Hypothesis

Age‑related loss of cystatin B removes a key brake on cathepsin C, unleashing cathepsin C‑dependent amyloid formation that mechanically ruptures lysosomal membranes. This rupture amplifies cathepsin leakage, aggravates neuroinflammation, and creates a feed‑forward loop that accelerates lysosomal dysfunction during brain aging.

Mechanistic Basis

• Cystatin B normally associates with lysosomes and inhibits cysteine cathepsins, especially cathepsin C (3).
• Recent work shows cathepsin C‑driven amyloidogenesis causes mechanical lysosomal membrane rupture, not mere permeabilization (1).
• In aging brain, cathepsin D rises while cathepsin L activity falls despite stable protein levels (2), shifting the protease balance toward cathepsin C‑like activities.
• Declining cystatin B therefore lifts inhibition on cathepsin C, increasing amyloid seed formation on the lysosomal lumen surface.
• Growing amyloid fibrils exert physical stress on the lysosomal membrane, leading to rupture as demonstrated by CatC inhibition preventing rupture while allowing swelling (1).
• Released cathepsins (especially cathepsin B) trigger mitochondrial dysfunction and pyroptosis in microglia (6), linking lysosomal failure to neuroinflammation.
• Cells attempt compensation via SKN‑1‑mediated lysosomal enlargement (7), but this response is overwhelmed when cystatin B loss persists.

Testable Predictions

1. Genetic or pharmacological restoration of cystatin B in aged neurons will reduce cathepsin C activity, decrease amyloid‑like deposits on lysosomes, and prevent mechanical lysosomal rupture.
2. In cystatin B‑deficient models, cathepsin C inhibition will rescue lysosomal integrity and lower microglial pyroptosis markers, even if cathepsin D levels remain high.
3. Overexpression of an amyloid‑blocking peptide that interferes with cathepsin C‑mediated fibril formation will mimic cystatin B rescue, preserving membrane stability without altering cathepsin expression levels.

Experimental Approach

• Use primary cultured neurons from young and adult mice; manipulate cystatin B via AAV‑mediated overexpression or CRISPR‑based knockdown.
• Measure cathepsin C activity with a fluorogenic substrate specific for cathepsin C (e.g., Z‑Arg‑Arg‑AMC) and assess amyloid‑like seeding using a thioflavin‑T‑based lysosomal assay.
• Evaluate lysosomal membrane integrity using galectin‑3 puncta formation and lysosomal swelling versus rupture assays (LysoTracker loss vs. cathepsin release).
• Quantify microglial activation and pyroptosis (caspase‑1 cleavage, IL‑1β release) in co‑culture systems.
• Apply cathepsin C specific inhibitor (e.g., LHVS) as a rescue control.
• Validate findings in aged cystatin B heterozygous mice vs. wild‑type controls, monitoring behavior and neurodegeneration markers.

If cystatin B loss is a upstream driver of cathepsin C‑mediated lysosomal rupture, restoring its function should break the cycle of amyloid seeding, membrane damage, and neuroinflammation, offering a novel therapeutic avenue for age‑related lysosomal decline.