Hypothesis

Lysosomal cystatin B acts as a gatekeeper that restrains cathepsin C–mediated amyloid‑induced lysosomal membrane permeabilization (LMP); age‑associated oxidative modification disrupts cystatin B lysosomal retention, unleashing a cathepsin C‑amyloid positive‑feedback loop that drives LMP, microglial neuroinflammation, and neurodegeneration.

Mechanistic Rationale

1. Oxidative modification of cystatin B – Reactive oxygen species (ROS) generated during aging cause S‑glutathionylation or disulfide‑linked crosslinking of cystatin B at conserved cysteine residues (Cys 3, Cys 26, Cys 30) (source). This alters its conformation, reducing affinity for lysosomal membrane anchors such as LAMP2A and the Hsp70 chaperone complex (source).

2. Loss of lysosomal cystatin B – Mislocalized cystatin B accumulates in the cytosol where it is less effective at inhibiting cathepsin C because the lysosomal lumen maintains a reducing environment that keeps cystatin B in its active conformation. Consequently, lysosomal cystatin B levels decline while total cellular cystatin B remains unchanged.

3. Unchecked cathepsin C activity – Free cathepsin C cleaves amyloid‑β precursors and amyloid‑β oligomers, generating positively charged fragments that electrostatically interact with lysosomal phospholipids, forming transient pores (source). The pores amplify LMP, releasing more cathepsins (especially cathepsin B) into the cytosol.

4. Feed‑forward amyloid amplification – Cytosolic cathepsin C also processes cystatin B itself, generating truncated isoforms that lack inhibitory potency, further diminishing the brake on cathepsin C activity.

5. Downstream consequences – Cytosolic cathepsin B triggers mitochondrial dysfunction and pyroptosis in microglia (source), while cathepsin D translocation to the cytosol (source) exacerbates proteolytic stress. Meanwhile, cathepsin L activity declines (source) possibly because cathepsin C‑mediated cleavage of its propeptide generates an inactive form, contributing to the observed cathepsin‑L deficit.

Testable Predictions

• Prediction 1: In aged mouse brain, lysosomal fractions will show a ≥30 % reduction in cystatin B immunoreactivity compared with young controls, while cytosolic cystatin B remains unchanged. Rescue of lysosomal cystatin B via a lysosome‑targeted CSTB construct (LAMP1‑signal peptide) will restore lysosomal cystatin B levels to >80 % of young levels.
• Prediction 2: Lysosome‑targeted CSTB overexpression will decrease cathepsin C activity in lysosomal isolates by ≥40 % (measured with a fluorogenic substrate) and reduce amyloid‑β‑induced LMP (assayed by galectin‑3 puncta formation) in primary neurons exposed to oxidative stress (H₂O₂).
• Prediction 3: Mice with lysosome‑targeted CSTB overexpression will exhibit attenuated microglial activation (Iba1⁺ cell count ↓25 %) and lower cytokine release (IL‑1β, TNF‑α ↓30 %) after chronic amyloid‑β infusion, despite comparable oxidative stress levels.
• Prediction 4: Pharmacological inhibition of cathepsin C (e.g., with GLY200) will mimic the protective effect of lysosomal CSTB overexpression, confirming that the deleterious pathway is cathepsin C‑dependent.

Falsification Criteria

If lysosomal cystatin B levels do not decline with age, or if restoring lysosomal cystatin B fails to reduce cathepsin C activity, LMP, or microglial inflammation, the hypothesis would be refuted. Conversely, confirmation would support a novel oxidative‑sensing checkpoint that couples cystatin B lysosomal retention to cathepsin C‑driven lysosomal integrity in aging.

Broader Implications

This model integrates oxidative stress, cysteine protease inhibition, and amyloid‑mediated membrane damage into a unified mechanism that explains why certain cathepsins rise (B, C, D) while L falls, and why cystatin B—despite being ubiquitous—becomes a critical liability in neurodegeneration. Targeting the lysosomal retention of cystatin B offers a tractable therapeutic avenue distinct from broad cathepsin inhibition.