Cathepsin B Contributes To Traumatic Brain Injury Induced Autophay And Cell Death Through Mitochondria-mediated Apoptotic Pathway

Objective: It has been reported that lysosomal proteases play important roles in ischemic and excitotoxic neuronal cell death. Our previous study also certificated that autophagy was activated and cathepsin B expression remarkably increased after traumatic brain injury (TBI). The present study was sought to investigate the effects of a selective cathepsin B inhibitor (CBI) on autophay, cell death and behavioral deficits in our TBI model.Methods: TBI model was established by weight drop device in adult mice based on procedures previously reported. A selective cathepsin B inhibitor (CBI) was administered with a single intracerebroventricular (i.v.c.) injection before TBI.Mice were pretreated with intraperitoneal injection of propidium iodide (PI) 1 h before sacrificed. PI-labeling was used to identify injured cells and the amount of PI-positive cells was counted. The cumulative loss of brain tissue was determined to explore whether PI-positive cells could represent TBI-induced cell loss. Motor test and Morris water maze were performed to detect whether TBI-induced cell loss would result in behavior deficits. We examined the levels of cathepsin B enzymatic activity and its expression, double labelling of damaged cells in the same brain slice using propidium iodide (PI) and anti-cathepsin B /anti-NeuN /anti-GFAP antibody, respectively. We also analyzed the mechanism of cathepsin B in TBI induced cell death and autophagy signaling pathways.Results: (1) PI-positive cells were found in all groups that underwent TBI. The number of PI-positive cells peaked in the 48 h time-point group. CBI treatment markedly reduced the number of PI-positive cells compared with saline vehicle groups at 12 h, 24 h, and 48 h post TBI (P<0.05). To clearly indentify the proportion of PI-postive cells, we combined PI labeling with DAPI nuclear staining. The results showed that CBI treatment markedly reduced the ratio of PI+/DAPI+ compared with saline group at 24 h and 48 h time-point post TBI (P<0.05). (2) An elevated enzymatic activity associated with TBI-induced increase in a mature form of cathepsin B. The majority of injured cortical cells detected at 48 h were PI+/cathepsin B+. In contrast, relatively few PI+/cathepsin B- and PI-/cathepsin B+ cells were observed in injured cortex. Treatment with CBI markedly reduced the number of PI+/cathepsin B+ cells compared with saline group. (3) PI was found to label cells positive for the neuronal-specific nuclear marker NeuN, while fewer GFAP positive cells were labelled by PI. (4) Agarose gel electrophoresis of DNA showed an evident smear due to non specific fragmentation that is known to occur after necrotic cell death. The possible formation of apoptotic ladders was also investigated, as a matter of fact the intensive DNA smear might mask the presence of internucleosomal fragmentation. (5) TBI-Induced lesion volume and behavioral deficits, Bcl-2/Bax protein expression levels, Bid cleavage, cytochrome c release, caspase-3 activation, as well as upregulation of microtubule-associated protein 1A/1B light chain 3 (LC3) and beclin 1 were also assessed. CBI reversed these changes in this study.Conclusion: (1) Neurons are more sensitive to cell death induced by TBI. (2) Pre-treatment with CBI remarkably attenuated TBI-induced cell death, lesion volume, motor and cognitive dysfunction, as well as activation of autophagy. (3) Cathepsin B contributes to TBI-induced cell death through the present programmed cell necrosis, mitochondria-mediated apoptotic pathway, and autophagic/lysosomal pathways.