Bcl-x pre-mRNA splicing regulates brain injury after neonatal hypoxia-ischemia

Thebcl-xgene appears to play a critical role in regulating apoptosis in the developing and mature CNS and following CNS injury. Two isoforms of Bcl-x are produced as a result of alternative pre-mRNA splicing: Bcl-xL(the long form) is anti-apoptotic, while Bcl-xS(short form) is pro-apoptotic. Despite the antagonistic activities of these two isoforms, little is known about how regulation of alternative splicing ofbcl-xmay mediate neural cell apoptosis. Here, we report that apoptotic stimuli (staurosporine or C2-ceramide) reciprocally altered Bcl-x splicing in neural cells, decreasing Bcl-xLwhile increasing Bcl-xS. Specific knockdown of Bcl-xSattenuated apoptosis. To further define regulatory elements that influenced Bcl-x splicing, a Bcl-x minigene was constructed. Deletional analysis revealed several consensus sequences within intron 2 that altered splicing. We found that the splicing factor, CUG-binding-protein-1 (CUGBP1), bound to a consensus sequence close to the Bcl-xL5′ splice site, altering the Bcl-xL/Bcl-xSratio and influencing cell death.In vivo, neonatal hypoxia–ischemia reciprocally altered Bcl-x pre-mRNA splicing, similar to thein vitrostudies. Manipulation of the splice isoforms using viral gene transfer of Bcl-xSshRNA into the hippocampus of rats before neonatal hypoxia–ischemia decreased vulnerability to injury. Moreover, alterations in nuclear CUGBP1 preceded Bcl-x splicing changes. These results suggest that alternative pre-mRNA splicing may be an important regulatory mechanism for cell death after acute neurological injury and may potentially provide novel targets for intervention.