目的 探究在小鼠海马齿状回(DG)神经元中特异性敲除Mecp2基因对成年海马神经发生(AHN)以及孤独症谱系障碍(ASD)样行为的影响,为基因异常导致的ASD治疗提供方向。方法 以Mecp2-flox小鼠为研究对象,分为Mecp2f/y组、Mecp2f/y-Con组、WT组和WT-Con组,每组6只。采用Western Blot检测MeCP2和脑源性神经营养因子(BDNF)蛋白表达水平。采用三箱交互、新物体识别、Y迷宫、Morris水迷宫、旷场、埋珠、自梳等行为学实验评估小鼠的ASD样行为。采用免疫荧光实验检测小鼠海马神经发生情况。结果 与Mecp2f/y-Con组及WT组小鼠相比,Mecp2f/y组小鼠社交能力指数、社交新奇指数均显著下降(P<0.05),新物体识别实验认知指数显著下降(P<0.001),Y迷宫新臂停留时间及进入次数显著下降(P<0.05),Morris水迷宫目标象限停留时间显著缩短(P<0.05)、平台穿越次数显著减少(P<0.05),旷场中央停留时间显著减少(P<0.05),埋珠数量、自梳次数及自梳时间均显著增加(P<0.05),Nestin+、Nestin+MCM2+、Tbr2+、Tbr2+MCM2+、DCX+、DCX+MCM2+和BrdU+NeuN+神经元细胞数量显著下降(P<0.05),BrdU+DCX+未成熟神经元细胞数量显著增加(P<0.01),BDNF蛋白表达水平显著下降(P<0.05)。结论 特异性敲除小鼠DG神经元中的Mecp2基因会影响AHN,诱发ASD样行为,为ASD的神经生物学机制提供了研究证据。
Abstract
Objective To investigate the effects of specific knockout of the Mecp2 gene in dentate gyrus (DG) neurons on adult hippocampal neurogenesis (AHN) and autism spectrum disorder (ASD)-like behaviors in mice, so as to provide potential therapeutic directions for ASD caused by genetic abnormalities. Methods Mecp2-flox mice were randomly divided into four groups: Mecp2f/y, Mecp2f/y-Con, wild-type (WT), and WT-Con groups,with 6 mice in each group. Western Blot was used to determine the protein expression levels of MeCP2 and brain-derived neurotrophic factor (BDNF).ASD-like behaviors were evaluated using the three-chamber sociability test, novel object recognition (NOR) test, Y-maze, Morris water maze (MWM), open field test, marble burying test, and self-grooming test. Immunofluorescence staining was utilized to assess hippocampal neurogenesis. Results Compared withMecp2f/y-Con group and WT group, Mecp2f/y mice exhibited significantly decreased sociability and social novelty indices (P<0.05), impaired cognitive performance indicated by a reduced recognition index in the NOR test (P<0.001), decreased duration and entries into the novel arm of the Y-maze (P<0.05),shortened target quatrant dwell time (P<0.05) and fewer platform crossings in the Morris water maze (P<0.05), reduced center dwell time in the open field test (P<0.05), and increased marble burying count, as well as elevated frequency and duration of self-grooming (P<0.05). Furthermore, significant reductions were observed in the number of Nestin+,Nestin+MCM2+,Tbr2+,Tbr2+MCM2+,DCX+,DCX+MCM2+ and BrdU+NeuN+ cells (P<0.05), while the number of BrdU+DCX+ immature neurons was significantly increased (P<0.01). The protein expression level of BDNF was significantly decreased in Mecp2f/y mice(P<0.05). Conclusion The specific knockout of the Mecp2 gene in mouse DG neurons disrupts AHN, and consequently induces ASD-like behaviors, which provide experimental evidence for the neurobiological mechanisms underlying ASD.
关键词
Mecp2基因 /
孤独症谱系障碍样行为 /
海马齿状回 /
海马神经发生 /
脑源性神经营养因子
Key words
Mecp2 gene /
autism spectrum disorder-like behaviors /
dentate gyrus /
adult hippocampal neurogenesis /
brain-derived neurotrophic factor
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基金
黑龙江省省属科研院所科研业务费项目(CZKYF2023-1-A010)
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