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The microRNA miR-124 suppresses seizure activity and regulates CREB1 activity.

Wang W, Wang X, Chen L, Zhang Y, Xu Z, Liu J, Jiang G, Li J, Zhang X, Wang K, Wang J, Chen G, Luo J - Expert Rev Mol Med (2016)

Bottom Line: Inhibition of neuronal firing by miR-124 was associated with the suppression of mEPSC, AMPAR- and NMDAR-mediated currents, which were accompanied by decreased surface expression of NMDAR.In addition, miR-124 injection resulted in decreased activity and expression of cAMP-response element-binding protein1 (CREB1). a key regulator in epileptogenesis.Immunoprecipitation studies confirmed that the CREB1 antibody effectively precipitated CREB1 and NMDAR1 but not GLUR1 from rat brain hippocampus.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology,Chongqing Key Laboratory of Neurology,The First Affiliated Hospital of Chongqing Medical University,1 Youyi Road,Chongqing 400016,China.

ABSTRACT
miR-124, a brain-specific microRNA, was originally considered as a key regulator in neuronal differentiation and the development of the nervous system. Here we showed that miR-124 expression was suppressed in patients with epilepsy and rats after drug induced-seizures. Intrahippocampal administration of a miR-124 duplex led to alleviated seizure severity and prolonged onset latency in two rat models (pentylenetetrazole- and pilocarpine-induced seizures), while miR-124 inhibitor led to shortened onset latency in pilocarpine-induced seizure rat models. Moreover, the result of local field potentials (LFPs) records further demonstrated miR-124 may have anti-epilepsy function. Inhibition of neuronal firing by miR-124 was associated with the suppression of mEPSC, AMPAR- and NMDAR-mediated currents, which were accompanied by decreased surface expression of NMDAR. In addition, miR-124 injection resulted in decreased activity and expression of cAMP-response element-binding protein1 (CREB1). a key regulator in epileptogenesis. A dual-luciferase reporter assay was used to confirm that miR-124 targeted directly the 3'UTR of CREB1 gene and repressed the CREB1 expression in HEK293T cells. Immunoprecipitation studies confirmed that the CREB1 antibody effectively precipitated CREB1 and NMDAR1 but not GLUR1 from rat brain hippocampus. These results revealed a previously unknown function of miR-124 in neuronal excitability and provided a new insight into molecular mechanisms underlying epilepsy.

No MeSH data available.


Related in: MedlinePlus

Effect of miR-124 on the expression of NMDAR1 and GLUR1 in the hippocampus. (a) Diagram of the CREB1-3′-UTR with potential binding-sites for miR-124. (b) Relative luciferase activity of reporters, including WT or mutant CREB1 and NMDAR1 3′-UTR co-transfected with NC or miR-124 mimics. *indicates significant difference at P  <  0.01, respectively. (c) Sample Western blots showing surface and total levels of GLUR1 subunits in normal (without seizure inducing) and pilocarpine-induced seizure rats without treatment (control), and in rats treated with mimics control and mimics. (d) Sample Western blots showing the surface and total levels of expression of NMDAR1 subunits in normal (without seizure inducing) and pilocarpine-induced seizure rats without treatment (control), and in rats treated with mimics control and mimics. (e) Summary showing the surface expression and the total and surface/total ratios of GLUR1 subunits. *P < 0.05, compared with the control. ▲P < 0.05, compared with the mimics control, n = 3. (f) Summary showing the surface expression and the total and surface/total ratios of NMDAR1 subunits. ▲P < 0.05, compared with the mimics control, n = 3.
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fig05: Effect of miR-124 on the expression of NMDAR1 and GLUR1 in the hippocampus. (a) Diagram of the CREB1-3′-UTR with potential binding-sites for miR-124. (b) Relative luciferase activity of reporters, including WT or mutant CREB1 and NMDAR1 3′-UTR co-transfected with NC or miR-124 mimics. *indicates significant difference at P  <  0.01, respectively. (c) Sample Western blots showing surface and total levels of GLUR1 subunits in normal (without seizure inducing) and pilocarpine-induced seizure rats without treatment (control), and in rats treated with mimics control and mimics. (d) Sample Western blots showing the surface and total levels of expression of NMDAR1 subunits in normal (without seizure inducing) and pilocarpine-induced seizure rats without treatment (control), and in rats treated with mimics control and mimics. (e) Summary showing the surface expression and the total and surface/total ratios of GLUR1 subunits. *P < 0.05, compared with the control. ▲P < 0.05, compared with the mimics control, n = 3. (f) Summary showing the surface expression and the total and surface/total ratios of NMDAR1 subunits. ▲P < 0.05, compared with the mimics control, n = 3.

Mentions: Using TargetScan (Release 4.2) online searching programs, we have identified CREB1 as the potential target of miR-124. A 100% matched sequence was found at the CREB1 mRNA 3′UTR (Fig. 5a).To confirm the hypothesis that miR-124 targets the 3′UTR region of CREB1, the entire 3′UTR region of CREB1 was cloned into the XhoI/NotI site of pmiR-RB-REPORT™ and co-transfected the HEK293T cells with this vector along with either the miR-124 mimics or its negative control. Using the luciferase reporter system, we found that co-transfection of miR-124 mimics along with the 3′UTR of WT CREB1 caused a significant decrease by over 50% in luciferase units compared to controls (Fig. 5b). However, co-transfecion of miR-124 mimics not significantly suppressed the luciferase activity of reporter genes containing 3′UTR of NMDAR1 compared with the control after statistics calculation (Fig. 5b). These results demonstrated that miR-124 targeted specifically the 3′UTR region of CREB1.Figure 5.


