| dc.description.abstract | The cause of mechanisms underlying perinatal brain injury is not fully known, but it results in a wide variety of neurological impairments in the affected children. When injured, neurons of the newborn brain are prone to undergo programmed cell death, apoptosis, since this genetic program is normally activated in some cells during development of the nervous system, even in the perinatal period. However, classical necrosis is also involved, especially when the injury is severe. The aim of this thesis was to elucidate the possible interaction between the classic necrotic and apoptotic cell death pathways in an animal model of perinatal brain injury. Specifically, we studied calpastatin, the endogenous inhibitor of calpains, a protease family known to be highly involved in necrosis. We also investigated two members of the Bcl-2 protein family, which are largely responsible for the outcome of the apoptotic process.Using a model for unilateral cerebral hypoxia-ischemia (HI) in 7-day-old rats, the expression of calpastatin was investigated by western blotting, immuno-histochemistry and quantitative RT-PCR. Animals were also treated with the calpain inhibitor cx-295 and evaluated for the calpain-dependent cleavage of calpastatin and fodrin (paper I). The effects of HI on the anti-apoptotic protein Bcl-2 and the pro-apoptotic Bax were studied using western blotting, immunohistochemistry and immuno-electron microscopy. An in vitro set-up was used to study the possible interactions between calpains and Bcl-2, Bax and caspase-12 after excitotoxic injury (paper II). Using the in vivo HI model, the correlation of serine (S) 24-phosphorylated Bcl-2 with apoptosis-related markers of cell death was characterized (paper III). Finally, the importance of the phosphorylation state of serine 24 of Bcl-2 was investigated in vitro, using protein mutagenesis techniques, transfection of BHK cells and chemical induction of apoptosis (paper IV). Calpain cleavage of calpastatin and fodrin occurred only in the hypoxic-ischemic hemisphere; not in the hypoxic one. Thus, during milder hypoxic conditions, calpastatin prevented full activation of calpains but during severe HI, calpastatin was degraded by calpains that then contributed to further degradation of cellular components. A decrease in total Bcl-2 occurred after HI, concurrent with an altered intracellular distribution. Bcl-2 was partly translocated to nuclei, while Bax was translocated to mitochondria after HI. Bcl-2 was cleaved and caspase-12 was released into the cytosol in vitro in a calpain-dependent manner, suggesting some degree of cross-talk in this model between apoptotic and necrotic cell death pathways. S24-phosphorylated Bcl-2 colocalized with mitochondria and nuclei in neurons with morphological signs of apoptosis and with other apoptotic markers. S24-phosphorylation of Bcl-2 preceded caspase-3 activation in injured cells, indicating an upstream role for Bcl-2 in the cell death pathway. The anti-apoptotic effect of Bcl-2 correlated with the presence of the S24-dephosphorylated form of Bcl-2. S24-phosphorylated Bcl-2 lost the ability to inhibit Bax, and furthermore, seemed to be transformed into a pro-apoptotic protein in itself. Calcineurin activity was critical for keeping Bcl-2 in its anti-apoptotic form. | en |
