MicroRNAs affect BCL-2 family proteins in the setting of cerebral ischemia

Abstract

The BCL-2 family is centrally involved in the mechanism of cell death after cerebral ischemia. It is well known that the proteins of the BCL-2 family are key regulators of apoptosis through controlling mitochondrial outer membrane permeabilization. Recent findings suggest that many BCL-2 family members are also directly involved in controlling transmission of Ca(2+) from the endoplasmic reticulum (ER) to mitochondria through a specialization called the mitochondria-associated ER membrane (MAM). Increasing evidence supports the involvement of microRNAs (miRNAs), some of them targeting BCL-2 family proteins, in the regulation of cerebral ischemia. In this mini-review, after highlighting current knowledge about the multiple functions of BCL-2 family proteins and summarizing their relationship to outcome from cerebral ischemia, we focus on the regulation of BCL-2 family proteins by miRNAs, especially miR-29 which targets multiple BCL-2 family proteins.

Keywords: BCL-2; Cerebral ischemia; Stroke; miR-29; microRNA.

Fig. 1

BCL-2 family members and those targeted by miR-29. A. The BCL-2 family is comprised of three subfamilies that contain one or more BCL-2 homology (BH) domains. The anti-apoptotic subfamily comprises proteins that contain all four BH domains. The multidomain pro-apoptotic subfamily lacks BH4 domains. Most members of these subfamilies also contain transmembrane domains (TM) and are therefore typically associated with membranes or pore formation in mitochondrial outer membranes. The BH3-only subfamily all contain the BH3 motif and most of them have no TM. The molecular weight of each protein is listed on the right (kDa). B. A single miRNA, miR-29, has many validated targets within the large BCL-2 family, including proteins in all three subfamilies.

Fig. 2

BCL-2 family-mediated cell death. A. The BCL-2 family regulates mitochondria-dependent apoptosis. BH3-only family members act as sentinels for many death stimuli including ischemia. They can mediate the activation of the multidomain pro-apoptotic BAX and BAK allowing oligomerization. Upon BAX and BAK oligomerization, the mitochondrial outer membrane is permeabilized releasing a apoptogenic proteins from the mitochondrial intermembrane space, such as cytochrome c, into the cytosol. Released cytochrome c triggers the activation of a downstream caspase cascade leading to cell death. BH3-only proteins can be sequestered by anti-apoptotic BCL-2 family members (e.g. BCL-2, MCL-1, BCL-xL,, etc.). B. The BCL-2 family regulates ER-mitochondria Ca²⁺ crosstalk at the MAM. The basic structure for release of Ca²⁺ at MAM is the IP3R on the ER, and VDAC and MCU on the mitochondrion. IP3R and VDAC are physically coupled by the chaperone GRP75/mortalin. Excessive increases in mitochondrial matrix Ca²⁺ triggers opening of the mitochondrial outer membrane permeability pore causing the release of cytochrome c and other pro-apoptotic factors into the cytoplasm. ER: endoplasmic reticulum; GRP75: glucose-regulated protein 75/mortalin; IP3R: inositol trisphosphate receptor; MAM: mitochondria-associated ER membrane; MCU: mitochondrial Ca²⁺uniporter; VDAC: voltage-dependent anion channel.

Fig. 3

Luciferase assays validate multiple Bcl-2 family members as targets of the miR-29 family. A. Bioinformatics search suggests miR-29 could target 5 members of the BCL-2 family. The seed sequences of BAK1, BBC3, BMF, BCL2L2 and MCL1 3′UTRs targeted by miR-29 are highly conserved across species (from TargetScan). B. Dual luciferase activity assays using co-transfection with a plasmid containing luciferase followed by the 3′UTRs (WT) of BCL-2 family mRNAs and plasmids encoding either pri-miR-29 wild type or pri-miR-29 with seed mutations (SM) demonstrate that miR-29ab, but not miR-29c, recognizes the 3′UTRs of all of these five BCL-2 family members, but of Bcl2L1, which is not a predicted target. C. The same assay performed with the wild type 3′UTRs or their seed mutants (SM) of the five BCL-2 family members shows that miR-29ab reduces luciferase activity, validating that miR-29ab targets all of them. Assays were performed 3 times in triplicate. *P<0.01 compared to the miR-29ab-SM or 3′UTRs-SM group (the data shown for BBC3 data comes from Ouyang et al. (2013c); the additional data is in press in Ouyang et al. (2013d)).