Hypothesis: Age‑associated loss of cystatin B removes a key brake on cysteine cathepsins, allowing their unchecked activity to promote lysosomal membrane damage and to catalyze the activation of leaked aspartyl cathepsin D, thereby intensifying inflammaging and neurodegeneration.

Mechanistic rationale Cystatin B is a potent endogenous inhibitor of cysteine cathepsins (B, L, H) that normally resides in the lysosomal lumen and cytosol. Recent work shows that lysosomal membrane permeabilization (LMP) during aging is driven by oxidative stress, iron overload, lipid peroxidation and lipofuscin, creating pores that leak cathepsins into the cytoplasm 1. In retinal pigment epithelial cells, iron overload impairs cathepsin D maturation and elevates the pro/mature ratio six‑fold 2. Cytosolic cathepsin B then activates NLRP3 inflammasomes in macrophages and microglia, driving IL‑1 release, mitochondrial dysfunction and pyroptosis 3. Broad autophagy‑lysosomal failure is a hallmark of neurodegenerative disease, with cathepsin dysregulation at its core 4.

Notably, the cystatin B–cysteine cathepsin axis is absent from these discussions. We propose that declining cystatin B with age lifts inhibition on cysteine cathepsins, which then:

1. Directly destabilize lysosomal membranes by cleaving lumenal glycoproteins or lipid‑anchored proteins, enlarging pores and increasing cathepsin D efflux.
2. Process procathepsin D in the cytosol or on damaged lysosomal surfaces, converting the inactive zymogen into its active aspartyl form despite impaired luminal maturation.
3. Create a feed‑forward loop where active cathepsin D further damages membranes, amplifying cystatin‑sensitive cysteine cathepsin release. Thus, cystatin B loss indirectly augments cytosolic cathepsin D activity, linking cysteine cathepsin dysregulation to the aspartyl protease‑driven inflammaging observed in aging tissues.

Testable predictions

• Cytosolic cystatin B levels will inversely correlate with active cathepsin D (but not total cathepsin D) in aged mouse tissues (brain, liver, retina) and human post‑mortem samples.
• Genetic or pharmacological restoration of cystatin B in aged mice will reduce cytoplasmic cathepsin D activity, lower galectin‑3 puncta (LMP marker), and attenuate NLRP3‑dependent IL‑1β release without altering lysosomal biogenesis.
• Conversely, neuron‑specific cystatin B knockdown in young mice will recapitulate age‑like phenotypes: increased pro/mature cathepsin D ratio, heightened ceramides, and exacerbated neurodegeneration in models of tauopathy.
• In vitro, recombinant cathepsin B/L will cleave procathepsin D at physiological pH only when cystatin B is absent, generating detectable active cathepsin D in cytosolic extracts.

Experimental approach Measure lysosomal integrity using galectin‑3‑GFP puncta and cathepsin D activity probes in primary microglia from wild‑type, cystatin B‑heterozygous, and cystatin B‑overexpressing mice under basal and iron‑overload conditions. Rescue experiments with cell‑permeable cystatin B mimetic peptides will test sufficiency. Lipid peroxidation and iron sensors will confirm that observed effects are upstream of LMP. Statistical analysis will employ two‑way ANOVA with post‑hoc Tukey tests; effect sizes and confidence intervals will be reported to ensure falsifiability.

If cystatin B deficiency indeed potentiates cytosolic cathepsin D via cysteine cathepsin priming, enhancing cystatin B function—or blocking its downstream cysteine cathepsin activity—should break the LMP‑inflammaging cycle, offering a therapeutic angle distinct from TFEB activation or ESCRT repair.