孤独症谱系障碍相关转录因子EB基因罕见变异导致神经元轴突发育障碍  

Rare variants of transcription factor EB gene associated with autism spectrum disorder lead to neuronal axon developmental disorder

作　　者：胡龙妃 谭洁琼[2] 胡章雪 HU Longfei;TAN Jieqiong;HU Zhangxue(Department of Pediatrics,Army Specialty Medical Center,Chongqing 400010,China;Medical Genetics Research Center,School of Life Sciences,Central South University,Changsha 410000,Hunan,China)

机构地区：[1]陆军特色医学中心儿科,重庆400010 [2]中南大学生命科学学院医学遗传学研究中心,湖南长沙410000

出　　处：《中南医学科学杂志》2023年第6期797-802,共6页Medical Science Journal of Central South China

基　　金：国家科技部科技创新2030-“脑科学与类脑研究”重大项目(2021ZD0201700);国家卫生健康委员会出生缺陷研究与预防重点实验室(湖南省妇幼保健院)开放课题(KF2021001)。

摘　　要：目的探究孤独症谱系障碍(ASD)中发现的转录因子EB(TFEB)基因3种变异对神经元轴突发育的影响及其机制。方法构建与ASD相关的3个TFEB突变体质粒和TFEB shRNA干扰质粒,细胞转染上述质粒后通过免疫荧光、细胞核质分离及蛋白免疫印迹检测TFEB变异是否改变其亚细胞定位;利用核糖体抑制剂CHX处理后经蛋白免疫印迹检测TFEB变异对其蛋白稳定性的影响;进行原代小鼠神经元培养并转染上述质粒,通过免疫荧光检测TFEB变异对神经元轴突生长发育的影响;使用实时定量PCR检测TFEB下游自噬-溶酶体基因的表达水平,以荧光素酶报告基因法测量TFEB的转录活性。结果ASD相关突变体p.R22Q及p.R465W导致TFEB在细胞核的定位增加,但变异并不改变蛋白的稳定性。同时,野生型(WT)TFEB的过表达可增加神经元轴突长度,而ASD相关的TFEB突变体则不能。这表明这些突变导致TFEB促进神经元轴突生长的功能受损。同时,自噬激活剂Rapamycin可恢复由TFEB干扰引起的神经元轴突缩短。TFEB WT的过表达增加了下游自噬-溶酶体相关靶基因(LAMP1、SQSTM1、CTSB、CTSD、CTSF、MAPLC3)的表达,但ASD相关的TFEB突变体降低了对相关靶基因的调节能力。结论TFEB变异可能导致自噬-溶酶体功能受损和神经元发育异常,为ASD发病机制和潜在治疗靶点提供了新的见解。Aim To investigate the effects of three variants of transcription factor EB(TFEB)found in autism spectrum disorder(ASD)on neuronal axon development and the mechanisms.Methods Three TFEB plasmid mutants associated with ASD and TFEB shRNA interference plasmids were constructed.After cell transfection with the above plasmids,immunofluorescence,cell nucleus-cytoplasm separation,and protein immunoblotting were used to detect whether TFEB mutations change its subcellular localization.The effect of TFEB mutations on its protein stability was detected by protein immunoblotting after treatment with ribosomal inhibitor CHX.Primary cultured mouse neurons were transfected with the above plasmids,and the effect of TFEB mutations on neuronal axon growth and development was detected by immunofluorescence.Quantitative real-time PCR was used to detect the expression level of TFEB downstream autophagy-lysosome genes,and the transcriptional activity of TFEB was measured by luciferase reporter gene assay.Results ASD-related mutants p.R22Q and p.R465W increased the localization of TFEB in the cell nucleus,but the mutations did not change the protein's stability.Meanwhile,overexpression of wild-type(WT)TFEB,but not ASD-related TFEB mutants,increased neuronal axon length.This indicates that these mutations impair TFEB's function in promoting neuronal axon growth.At the same time,autophagy activator Rapamycin can restore the axon shortening caused by TFEB interference.Overexpression of TFEB WT increased the expression of autophagy-lysosome-related downstream target genes(LAMP1,SQSTM1,CTSB,CTSD,CTSF,MAPLC3),but ASD-related TFEB mutants reduced their regulatory ability on related target genes.Conclusion TFEB mutations may lead to autophagy-lysosome dysfunction causing abnormal neuronal axon development,which may be one of the mechanisms associated with ASD.

关 键 词：孤独症谱系障碍 TFEB 自噬-溶酶体 

分 类 号：Q987[生物学—遗传学]