Astrocyte-enriched miR-29a targets PUMA and reduces neuronal vulnerability to forebrain ischemia

Abstract

Following transient forebrain ischemia, astrocytes play a key role in determining whether or not neurons in the hippocampal CA1 sector go on to die in a delayed fashion. MicroRNAs (miRNAs) are a novel class of RNAs that control gene expression at the post-transcriptional level and the miR-29 family is highly expressed in astrocytes. In this study we assessed levels of miR-29 in hippocampus following forebrain ischemia and found that after transient forebrain ischemia and short periods of reperfusion, miR-29a significantly increased in the resistant dentate gyrus, but decreased in the vulnerable CA1 region of the hippocampus. We demonstrate that miR-29a targets BH3-only proapoptotic BCL2 family member PUMA by luciferase reporter assay and by Western blot. Comparing primary neuron and astrocyte cultures, and postnatal brain, we verified the strongly astrocytic expression of miR-29a. We further found that miR-29a mimic protects and miR-29a inhibitor aggravates cell injury and mitochondrial function after ischemia-like stresses in vitro. Lastly, by overexpressing and reducing miR-29a we demonstrate the protective effect of miR-29a on CA1 delayed neuronal death after forebrain ischemia. Our data suggest that by targeting a pro-apoptotic BCL2 family member, increasing levels of miR-29a might emerge as a strategy for protection against ischemia-reperfusion injury.

Keywords: PUMA; astrocyte; microRNA.

Fig. 1

A. Schematic representation of the genomic organization of mouse miR-29. The two clusters are on chromosomes 1 and 6. B. Sequences of mature wild type (WT) and seed mutated (SM) miR-29a, miR-29bb and miR-29c. SMs are used as negative controls. C. Vector MWX-PGK-IRES-GFP to express pri-miR-29 and its SM. D. Sequence and alignment of the miR-29-binding sites in the 3′UTR of PUMA. BBC3 has two predicted target sites (from TargetScan): the first (316–322) is less broadly conservative than the second one (401–407). E. Two wild type (BBC3-1 and BBC3-2) and seed mutant (BBC3-1-SM and BBC3-2-SM) in the 3′UTR of PUMA. F. Renilla luciferase reporter vector phRL-TK to express 3′UTRs of PUMA.

Fig. 2. Levels of miR-29 and distribution of PUMA in hippocampus after 10 min forebrain ischemia

By RT-qPCR levels of miR-29a (A), miR-29b (B) and miR-29c (C) were assessed in hippocampal DG and CA1 area after 10 min forebrain ischemia and 0 to 5 h reperfusion. Hippo: hippocampus. N=4–6 animals in each group. *P<0.05 compared to DG group at the same time point. D. Sections from rat brain fixed 1 h after sham or 10 min forebrain ischemia, and double labeled for PUMA (green) and the astrocyte marker GFAP (red). GFAP co-labeled for PUMA shows yellow/orange color in merged panel. The CA1 pyramidal neuron cell layer is indicated by CA1- on the left side of the micrographs.

Fig. 3

miR-29 targets PUMA. A. Dual luciferase activity assays using co-transfection with wild type 3′UTR of BBC3 and pri-miR-29 (WT) or pri-miR-29 mutant (SM). B. Dual luciferase activity assays using co-transfection with miR-29 and the wild type 3′UTR (WT) of BBC3, and its first seed mutant (SM-1) or second seed mutant (SM-2) as well as wild type pri-miR-29. Assays were performed 3 times in triplicate. P<0.01 compared to the *vector control group or to the ^#3′UTR-WT group.

Fig. 4

miR-29a is dominant in hippocampus and enriched in astrocytes. A. Relative levels of miR-29a, b, and c in normal rat hippocampus. N=4–6/group. B. Relative miR-29a levels in primary cultures of cortical neurons and astrocytes from 3 to 30 days in vitro, and postnatal day 3 to 60 brain cortex. All experiments were performed 3 times in triplicate. All values were normalized to neuronal miR-29a level at 3 days.

Fig. 5. Effect of miR-29a on in vitro ischemia in astrocytes

A, B. Dose-response of miR-29a levels to transfection with increasing amounts of miR-29a mimic (A) or inhibitor (B) in primary cultures of astrocytes, relative to control (Ctrl=1). Arrows indicate the dose we used for the following experiments. C. Representative immunoblots show PUMA protein levels in primary astrocytes without stress or after 6 h GD after transfection with control, mimic, or inhibitor. D. The graph shows the quantification of the Western blots. E. Effect of miR-29a mimic or inhibitor on astrocyte injury induced by 24 h glucose deprivation (GD). F. Effect of miR-29a mimic or inhibitor on cell injury induced by 7.5 h oxygen glucose deprivation (OGD) in primary astrocyte cultures. G. Effect of miR-29a mimic and inhibitor on mitochondrial membrane depolarization in astrocytes subjected to 3 h GD. Depolarization is indicated by decreased TMRE fluorescence. H. Effect of miR-29a mimic or inhibitor on ROS in astrocytes subjected to 3 h GD. Increasing HEt fluorescence indicates increasing ROS. Fluorescence values are normalized to the starting fluorescence = 1.0. Ctrl: transfection control. All experiments were performed 3 times in triplicate. *P<0.01 compared to Ctrl.

Fig. 6

Effect of miR-29a on CA1 delayed neuronal damage after transient forebrain ischemia. A, B. Hippocampal levels of miR-29a in rats treated with pri-miR-29a plasmid (A) or miR-29a antagomir (B). N=6/group, *P<0.01 compared to seed mutant (SM) or Ctrl group. C, D. Western blots show PUMA protein levels in the hippocampus of rats pretreated with pri-miR-29a plasmid (C), or miR-29a antagomir (D). Representative immunoblots are shown above the graphs. N=6 rats in each group. *P<0.01 compared to SM or Ctrl group. E. Representative cresyl violet-stained coronal sections demonstrate selective loss of CA1 hippocampal neurons (between white arrows) at 6 days reperfusion after 10 min forebrain ischemia. F. Loss of CA1 neurons was quantified by cresyl violet staining density. N=12/group, P<0.01 compared to sham (*) or to ischemic control/antagomir group (#).