The Effect Of MRTF-A Protecting Synapses Injury–induced By Aβ-(25-35) And Its Mechanisms

Objectives: To investgate the effect of MRTF-A on rat’s cortex synapses injury–inducedby Aβ25-35and its mechanisms.Methods:1. To estimate the behavior, the expressions of neurofilament, MRTF-A and CaMKⅡon a rat’s Alzheimer model: the rat’s Alzheimer model was established by ventricleinjection of Aβ25-35(AP:-3.8mm, ML:±3.0mm, DV:-3.2mm), and then the rats’ behaviorwas determined by the method of morris water maze，and the expressions ofneurofilament medium, MRTF-A, CaMKⅡand were respectively detected byImmunohistochemistry, Immunofluorescence, laser confocal scanning microscopy.2. Effect of MRTF-A on neurofilament medium expression of primary cortex neuronspretreated with Aβ25-35: primary cortex neurons were divided into control group, Aβ25-35treated-group and MRTF-A transfected-group. MRTF-A transfected-group wastransfected WT-MRTF-A plasmid for48h, then receiving20μΜ Aβ25-35for24h; whilecontrol group and Aβ25-35treated-group were transfected vetor, then receiving equalvolume of stroke-physiological saline solution. The neurofilament medium of cortexneurons was observed under a laser confocal scanning microscopy.3. Effect of MRTF-A on Arc expression was analyzed by RT-PCR and Western blotanalysis: The primary cortex neurons were divided into control group, Aβ25-35treated-group and MRTF-A transfected-group. MRTF-A transfected-group wastransfected WT-MRTF-A plasmid for48h, then receiving20μΜ Aβ25-35for24h; whilecontrol group and Aβ25-35treated-group were transfected vetor, then receiving equalvolume of stroke-physiological saline solution. Arc expression at mRNA and proteinlevels in all groups was analyzed by the methods of RT-PCR and Western blotrespectively.4. To investigate whether the upregulation of MRTF-A on Arc expressions was viatrigging CArG box on Arc genic promoter：the primary cortex neurons were dividedinto vetor group, control group, Aβ25-35treated-group and MRTF-A transfected-group.Vetor group was transfected vetor plasmid (0.4μg); control group was co-transfectedwith Arc luciferase reporter plasmid (0.2μg) and vetor plasmid (0.2μg) or DN-Arc-luciferase reporter plasmid (0.2μg) and vetor plasmid (0.2μg); Aβ25-35treated-group was the same with control group; MRTF-A transfected-group wasco-transfected with Arc luciferase reporter plasmid (0.2μg) and WT-MRTF-A plasmid(0.2μg) or DN-Arc luciferase reporter plasmid (0.2μg) and WT-MRTF-A plasmid(0.2μg). After48h transfection, Aβ25-35treated-group and MRTF-A transfected-groupwere treated with20μM Aβ25-35for24h, the rest two groups received equal volume ofstroke-physiological saline solution. Arc luciferase reporter activity and DN-Arcluciferase reporter activity were analyze by Dual-Luciferase Reporter Assay System.5.To determine wether the upregulation of MRTF-A on Arc expression is related toCaMKⅡ: KN-62was used to selectively inhibite CaMK Ⅱ.Cultured cortex neuronswere divided into control group, Aβ25-35treated-group and MRTF-A transfected-group,different concentrations of KN-62(5mM,10mM,20mM)-treated groups. EndogenousArc protein expression was analyzed by Western blot.Results:1. The rat behavior results suggested that Aβ25-35ventricle injection in rats significantlydecreased learning and memory abilities. Rat’s brain injected with Aβ25-35expressed lessneurofilament medium compared with control group, but obviously increasing MRTF-Aand CaMK Ⅱexpressions.2. In vitro cultured cortex neurons, MRTF-A overexpression increased neurofilamentmedium expression and neuronal structure integrity. RT-PCR and Western blot testsindicated: MRTF-A overexpression significantly reversed Aβ25-35downregulating Arc(Activity-regulated cytoskeletal-associated protein) expression (p<0.05). Otherwise,according to the results of Arc luciferase reporter or DN-Arc luciferase reportertranscription activity, MRTF-A significantly increased the Arc luciferase reportertranscription activity (p<0.05), but had no effect on transcription activity of DN-Arcluciferase reporter.3. Western blot test results showed that KN-62significantly inhibited MRTF-A inducedArc expression in a dose-dependent manner (p<0.05or p<0.01).Conclusion:1. AD model rats indicate obvious reduction in the ability of learning and memory,increasing MRTF-A and CaMK Ⅱ expression, indicating MRTF-A and CaMK Ⅱmaybe involved in the pathological process of AD. 2. MRTF-A sustained neuronal structure integrity of cortex neurons injuried by Aβ25-35.MRTF-A overexpression, via trigging CArG box, enhanced the expression of synapticplasticity-related protein Arc in mRNA and protein levels respectively.3. CaMKⅡ selective inhibitors KN-62signicantly upregulated Arc expression,suggesting CaMK Ⅱ is involved in MRTF-A upregulating Arc.