Loss of miR-29b following acute ischemic stroke contributes to neural cell death and infarct size

Abstract

Glutathione depletion and 12-lipoxygenase-dependent metabolism of arachidonic acid are known to be implicated in neurodegeneration associated with acute ischemic stroke. The objective of this study was to investigate the significance of miR-29 in neurodegeneration associated with acute ischemic stroke. Neural cell death caused by arachidonic acid insult of glutathione-deficient cells was preceded by a 12-lipoxygenase-dependent loss of miR-29b. Delivery of miR-29b mimic to blunt such loss was neuroprotective. miR-29b inhibition potentiated such neural cell death. 12-Lipoxygenase knockdown and inhibitors attenuated the loss of miR-29b in challenged cells. In vivo, stroke caused by middle-cerebral artery occlusion was followed by higher 12-lipoxygenase activity and loss of miR-29b as detected in laser-captured infarct site tissue. 12-Lipoxygenase knockout mice demonstrated protection against such miR loss. miR-29b gene delivery markedly attenuated stroke-induced brain lesion. Oral supplementation of α-tocotrienol, a vitamin E 12-lipoxygenase inhibitor, rescued stroke-induced loss of miR-29b and minimized lesion size. This work provides the first evidence demonstrating that loss of miR-29b at the infarct site is a key contributor to stroke lesion. Such loss is contributed by activity of the 12-lipoxygenase pathway providing maiden evidence linking arachidonic acid metabolism to miR-dependent mechanisms in stroke.

Figures

Figure 1

Arachidonic acid-induced death of glutathione-deficient neural cells is associated with loss of miR-29b. Arachidonic acid-induced cell death in glutathione-depleted HT4 neural cells (A) or primary cortical neurons (C) at 24 hours. In both cases, cells were either challenged (solid bar) or not (open bar, control) with L-buthionine-sulfoximine (50 μmol/L; BSO) and arachidonic acid (50 μmol/L; AA) and collected at the indicated time points for lactate dehydrogenase (LDH) assay. Cell death was associated with specific loss of miR-29b (B–D) in both cell types. Results are mean±s.d. *P<0.05 compared with control.

Figure 2

Mimic delivery aimed at compensating for loss of miR-29b after arachidonic acid insult protected against loss of mitochondrial membrane potential and cell death. (A) Real-time PCR analysis of miR-29b after transfection of miR-29b mimic. (B) After 12 hours of L-buthionine-sulfoximine (BSO) and arachidonic acid (AA) challenge, cells were stained with JC-1 dye and analyzed using a flow cytometer. (C and D) miR-29b delivery attenuated loss of cell viability caused by BSO+AA. Viability was measured after 24 hours of BSO and AA challenge by lactate dehydrogenase (LDH) leakage (C) or calcein-AM (green, live cells) and propidium iodide (red, dead cells) staining (D). Magnification, × 10 results are mean±s.d. P<0.05, *effect of BSO+AA, †effect of miR-29b mimic.

Figure 3

Inhibition of miR-29b exacerbated arachidonic acid-induced loss of mitochondrial membrane potential and cell death. (A) Real-time PCR analysis of miR-29b expression after transfection of cells with miR-29b inhibitor. (B) miR-29b inhibitor-transfected cells (72 hours) were challenged for 12 hours. Next, cells were stained with JC-1 dye and analyzed using a flow cytometer. (C and D) cells transfected with miR-29b inhibitor potentiated loss of cell viability. Viability was measured after 24 hours of challenge by lactate dehydrogenase (LDH) leakage (C) or calcein-AM (green, live cells) and propidium iodide (red, dead cells) staining (D). Magnification, × 10. Results are mean±s.d. P<0.05 *effect of L-buthionine-sulfoximine (BSO) + arachidonic acid (AA), †effect of miR-29b inhibitor.

