132 Age-dependent changes in p18 expression in brain of wild-type and angleman syndrome mice

morphology relating to granulovacuolar degeneration, a pathological hallmark of Alzheimer’s Disease (AD). Our laboratory previously identified a link between traumatic brain injury (TBI) and AD by showing that calpain-2 activation is involved in tau phosphorylation and oligomer accumulation. This study will investigate effects of TBI-induced calpain activation on Hsp70.1 and determine involvement of calpain-1 or calpain-2 in lysosomal stability. Methods used Controlled cortical impact model of TBI was used on wild-type (WT) and calpain-1 KO (C1KO) C57Bl/6 mice. A 5 mm craniotomy was performed with anaesthesia, and a penetrating injury was induced on cortex lateral to the sagittal suture. Single injection of a selective calpain-2 inhibitor (C2I, 0.3 mg/kg) was made intraperitoneally 1 hour after TBI. For sham surgery, mice were subjected to craniotomy only. Brains were isolated 24 hour after TBI and cortical tissue surrounding injury was homogenised in homogenization buffer containing a protease and phosphatase inhibitor cocktail. After two rounds of centrifugation, P2 membrane (pellet) fraction was resuspended in lysis buffer. Primary antibody for western blot (WB) was Hsp70.1 (1:3000). Summary of results WB showed breakdown products of Hsp70.1 after TBI with apparent Mw of 50, 37 and 25 kDa. Quantification of WB indicated that ratio of breakdown products to full-length Hsp70.1 was significantly higher in WT and C1KO mice after TBI, as compared to sham (n=3 animals. p<0.05 sham vs WT. p<0.01 sham vs C1KO). Post-TBI injection of C2I appeared to prevent Hsp70.1 truncation. Conclusions TBI resulted in rapid calpain-2-mediated Hsp70.1 truncation, as selective inhibition of calpain-2 decreased Hsp70.1 cleavage, which was evident in absence of calpain-1. Our results support the critical role of calpain-2 in neuronal death and provide an additional link between TBI and AD through calpain-2-mediated cleavage of Hsp70.1, and resulting lysosomal destabilisation.