Cysteine Proteases as Therapeutic Targets for Neurodegenerative Diseases

The calpain-cathepsin hypothesis (CCH) predicates elevation of calpain-1 (CAPN1) and cathepsin-B (CTSB) as an underlying mechanism in the pathogenesis of Alzheimer’s disease (AD) and related dementia, traumatic brain injury (TBI), and ischemic stroke. The hypothesis is supported by studies with small molecule inhibitors, such as NYC-438, that reduce cognitive deficits in AD mouse models. Though they display efficacy, NYC-438, a nonselective CAPN1/CTSB inhibitor and selective CAPN1 inhibitors reported in the literature exhibit poor brain bioavailability. We hypothesized that the CCH could account for dysfunction of the blood-brain barrier (BBB) and, in particular, brain endothelial cell (BEC) dysfunction. To test this theory and further characterize selective vs nonselective targeting of CAPN1 vs CTSB, we developed selective small molecule inhibitors, and characterized their neuroprotective efficacy in in vitro ischemia-reperfusion injury and neuroinflammatory attenuation in an in vivo mTBI mouse model of oxidative-stress (OS). Various inhibition strategies provided the expected dose-dependent neuroprotection in primary neurons and mitigated the post-mTBI neuroinflammatory surge seen in the OS-mice. Furthermore, we revealed that our in vivo model presents with significant BBB-dysfunction, evidenced by increased sodium fluorescein extravasation and MMP-9 as well as loss in tight junction protein and eNOS. These effects were mitigated by a single injection of our inhibitors 24 hours post trauma. We then isolated BECs from WT and OS mice and saw enhanced susceptibility in the OS-BECs after ischemia-reperfusion injury, suggesting a role for oxidative stress and lipid peroxidation in exacerbating CAPN1/CTSB mediated BBB damage. BECs from WT and OS mice provide a platform to assess the role of CCH in cell viability and tight junction proteins, and provides support for targeting CAPN1/CTSB in protecting the BBB, either in early life trauma, such as TBI, or in ADRD itself. This work establishes CCH inhibition as a potential prophylactic therapeutic strategy to protect the neurovascular unit.