FMR1基因错义突变P626L导致DGCR8-microRNA通路的异常及其调控神经元退行性病变的分子机制

Fragile X mental retardation gene (FMR1，encoding fragile X mental retardation protein FMRP) plays an important role in brain development and synaptic plasticity. FMR1 is associated with neurodevelopmental and neurodegenerative disorders. It has been demonstrated that FMR1 pre-mutation can lead to fragile X-associated tremor and (or) ataxia syndrome (FXTAS), in which DGCR8-mediated microRNA pathway is involved. However, the molecular pathogenic mechanism underlying the FMR1 mutation in FXTAS is still poorly known. Our previous study identified a FMR1 missense mutation (P626L) in a FXTAS patient. Further analysis showed that the P626L mutation could downregulate DGCR8 expression at posttranscriptional level, suggesting a pathogenic effect of this mutation in FXTAS. The project will further investigate the role of wild-type and mutant FMRP in regulating the DGCR8-mediated microRNA pathway in vitro. We will construct a transgenic mouse containing this mutation and analyze the regulatory role of the P626L mutation in the expressions of microRNAs and their target genes by affecting DGCR8 expression at molecular and cellular levels. We will also analyze the changes in brain morphology and behavior in the transgenic mice. This study will reveal the molecular mechanisms underlying the neurodegenerative disease resulted from the FMR1 mutation.

脆性X智力低下基因（FMR1）编码蛋白FMRP具有调节脑发育和神经元突触可塑性作用。除了导致脆性X综合征外，目前已证实FMR1基因前突变携带者可产生一种退行性疾病- - 脆性X 相关震颤和(或)共济失调综合征（FXTAS），其中RNA结合蛋白DGCR8所参与的miRNA加工途径可能在FXTAS中发挥重要作用，但分子机制不明。在前期工作中我们首次发现一例FXTAS患者只有FMR1基因编码区错义突变（P626L），并证实该突变在转录后水平上负性调控DGCR8的表达，提示该突变通过与FMR1基因前突变不同的调控途径参与FXTAS发生。本项目将进一步研究正常及突变FMRP对DGCR8的表达调控途径，并且通过构建突变转基因小鼠，分析该突变通过DGCR8改变miRNAs及其下游靶基因的表达情况，还将研究转基因小鼠脑组织形态结构和运动协调性变化，以期揭示FMR1基因突变在神经元退行性病变中的分子机制。

FMR1基因编码区发生错义突变时可影响多种生物学功能。本研究首次发现，FMR1基因错义突变P626L可影响FMRP与GRK2结合，在野生型小鼠纹状体中，FMRP与GRK2在胞浆中结合，不影响D1受体与多巴胺的相互作用；在P608L突变小鼠纹状体中，FMRP与GRK2结合能力下降，导致GRK2转移至细胞膜上与D1受体结合，引起D1受体磷酸化水平增高，从而引起运动障碍。该过程揭示了FMR1基因错义突变P626L在FXTAS中可能的发病机制。同时首次发现FMRP保守核定位信号（NLS）内的氨基酸132残基在不同物种间具有差异；证实了人类FMRP-D132E减少其核定位，而小鼠FMRP-E132D促进其核定位。人类FMRP可与poly（A）结合蛋白1（PABP1）以RNA依赖性方式相互作用；相反，小鼠FMRP不能与PABP1相互作用。本研究表明人类FMRP蛋白与氨基酸D132残基以蛋白质-RNA-蛋白质相互作用，这可能暗示FMRP残基转换和神经进化之间的联系。