The Effect And Mechnanism Of MRTF-A On Neuronal Apoptosis And Autophagy In Tg2576 Mice

Objective:The present study was to explore the effect of MRTF-A on neurons autophagy and apoptosis and its possible mechanisms in Tg2576 mouse hippocampus.Methods:Part 1 Effect of MRTF-A on the learning and memory function of Tg2576 mice1.To investigate the function of learning and memory and the level of endogenous hippocampus MRTF-A of Tg2576 mice Tg2576 mice and wild-type C57 BL mice(control)were selected after 7 days feeding adaptation.We first access the behavioral manifestations by Morris water maze and conditioned fear test,fresh hippocampus tissue were isolated at the end of the experiment.Real-time PCR and Western blot were employed to detect m RNA and protein levels of MRTF-A.2.Effect of MRTF-A overexpression on learning and memory function in Tg2576 mice MRTF-A lentiviral vector was constructed with a titer of 2 × 108 TU / ml.Tg2576 mice were randomly assigned and treated 4?l lentivitus by intracerebroventricular administration respectively,control group were treated with the same volume negative lentivitus.MRTF-A level in the hippocampus region were analyzed on the 7,14,21,28 day after the injection of lentivirus at the optimal volume.Congnitive functions were evaluated in conditioned fear test and open field test.Part 2 Effect of MRTF-A on neuroual apoptosis and autophagy of Tg2576 mice1.Effect of MRTF-A on neuronal apoptosis and autophagy in the hippocampus of Tg2576 mice Mice were randomly divided into control group(C57BL mice),model group(Tg2576mice)and experimental group(Tg2576 mice).Mice in control group and model group were treated with 4ul negative lentivirus,while mice in the experimental group weretreated with 4ul MRTF-A lentivirus solution.Brain tissues were taken for HE staining to observe morphological changes and TEM(transmission eletron microscope)were employed to observe autophagic bodies in the neuron of hippcampus on day 28 after the injection.2.Effect of MRTF-A expression on neuronal apoptosis and autophagy related genes in the hippocampus of Tg2576 mouse Animals were divided and treated as the third section.Apoptosis-related genes Bcl-2,mcl-1 and autophagy-related genes Atg5,Atg6,Atg7 m RNA levels in the hippocampus were measured 28 days after the lentivirus injection by Real-time PCR;Western blot were employed to exam the protein levels of apoptosis-related genes Bcl-2,mcl-1 and autophagy-related genes Beclin1,LC3.Result:1.Effect of MRTF-A on the learning and memory function of Tg2576 mice:(1)Learning and memory defects and the change of MRTF-A expresionin Tg2576 mice:Compared with the same week-old wild-type C57 BL mice,Tg2576 mice spent more time to reach the platform in the Morris water maze.In conditioned fear test,the percentage of freezing time of Tg2576 mice is significantly less than the wild-type C57 BL mice(P<0.01).And the endogenous MRTF-A expression level in hippocampus of Tg2576 mice is lower than that in wild-type C57 BL mice(P<0.01).(2)Overexpression of MRTF-A attenuated learning and memory impairment in Tg2576 mice: Weak fluorescence was observed after hippocampus injection of 1ul lentivirus,a single injection of 2ul and 4ul lentivirus expressed obvious fluorescence,fluorescence in hippocampus at the dose of 4ul was significantly higher than other groups,therefore the dose of 4ul was selected as the injection volume in the next experiment.Further validation of MRTF-A lentivirus overexpression in mice brain showed that,compared with the negative control lentivirus,MRTF-A expression level in the brain of lentiviral injection group reached their highest concentration at day 7 after injection,and decreased slowly until day 28,but still higher than that of the wild-type C57 BL mice.The tatal distance of movement in open field testdid not change on day 7,14,21,28 after lentivirus injection,while the time spent in center space was shortened on day 28.Conditioned fear test found that MRTF-A overexpression in hippocampus improved percentage of frezzing time on day 28 after lateral cerebral ventricle injection compared to the control group.2.Effect of MRTF-A on neuroual apoptosis and autophagy of Tg2576 mice:(1)HE staining on day 28 after lateral cerebral ventricle injection found that,LV-MRTF-A intervention can significantly reduce damage of the hippocampus neuron in Tg2576 mice.TEM observation found there were autophagic vacuoles surround with visible bilayers after MRTF-A treatment.(2)LV-MRTF-A interventions increased Bcl-2,mcl-1 expression,Atg5,Atg6,Atg7 m RNA expression increased,LC3 ? /LC3 ? ratio and Beclin 1expression.Conclusion:These findings suggest that Tg2576 mice shows learning and memory defects which is accompanied with low level of MRTF-A level in the hippocampus.Overexpression MRTF-A attenuates memory impairments.This may be related to MRTF-A's upregulation of anti-apoptotic gene Bcl-2,mcl-1 and autophagy-related genes Atg5,Atg6,Atg7,further inhibites hippocampus neuroral apoptosis and promote its autophagy in Tg2576 mice.