Non-lethal active caspase-3 expression in Bergmann glia of postnatal rat cerebellum

Abstract

Caspase-3, an apoptotic executor, has been shown in recent years to mediate non-lethal events like cellular proliferation and differentiation, primarily in studies related to non-neural tissue. In central nervous system development, the role of active caspase-3 is still unclear. We provide the first evidence for a potential new role of active (cleaved) caspase-3 in promoting differentiation of Bergmann glia. This study was predicated on the hypothesis that active caspase-3 is important for the differentiation of glia. We addressed the hypothesis through the following specific aims: (1) to establish the expression of active caspase-3 in glia; (2) to determine the developmental phenotype of the active caspase-3-expressing glia; and (3) to confirm that active caspase-3 expression is not mediating an apoptotic event. Through a temporal investigation from postnatal day 8 to 21, we observed that Bergmann glia express active caspase-3 without compromising their survival. Potential apoptotic fate of active caspase-3-positive Bergmann glia were ruled out based on immunohistochemical exclusion of phosphatidylserine exposure (Annexin V), DNA fragmentation (TUNEL), and DNA compaction (TOPRO-3). More than 90% of the active caspase-3-positive cells lacked colabeling for one of the apoptotic markers. Correlative studies using a proliferation marker Ki67 and a differentiation marker brain lipid-binding protein suggest that the expression of active caspase-3 was mostly associated with differentiating rather than proliferating Bergmann glia at all ages. Thus, this study supports the hypothesis that active caspase-3 may be regulating both differentiation of Bergmann glia by allowing the cells to exit the cell cycle and their morphogenesis.