Table_2_MECP2 Mutation Interrupts Nucleolin–mTOR–P70S6K Signaling in Rett Syndrome Patients.pdf

Rett syndrome (RTT) is a severe and rare neurological disorder that is caused by mutations in the X-linked MECP2 (methyl CpG-binding protein 2) gene. MeCP2 protein is an important epigenetic factor in the brain and in neurons. In Mecp2-deficient neurons, nucleoli structures are compromised. Nucleoli are sites of active ribosomal RNA (rRNA) transcription and maturation, a process mainly controlled by nucleolin and mechanistic target of rapamycin (mTOR)–P70S6K signaling. Currently, it is unclear how nucleolin–rRNA–mTOR–P70S6K signaling from RTT cellular model systems translates into human RTT brain. Here, we studied the components of nucleolin–rRNA–mTOR–P70S6K signaling in the brain of RTT patients with common T158M and R255X mutations. Immunohistochemical examination of T158M brain showed disturbed nucleolin subcellular localization, which was absent in Mecp2-deficient homozygous male or heterozygote female mice, compared to wild type (WT). We confirmed by Western blot analysis that nucleolin protein levels are altered in RTT brain, but not in Mecp2-deficient mice. Further, we studied the expression of rRNA transcripts in Mecp2-deficient mice and RTT patients, as downstream molecules that are controlled by nucleolin. By data mining of published ChIP-seq studies, we showed MeCP2-binding at the multi-copy rRNA genes in the mouse brain, suggesting that rRNA might be a direct MeCP2 target gene. Additionally, we observed compromised mTOR–P70S6K signaling in the human RTT brain, a molecular pathway that is upstream of rRNA–nucleolin molecular conduits. RTT patients showed significantly higher phosphorylation of active mTORC1 or mTORC2 complexes compared to age- and sex-matched controls. Correlational analysis of mTORC1/2–P70S6K signaling pathway identified multiple points of deviation from the control tissues that may result in abnormal ribosome biogenesis in RTT brain. To our knowledge, this is the first report of deregulated nucleolin–rRNA–mTOR–P70S6K signaling in the human RTT brain. Our results provide important insight toward understanding the molecular properties of human RTT brain.

雷特综合征（Rett syndrome, RTT）是一种严重且罕见的神经疾病，由X连锁的MECP2（methyl CpG-binding protein 2，甲基CpG结合蛋白2）基因突变所致。MeCP2蛋白是脑内及神经元中重要的表观遗传因子。在Mecp2缺陷神经元中，核仁结构会受到破坏。核仁是核糖体RNA（ribosomal RNA, rRNA）转录与成熟的活跃位点，这一过程主要由核仁素（nucleolin）及雷帕霉素靶蛋白（mechanistic target of rapamycin, mTOR）-P70S6K信号通路调控。
目前尚不清楚，来自雷特综合征细胞模型的核仁素-rRNA-mTOR-P70S6K信号通路如何转化至人类雷特综合征脑组织中。本研究针对携带常见T158M与R255X突变的雷特综合征患者脑组织，对核仁素-rRNA-mTOR-P70S6K信号通路的组成成分展开了研究。对T158M突变患者脑组织的免疫组织化学检测显示，核仁素的亚细胞定位出现紊乱；而与野生型（wild type, WT）相比，Mecp2缺陷的纯合雄性或杂合雌性小鼠脑组织中并未出现该现象。
我们通过蛋白质印迹分析证实，雷特综合征患者脑组织中的核仁素蛋白水平发生了改变，但在Mecp2缺陷小鼠脑组织中并未观察到此变化。进一步地，我们针对Mecp2缺陷小鼠与雷特综合征患者体内的rRNA转录本表达情况进行了研究，这类转录本属于核仁素调控的下游分子。通过对已发表的染色质免疫沉淀测序（Chromatin Immunoprecipitation Sequencing, ChIP-seq）研究数据进行挖掘，我们发现小鼠脑组织中存在MeCP2结合多拷贝rRNA基因的现象，这提示rRNA可能是MeCP2的直接靶基因。
此外，我们在人类雷特综合征脑组织中观察到mTOR-P70S6K信号通路功能受损，该分子通路是rRNA-核仁素分子轴的上游调控通路。与年龄、性别匹配的对照个体相比，雷特综合征患者体内活性形式的mTOR复合物1（mTORC1）或mTOR复合物2（mTORC2）的磷酸化水平显著升高。对mTORC1/2-P70S6K信号通路的相关性分析发现，该通路存在多个偏离正常对照组织的节点，这些异常可能导致雷特综合征脑组织中核糖体生物发生过程异常。
据我们所知，本研究是首次报道人类雷特综合征脑组织中核仁素-rRNA-mTOR-P70S6K信号通路失调。本研究结果为阐明人类雷特综合征脑组织的分子特性提供了重要见解。