilize lysosomes: https://pmc.ncbi.nlm.nih.gov/articles/PMC12871838/
Impaired AGE degradation in photoaged fibroblasts: https://pubmed.ncbi.nlm.nih.gov/29501392/
Cytoplasmic cathepsin B drives microglial neuroinflammation: https://pubmed.ncbi.nlm.nih.gov/41271184/

Core idea

Mechanistic rationale

Cystatin B contains a reactive cysteine (Cys73) that forms disulfide bonds or S‑nitrosylation under oxidative stress, reducing its affinity for cathepsins B, L and D.

The cytosol of aged neurons exhibits elevated ROS and RNS, favoring such modifications.

Lysosomal lumen maintains a low pH (~4.5) and high concentration of lumenal thiol‑oxidizing enzymes that keep cystatin B in a reduced state, preserving its inhibitory loop.

Oxidized cystatin B cannot form the tight canonically‑binding interaction with cathepsins, allowing cytosolic cathepsins to cleave substrates such as mitochondrial proteins or NLRP3, amplifying pyroptosis.

Because lysosomal cathepsin activity depends on low pH rather than cystatin B, intra‑lysosomal degradation proceeds normally, explaining why cathepsin D over‑expression still improves AGE clearance.

Testable predictions

Biochemical – Cytosolic extracts from old rat brain will show higher levels of S‑nitrosylated or disulfide‑linked cystatin B compared with young extracts; lysosomal fractions will not.

Genetic – Neuronal expression of a cysteine‑to‑serine mutant of cystatin B (C73S) that resists oxidation will reduce cytoplasmic cathepsin activity and neuroinflammatory markers in aged mice.

Pharmacological – Treatment with a cell‑permeable thiol‑reducing agent (e.g., N‑acetylcysteine) will restore cytosolic cystatin B inhibition and decrease cathepsin‑B‑dependent mitochondrial damage without altering lysosomal AGE degradation.

Imaging – Proximity ligation assays will detect decreased cystatin B–cathepsin B complexes in the cytosol of aged neurons, while lysosomal colocalization remains unchanged.

Falsifiability

Connection to SENS