Inhibition of histone deacetylase 3 by MiR-494 alleviates neuronal loss and improves neurological recovery in experimental stroke

Abstract

HDAC3 is an essential negative regulator of neuronal plasticity and memory formation. Although a chemical inhibitor has been invented, little is known about its endogenous modulators. We explored whether miR-494 affects HDAC3-mediated neuronal injury following acute ischemic stroke. A substantial increase in plasma miR-494 was detected in AIS patients and was positively associated with the mRS at one year after symptom onset. The miR-494 levels were transiently increased in the infarcted brain tissue of mice. In contrast, miR-494 levels were reduced in neurons but increased in the medium after OGD. Intracerebroventricular injection of miR-494 agomir reduced neuronal apoptosis and infarct volume at the acute stage of MCAO, promoted axonal plasticity and long-term outcomes at the recovery stage, suppressed neuronal ataxin-3 and HDAC3 expression and increased acetyl-H3K9 levels in the ipsilateral hemisphere. In vitro studies confirmed that miR-494 posttranslationally inhibited HDAC3 in neurons and prevented OGD-induced neuronal axonal injury. The HDAC3 inhibitor increased acetyl-H3K9 levels and reversed miR-494 antagomir-aggravated acute cerebral ischemic injury, as well as brain atrophy and long-term functional recovery. These results suggest that miR-494 may serve as a predictive biomarker of functional outcomes in AIS patients and a potential therapeutic target for the treatment of ischemic stroke.

Keywords: HDAC3; Ischemic stroke; miR-494; neuron.

Figure 1.

MiR-494 was increased in the plasma of AIS patients and the supernatant of primary neurons. (a) Expression of miR-494 in plasma of AIS patients (n = 76) and control volunteers (n = 52) was measured by RT-PCR. Student’s t test. (b) Pearson correlation coefficient between plasma miR-494 level and mRS after one year. n = 32. (c) Comparison of miR-494 levels in the plasma of AIS patients with mRS ≥2 (n = 13) and patients with mRS <2 (n=19). Student’s t test. (d) MiR-494 levels in the ipsilateral brain tissue of a mouse model of MCAO 45 min after ischemia and one day and three days after reperfusion. n = 6 per group. One-way ANOVA with Tukey–Kramer post hoc test. (e–f) MiR-494 levels in primary neurons and supernatant at different time points after OGD were determined by real-time PCR, with U6 used as an internal control. n = 6/group. One-way ANOVA with Tukey–Kramer post hoc test. *P < 0.05, **P < 0.01 vs. control group.

Figure 2.

MiR-494 protected the brain against mouse ischemic injury in the acute stage and recovery stage. (a) Experimental protocol. (b) FAM-labeled miR-494 agomir with green fluorescence was used to assess neuronal uptake (red) after ICV delivery. Green fluorescence was mainly distributed in neurons (arrows) in the brain tissue. (c) QuantitativePCR was performed to verify the efficacy of miR-494 agomir and antagomir transfection. n = 6/group. (d) Cerebral infarct volume and brain edema evaluated by TTC staining of coronal brain sections. n = 10–12/group. (e–f) Neuronal apoptosis in the ipsilateral cortex as detected by TUNEL/NeuN immunofluorescence double staining. n = 6 per group. (g) Immunoblot of MAP-2 in the cortex in the recovery stage. n = 6/group. MCAO + control denotes mice subjected to 45 min ischemia followed by reperfusion for three days, with control administered by ICV injection immediately after ischemia; MCAO + miR-494 denotes mice subjected to 45 min ischemia followed by reperfusion for three days, with miR-494 agomir administered by ICV injection immediately after ischemia. *P < 0.05, **P < 0.01, ***P < 0.001 vs. sham group. ^#P < 0.05 vs. MCAO + control group. One-way ANOVA with Tukey–Kramer post hoc test.

Figure 3.

MiR-494 downregulated HDAC3 expression in neurons of MCAO mice. (a) Protein expression levels of HDAC3 and acetyl-histone H3 (Lys9/Lys14), histone 3 acetyl-histone H4 (Lys5), and histone 4 in the ipsilateral cortex were assessed by Western blot (n = 6/group). (b) The localization of HDAC3 in neurons was assessed by HDAC3/NeuN immunofluorescence double staining (n = 6/group). ***P < 0.001 vs. sham group; ^#P < 0.05 vs. MCAO + control group. One-way ANOVA with Tukey–Kramer post hoc test.

Figure 4.

MiR-494 downregulated HDAC3 in cortical primary neurons. (a–b) Expression of ATXN3 and HDAC3 in mouse primary neurons was measured by RT-PCR following transfection with miR-494 agomir or antagomir (n = 6/group) for 48 h. (c) LDH release in neurons transfected with miR-494 agomir for 48 h and then exposed to OGD/R for 2 h/24 h (n = 6/group). (d) MAP-2 expression in neurons transfected with miR-494 agomir for 48 h and then exposed to OGD/R 2 h/ 24 h was detected by Western blot (n = 6/group). (e) MAP-2 expression in neurons following OGD was detected by immunofluorescence (n = 6/group). Neurons were transfected with control, miR-494 agomir, or miR-494 antagomir for 48 h and then exposed to OGD/R 2 h/24 h, with the addition of RGFP966 at the time of reoxygenation. OGD/R: oxygen and glucose deprivation/reoxygenation. *P < 0.05, **P < 0.01 vs. control group. ^#P < 0.05 vs. control + OGD group. One-way ANOVA with Tukey–Kramer post hoc test.

Figure 5.

HDAC3 inhibition reversed miR-494 antagomir-aggravated acute cerebral ischemic injury. Mice were subjected to 45 min ischemia followed by reperfusion for three days, with miR-494 antagomir administered by ICV injection immediately after ischemia and RGFP966 injected intraperitoneally immediately following reperfusion. (a) Expression levels of HDAC3, acetyl-histone H3 (Lys9/Lys14), histone 3 acetyl-histone H4 (Lys5), and histone 4 in the ipsilateral cortex were assessed by Western blot. n = 6/group. (b) Cerebral infarct volume and brain edema evaluated by TTC staining of coronal brain sections. n = 10–11/group. ^&P < 0.05, ^&&&P < 0.001 vs. MCAO+ miR-494 antagomir group. Student’s t test.

Figure 6.

HDAC3 inhibition reversed the miR-494 antagomir-worsened long-term outcome. (a) Experimental protocol. Mice were subjected to 45 min ischemia followed by reperfusion for 28 days, with control, miR-494 agomir, or miR-494 antagomir administered by ICV injection immediately after ischemia and RGFP966 injected intraperitoneally immediately following reperfusion. (b) Brain tissue loss at 28 days following stroke was calculated. One-way ANOVA with Tukey–Kramer post hoc test. (c) Regional CBF was monitored using two-dimensional laser speckle imaging techniques. Representative images and quantification of CBF before, during, and after transient MCAO. (d) MBP was recorded by tail cuff plethysmography using a physiological pressure transducer coupled to a data-acquisition system. (e) Motor function recovery was detected by the balance beam test. (f) Computer printouts of superimposed swimming trajectories of mice trained in light to find an invisible platform after starting in any of the four cardinal points (north, east, south, west) at the circumference of the pool. (g) Learning and memory ability were detected by escape latency in the Morris water maze. n = 9–12/group. Two-way RM ANOVA followed by the Bonferroni post hoc correction. *P < 0.05, ***P < 0.001 vs. sham group; ^#P < 0.05 vs. MCAO + control group; ^&P < 0.05 vs. MCAO + miR-494 antagomir group.