Figure 4

12-lipoxygenase (12-LOX) inhibition protected cells against L-buthionine-sulfoximine (BSO) and arachidonic acid (AA)-induced loss of miR-29b, miR-29c and cell death. (A) Transfection with 12-LOX siRNA decreased 12-LOX mRNA and (B) protein expression in HT4 cells. *, lower in 12-LOX siRNA group compared with control siRNA group. (C)12-LOX siRNA attenuated BSO- and AA-induced loss of cell viability. (D) 12-LOX siRNA-transfected cells (solid bar) were resistant to BSO- and AA-induced loss of miR-29b and miR-29c expression compared with control siRNA-transfected cells (open bar). (E) α-Tocotrienol (TCT, 0.25 μmol/L) or Baicalein (BL15, 2.5 μmol/L) treatments prevented loss of miR-29b and miR-29c in BSO- and AA-challenged cells. Results are mean ±s.d. P<0.05 *compared with control, †compared with BSO and AA.

Figure 5

Acute ischemic stroke-induced brain lesion was attenuated by delivery of miR-29b mimic to the brain. Stroke was induced in C57BL/6 mice (A) by 90 minutes of middle-cerebral artery occlusion (MCAO). After 48 hours of stroke, coronal slices of brain tissue were collected using a mouse brain matrix. OCT-embedded slices were subsequently cut in 12-μm-thick sections. Matched area (1 × 106 μm2) of contralateral or stroke-affected somatosensory cortex was laser captured into direct lysis extraction buffer for analyses of microRNA expression. (A) miR-29b and miR-29c, but not miR-29a, were downregulated at the infarct site. pLenti-III-mmu-miR-29b (miR-29b mimic) or pLenti-III-miR-GFP (con-miR) lentiviral vector delivered to cortex transduced 25–35 mm3 of brain tissue covering the area at risk for stroke. B and C demonstrates productive gene delivery after 72 hours of injection. (B) Targeted delivery of pLenti-III-mir-GFP control miRNA (108 IU/mL) to the MCA supplied S1 cortex of C57/BL6 mouse was achieved by stereotaxic injection via Hamilton Syringe connected to a nano-injector. At 72 hours after delivery, mouse brain was collected and sectioned to visualize successful transfection as evidenced by GFP in S1 cortex After 72 hours of such gene delivery, mice were subjected to MCAO. (D) Shows that delivery of miR-29b mimic abrogated stroke-induced loss of miR-29b at the infarct site. (E) Representative 11.7 T MRI (48 hours) image and lesion area quantitation showing protection in response to delivery of miR-29b mimic. P<0.05 *control versus infarct with in group, †compared with infarct hemisphere of con-miR.

Figure 6

Delivery of miR-29b mimic to the brain improves (BL) poststroke sensorimotor function and attenuates stroke-induced neurodegeneration. (A) Representative track plots from baseline and 48 hours after stroke with control or miR-29b mimic delivery. (B) Delivery of miR-29b mimic significantly improved 48 hours poststroke distance traveled, mean speed, and time mobile as compared with control. (C) Stroke-induced neurodegeneration was attenuated in response to miR-29b mimic delivery. Fluoro-Jade+-degenerating neurons were fewer in the infarct hemisphere of miR-29b mimic group (green, Fluoro-Jade; blue, DAPI-counterstained nuclei). P<0.05 *control versus infarct with in group, †compared with infarct hemisphere of con-miR.

Figure 7

Orally supplemented TCT protected against acute ischemic stroke by sparing loss of miR-29b and 29c. C57BL/6 mice (n=6) were orally gavaged with vitamin-E-stripped corn oil (PBO) or α-tocotrienol (TCT, 50 mg/kg body weight) for 10 weeks. Next, mice were subjected to middle-cerebral artery occlusion (MCAO) for 90 minutes. (A) TCT supplementation protected against loss of miR-29b and miR-29c at the infarct site. Compared with data shown in Figure 5A, the placebo data (PBO) are from older (15 weeks) mice because of the 10-week-long supplementation. (B) Representative 11.7 T MRI (48 hours) image and lesion area quantitation showing protection in response to TCT supplementation. Data are mean ±s.d. P<0.05 *control versus infarct within group, †compared with infarct hemisphere of PBO group.