Activation of Cytosolic Cathepsin B Activity in the Brain by Traumatic Brain Injury and Inhibition by the Neutral pH Selective Inhibitor Probe Z-Arg-Lys-AOMK

Cathepsin B has been shown to contribute to deficits in traumatic brain injury (TBI), an important risk factor for Alzheimer's disease (AD). Cathepsin B is elevated in TBI and AD patients, as well as in animal models of these conditions. Knockout of the Cathepsin B gene results in amelioration of TBI-induced motor dysfunction and improvement of AD memory deficit in mice. The mechanism of Cathepsin B pathogenesis in these brain disorders has been hypothesized to involve its translocation to the cytosol from its normal lysosomal location. This study, therefore, evaluated brain cytosolic Cathepsin B activity in the controlled cortical impact (CCI) mouse model of TBI. CCI-TBI resulted in motor deficits demonstrated by the rotarod assay, brain tissue lesions, and disorganization of the hippocampus. Significantly, CCI-TBI increased cytosolic Cathepsin B activity in the brain cortex in the ipsilateral brain hemisphere that received the CCI-TBI injury, with a concomitant decrease in the lysosomal fraction. Cathepsin B activity was monitored using the substrate Z-Nle-Lys-Arg-AMC which specifically detects Cathepsin B activity but not Other cysteine proteases. The normal lysosomal distribution of Cathepsin B was observed by its discrete localization in brain cortical cells. CCI-TBI resulted in a more diffuse cellular distribution of Cathepsin B consistent with translocation to the cytosol. Further studies utilized the novel neutral pH-selective inhibitor, Z-Arg-Lys-AOMK, that specifically inhibits Cathepsin B at neutral pH 7.2 of the cytosol but not at acidic pH 4.6 of lysosomes. Daily administration of Z-Arg-Lys-AOMK (IP), beginning 1 day before CCI-TBI, resulted in the reduction of the increased cytosolic Cathepsin B activity induced by CCI-TBI. The inhibitor also reduced Cathepsin B activities in homogenates of the brain cortex and hippocampus which were increased by CCI-TBI. Furthermore, the Z-Arg-Lys-AOMK inhibitor resulted in the reduction of motor function deficit resulting from CCI-TBI. These findings demonstrate the activation of cytosolic Cathepsin B activity in CCI-TBI mouse brain injury.