The microRNA miR-124 suppresses seizure activity and regulates CREB1 activity.

Wang W, Wang X, Chen L, Zhang Y, Xu Z, Liu J, Jiang G, Li J, Zhang X, Wang K, Wang J, Chen G, Luo J - Expert Rev Mol Med (2016)

Effect of miR-124 on the expression of NMDAR1 and GLUR1 in the hippocampus. (a) Diagram of the CREB1-3′-UTR with potential binding-sites for miR-124. (b) Relative luciferase activity of reporters, including WT or mutant CREB1 and NMDAR1 3′-UTR co-transfected with NC or miR-124 mimics. *indicates significant difference at P  <  0.01, respectively. (c) Sample Western blots showing surface and total levels of GLUR1 subunits in normal (without seizure inducing) and pilocarpine-induced seizure rats without treatment (control), and in rats treated with mimics control and mimics. (d) Sample Western blots showing the surface and total levels of expression of NMDAR1 subunits in normal (without seizure inducing) and pilocarpine-induced seizure rats without treatment (control), and in rats treated with mimics control and mimics. (e) Summary showing the surface expression and the total and surface/total ratios of GLUR1 subunits. *P < 0.05, compared with the control. ▲P < 0.05, compared with the mimics control, n = 3. (f) Summary showing the surface expression and the total and surface/total ratios of NMDAR1 subunits. ▲P < 0.05, compared with the mimics control, n = 3.
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fig05: Effect of miR-124 on the expression of NMDAR1 and GLUR1 in the hippocampus. (a) Diagram of the CREB1-3′-UTR with potential binding-sites for miR-124. (b) Relative luciferase activity of reporters, including WT or mutant CREB1 and NMDAR1 3′-UTR co-transfected with NC or miR-124 mimics. *indicates significant difference at P  <  0.01, respectively. (c) Sample Western blots showing surface and total levels of GLUR1 subunits in normal (without seizure inducing) and pilocarpine-induced seizure rats without treatment (control), and in rats treated with mimics control and mimics. (d) Sample Western blots showing the surface and total levels of expression of NMDAR1 subunits in normal (without seizure inducing) and pilocarpine-induced seizure rats without treatment (control), and in rats treated with mimics control and mimics. (e) Summary showing the surface expression and the total and surface/total ratios of GLUR1 subunits. *P < 0.05, compared with the control. ▲P < 0.05, compared with the mimics control, n = 3. (f) Summary showing the surface expression and the total and surface/total ratios of NMDAR1 subunits. ▲P < 0.05, compared with the mimics control, n = 3.
Mentions: Using TargetScan (Release 4.2) online searching programs, we have identified CREB1 as the potential target of miR-124. A 100% matched sequence was found at the CREB1 mRNA 3′UTR (Fig. 5a).To confirm the hypothesis that miR-124 targets the 3′UTR region of CREB1, the entire 3′UTR region of CREB1 was cloned into the XhoI/NotI site of pmiR-RB-REPORT™ and co-transfected the HEK293T cells with this vector along with either the miR-124 mimics or its negative control. Using the luciferase reporter system, we found that co-transfection of miR-124 mimics along with the 3′UTR of WT CREB1 caused a significant decrease by over 50% in luciferase units compared to controls (Fig. 5b). However, co-transfecion of miR-124 mimics not significantly suppressed the luciferase activity of reporter genes containing 3′UTR of NMDAR1 compared with the control after statistics calculation (Fig. 5b). These results demonstrated that miR-124 targeted specifically the 3′UTR region of CREB1.Figure 5.

Bottom Line: Inhibition of neuronal firing by miR-124 was associated with the suppression of mEPSC, AMPAR- and NMDAR-mediated currents, which were accompanied by decreased surface expression of NMDAR.In addition, miR-124 injection resulted in decreased activity and expression of cAMP-response element-binding protein1 (CREB1). a key regulator in epileptogenesis.Immunoprecipitation studies confirmed that the CREB1 antibody effectively precipitated CREB1 and NMDAR1 but not GLUR1 from rat brain hippocampus.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology,Chongqing Key Laboratory of Neurology,The First Affiliated Hospital of Chongqing Medical University,1 Youyi Road,Chongqing 400016,China.

ABSTRACT
miR-124, a brain-specific microRNA, was originally considered as a key regulator in neuronal differentiation and the development of the nervous system. Here we showed that miR-124 expression was suppressed in patients with epilepsy and rats after drug induced-seizures. Intrahippocampal administration of a miR-124 duplex led to alleviated seizure severity and prolonged onset latency in two rat models (pentylenetetrazole- and pilocarpine-induced seizures), while miR-124 inhibitor led to shortened onset latency in pilocarpine-induced seizure rat models. Moreover, the result of local field potentials (LFPs) records further demonstrated miR-124 may have anti-epilepsy function. Inhibition of neuronal firing by miR-124 was associated with the suppression of mEPSC, AMPAR- and NMDAR-mediated currents, which were accompanied by decreased surface expression of NMDAR. In addition, miR-124 injection resulted in decreased activity and expression of cAMP-response element-binding protein1 (CREB1). a key regulator in epileptogenesis. A dual-luciferase reporter assay was used to confirm that miR-124 targeted directly the 3'UTR of CREB1 gene and repressed the CREB1 expression in HEK293T cells. Immunoprecipitation studies confirmed that the CREB1 antibody effectively precipitated CREB1 and NMDAR1 but not GLUR1 from rat brain hippocampus. These results revealed a previously unknown function of miR-124 in neuronal excitability and provided a new insight into molecular mechanisms underlying epilepsy.

No MeSH data available.


Related in: MedlinePlus