Gene-Regulation of the BCL-2 Family in a Cell Model of the Ischemic Penumbra

Apoptosis is a regulated mechanism of cell death characterised by distinct morphological changes that are induced through either intrinsic or extrinsic pathways. The Bcl-2 family of proteins are central components of the intrinsic pathway of apoptosis; the balance between pro-survival and pro-death members dictates the fate of a cell. Aberrant apoptosis is a classic feature of cell death in the ischemic penumbra following a stroke. Studies have identified post-translational regulation of BCL-2 family members to underlie neuronal death in response to ischemia. However, the early regulatory events, such as the gene dependent regulation of the BCL-2 family in response to ischemia, have not been extensively studied. This project aimed to investigate the transcriptional and post-transcriptional regulation, at the miRNA level, of the BCL-2 family in an in vitro model of the ischemic penumbra as a potential mechanism involved in the control of ischemia-induced apoptosis. Using a limited mRNA screen of the BCL-2 family the results show that this family is differentially regulated at the transcriptional level, following ischemic-like insults, to generate a profile that favours cell death. In particular the pro-apoptotic BH3-only member NOXA is significantly upregulated at the mRNA level before detection of neuronal cell death. The results also show that the BCL-2 family are susceptible to post-transcriptional regulation, indicated by a mismatch between mRNA and protein levels. Microarray analysis of in vivo and in vitro ischemic samples shows that miRNAs are differentially regulated following ischemia. This study identified miR-29b-2*, miR-19b and miR-339-Sp to be significantly upregulated following ischemia in both in vivo and in vitro models of stroke, before and after the detection of neuronal cell death. Consequently, this data demonstrates that miR-29b-2*, miR-19b and miR-339-Sp could be important regulatory elements that co-ordinate the neuronal death response to ischemic insults in the penumbra, and hence be possible targets for future stroke therapy.