RNA m6A去甲基化酶ALKBH5基因敲除对老年小鼠小脑形态和功能的影响  

Effects of RNA M6A demethylase ALKBH5 gene deficiency on morphology and function of cerebellum in aged mice

作　　者：费祎 马春卉 李晴 宋伟[2] 佟伟民[1] 牛亚梅[1] Fei Yi;Ma Chunhui;Li Qing;Song Wei;Tong Weimin;Niu Yamei(Department of Pathology,Institute of Basic Medical Sciences Chinese Academy of Medical Science,School of Basic Medicine Peking Union Medical College,Beijing 100005,China;Department of Biochemistry and Molecular Biology,State Key Laboratory of Medical Molecular Biology,Institute of Basic Medical Sciences Chinese Academy of Medical Sciences,School of Basic Medicine Peking Union Medical College,Beijing 100005,China)

机构地区：[1]中国医学科学院基础医学研究所暨北京协和医学院基础学院病理学系,北京100005 [2]中国医学科学院基础医学研究所暨北京协和医学院基础学院、医学分子生物学国家重点实验室、生物化学与分子生物学系,北京100005

出　　处：《中华病理学杂志》2023年第6期606-611,共6页Chinese Journal of Pathology

基　　金：中国医学科学院医学与健康科技创新工程(2021-I2M-1-020)。

摘　　要：目的观察RNA m6A去甲基化酶ALKBH5基因敲除之后对老年小鼠小脑形态和功能的影响,探究ALKBH5在小脑退行性病变中的作用。方法免疫印迹实验检测不同年龄C57BL/6野生型小鼠(包括2月龄、6月龄、12月龄、18月龄)小脑中ALKBH5的蛋白水平。通过免疫组织化学实验检测中年(12月龄)及老年(18月龄)野生型小鼠和ALKBH5基因敲除(ALKBH5^(-/-))小鼠中NeuN、Calbindin-D28K、MAP2、胶质纤维酸性蛋白等蛋白的表达情况。平衡木实验和步态实验检测小鼠平衡能力以及运动协调能力。结果ALKBH5蛋白在小脑中的表达水平随着小鼠生理性衰老的进行而逐渐升高。在中年小鼠中,野生型和ALKBH5^(-/-)小鼠的体重、脑重以及小脑的形态大小没有明显差别,同时颗粒神经元、浦肯野细胞及神经元树突的形态和数量均未见明显改变。在老年小鼠中,相较于野生型小鼠,ALKBH5^(-/-)小鼠的体重、全脑重和小脑重分别下降15%、10%和21%(P<0.05)。ALKBH5在浦肯野细胞中的表达高于其他类型的神经细胞,与之对应在老年ALKBH5^(-/-)小鼠中浦肯野神经元的数量下降40%,树突的长度和密度也明显减少(P<0.01)。老年ALKBH5^(-/-)小鼠通过平衡木相较于同龄的野生型小鼠所用的时间增加70%,且出现步态不稳的情况(P<0.01)。结论RNA m6A去甲基化酶ALKBH5缺失会造成老年小鼠小脑萎缩、浦肯野神经元缺失与受损,进而损害其运动协调和平衡能力。上述结果提示RNA m6A甲基化失衡会导致小脑的神经退行性病变。Objective To investigate the effects of RNA m6A demethylase ALKBH5 gene deficiency on cerebellar morphology and function in the aged mice,and to explore the role of ALKBH5 in cerebellar degeneration.Methods Western blot was performed to detect the protein level of ALKBH5 in the cerebellum of wild-type mice of various ages.The expression of NeuN,Calbindin-D28K,MAP2,GFAP and other proteins in the cerebella of middle-aged(12-month-old)and aged(18-month-old)wild-type mice and ALKBH5^(-/-)mice was examined using immunohistochemistry.The balance beam test and gait analysis were performed to test the balance ability and motor coordination of the mice.Results With aging of the mice,the expression of ALKBH5 in the cerebellum increased gradually in an age-dependent manner.In the aged mice,but not middle-aged mice,the body weight,whole brain weight and cerebellum weight of ALKBH5^(-/-)mice decreased by 15%,10%and 21%,respectively(P<0.05).The expression of ALKBH5 in the Purkinje cells was much higher than that in other types of neural cells.Correspondingly,ALKBH5-deficiency caused 40%reduction in the number of Purkinje cells,as well as the length and density of neuronal dendrites in the aged mice(P<0.01).In addition,the time for the aged ALKBH5^(-/-)mice to pass the balance beam was 70%longer than that of the wild type mice of the same age,with unstable gaits(P<0.01).Conclusions Gene deficiency of RNA m6A demethylase ALKBH5 causes cerebellar atrophy,Purkinje neuron loss and damage in the aged mice.These changes eventually affect mice′s motor coordination and balance ability.These results suggest that imbalanced RNA m6A methylation may lead to neurodegenerative lesions in the cerebellum of mice.

关 键 词：小脑 浦肯野细胞 RNA m6A甲基化 ALKBH5 

分 类 号：R741[医药卫生—神经病学与精神病学]