The blots were developed with Pico Chemiluminescence substrate

after a procedure that transects the SN and prevents re-innervation. Our in vitro 20516370 serumwithdrawal model detected significant levels of C-CASP2 in TUNEL+ DRGN and DRG glia consistent with our in vivo observations, linking DRGN and DRG glia death with CASP2. Although earlier reports suggest that CASP2 is processed after a variety of stimuli but was not required for death of sensory DRGN, these studies were performed in postnatal 2-day old rats, which are NGF-dependent for their survival. This is contrast to our current study where we 18794083 use adult DRGN, which are independent of NGF for their survival and generally less reliant on trophic support and show that DRGN death is regulated by CASP2 in both in vivo and in vitro serum withdrawal paradigms. Furthermore, we found little or no C-CASP3 immunoreactivity in serum-starved DRGN, while an abundance of C-CASP2 immunoreactivity was observed in these neurons, further vindicating our assertion that CASP2 is the major caspase responsible for death of DRGN after neurotrophic factor withdrawal. The sub-cellular localization of C-CASP2 and the mechanism of activation of CASP2 may be linked and differs from cell type and death stimulus. Although the majority of CASP2 is found in the cytoplasm some contradictory findings report that proCASP2 is localized to both cytoplasm and nucleus and that processing occurs in both compartments. CASP2 is constitutively localised to the nucleus by interaction of its CP 868596 chemical information nuclear localization signal with both its prodomain signal and the importin mediator. Our results are consistent with these observations since they demonstrate that, although C-CASP2 is both nuclear and cytoplasmic in DRGN, significantly greater levels were detected in the nucleus after serum withdrawal. Colussi and co-workers Caspase-2 Mediates DRGN Apoptosis also reported that CASP2 was localised in the cytoplasm, mitochondria and nuclei of normal Jurkat cells, while C-CASP2 was only present in the nucleus of apoptotic cells. Thus the nucleus appears to be the site of CASP2 cleavage and activation, both of which remain nuclear even after the induction of cell death. Of potential translational importance, we have also demonstrated that silencing of CASP2 expression by siRNA-mediated knockdown of CASP2 mRNA protected DRGN from apoptosis after serum withdrawal. By contrast, CASP3 knockdown failed to rescue DRGN from apoptosis. Therefore, we suggest that CASP2 plays a major role in the execution of apoptosis in axotomised adult DRGN after neurotrophic factor depletion. Despite CASP2 sharing features of both initiator and effector caspases, processed rapidly in response to both intra- and extra-cellular signalling events occurring early during apoptosis in many cell types, its role in apoptosis has been contentious. For example, O’Reilly et al. reported that DRGN in postnatal 2 dayold CASP-2 deficient mice did not die after NGF withdrawal. However, this contradicts another study, which reported that decreasing CASP-2 levels by anti-sense technology, delayed apoptosis induced by trophic factor deprivation in sympathetic neurons. Others have also shown that CASP2 triggers apoptosis of serum-deprived PC12 cells and cortical neurons, while CASP2 inhibitors and siRNA-mediated knock down of CASP2 protects these neurons from death induced by serum deprivation. Our current results however, agree with the vast body of evidence that suggest that CASP2 regulates the death of DRGN. In conclusion, we have demonstrated an