Differential PARP cleavage: an indication of heterogeneous forms of cell death and involvement of multiple proteases in the infarct of focal cerebral ischemia in rat.

OBJECTIVE

Poly (ADP-ribose) polymerase (PARP) is a nuclear repair enzyme whose role is widely depicted in various physiological and pathological processes. In the present study, we wanted to check the status of PARP and the role of various cell death proteases involved in apoptotic and non-apoptotic forms of cell death during transient focal cerebral ischemia in rat model. The activation of these proteases can result in the production of PARP fragments which can be treated as specific signature fragments to the particular protease involved in the pathology and hence the type of cell death.

RESULTS

In the ischemic samples, we observed activation of calpain, cathepsin-b, caspase-3, and granzyme-b which were known to act on and cleave PARP to produce specific signature fragments by Western blot and immunohistochemical analysis. Cresyl violet staining showed the presence of apoptotic and necrotic cell deaths. Further we observed interaction of AIF and gra-b with PARP in double immunofluorescence and co-immunoprecipitation experiments.

CONCLUSIONS

Activation of calpains, cathepsin-b, caspase-3, and granzyme-b correlated with either apoptotic or necrotic cell deaths in cresyl violet staining. The appearance of PARP signature fragments gives a clear idea on the involvement of particular protease in the pathology. Appearance of signature fragments like 89- and 50-kDa indicates the involvement of apoptotic and necrotic cell death in the pathology. Further interaction of AIF and gra-b with PARP also indicates the involvement of non-apoptotic modes of cell death during the pathology of focal cerebral ischemia.