CUL4B/ROS在智力障碍中的新型分子机制及其干预策略研究

Mental retardation research is one of the most important topics in the medical research. Recent studies have revealed that germ-line mutations in CUL4B can cause mental retardations in humans. CUL4B is a member of the cullins family, which provides "scaffolding" for the largest known E3 ubiquitin ligases, the cullin-RING ubiquitin ligases. We have demonstrated that Peroxiredoxin III （PrxIII） is a novel specific substrate of the CUL4B ligase complex with use of two-dimensional gel electrophoresis coupled with mass spectrometry. There is a significant decrease in cellular reactive oxygen species (ROS) production in CUL4B-silenced cells, which is associated with accumulation of PrxIII. Furthermore, .the knockout mice experiments suggest CUL4B is involved in the cortex neuronal synaptic plasticity. The majority of research on ROS has focused on their cellular toxicities. However, recent studies suggest that proliferative, self-renewing multipotent neural stem cells maintain a high ROS status and are highly responsive to ROS stimulation. Based on all these new findings, we proposed the hypothesis: the decreased ROS might interfere with the neurogenesis of the neural stem cells and then cause the damages of cortex neuronal synaptic plasticity. We would use the Cul4b knockout mice model to identify the ROS/PrxIII functions in the self-renewal and multipotency of the neural stem cells, to elucidate the ROS/PrxIII functions in the neuronal synaptic plasticity, and then further try to improve the neuronal synaptic plasticity with the use of the optogenetics technique. Therefore, our research would have significant implications for the pathogenesis observed in mental retradation patients and also provide a new light for the new intervention strategies for these kinds of diseases.

智力障碍是医学研究的重要课题之一。多个种族家系发现CUL4B基因突变导致智力障碍。CUL4B作为支架蛋白参与组成E3 泛素连接酶。申请者前期研究发现一个新现象，CUL4B 缺失导致抗氧化蛋白PrxIII累积，引起细胞内活性氧(ROS)水平下降；细胞和动物实验提示CUL4B参与调控皮层神经元的突触可塑性。基于此，并结合国内外有关ROS对神经系统影响的新认识，申请者提出本课题假说：异常下降的ROS损伤神经干细胞, 破坏皮层神经元突触可塑性，导致智力障碍。本课题将围绕该假说，利用Cul4b脑组织敲除小鼠，明确CUL4B/ROS调控神经干细胞自我更新和多向分化的核心环节，确定CUL4B/ROS对皮层神经元突触可塑性的影响和机制。并在此基础上，针对已经建立的单基因突变疾病模型小鼠，探索利用光遗传学策略改善突触可塑性。研究的实施将有助于进一步揭示智力障碍的发病机理，为疾病的干预治疗提供有价值的新策略。

有关智力障碍致病基因CUL4B的功能和致病机理的研究具有重要的科学意义和临床价值。课题组自2013年系统研究了CUL4B 丧失功能突变导致CUL4B E3泛素连接酶功能破坏造成的影响。经过四年的努力，课题组 （1）系统完善了基于二维电泳－质谱联用技术的差异蛋白质筛选鉴定体系，针对细胞，组织及血清等各体系均发展优化了对应的技术路线。此外，针对LED蓝光影响神经细胞分化的研究结果，建立了初步的技术平台。（2）发现Jab1是CUL4B 泛素连接酶的特异性底物，并解释了CUL4B 突变引起Jab1累积导致神经发育异常的分子机制。（3）利用神经系统Cul4b基因敲除小鼠(Cul4bNestin-Cre)模型，系统研究了CUL4B及其特异性底物PrxIII对神经发育过程的影响。在Cul4bNestin-Cre小鼠前脑组织及分离培养的神经干细胞中均发现PrxIII蛋白累积，ROS水平降低。CUL4B通过PrxIII结合PTEN，影响AKT信号通路，导致神经干细胞自我更新能力降低及定向分化过程异常。（4）CUL4B突变患者除了智力发育迟缓以外，还表现出单核细胞增多等血液系统异常的临床症状。课题组发现miR－26a－5p 和miR－23b－3p下调，导致靶基因PrxIII异常高表达，细胞内ROS下降影响造血细胞分化。以上结果一定程度上阐明了CUL4B基因丧失功能突变的致病机理。（5）前期工作提示CUL4B基因对心脏发育过程中的关键基因MEF2C可能有一定的调节作用。课题组的研究工作显示miRNA-135b-5p和miR-499a-3p异常上调会抑制MEF2C基因表达，从而引起血管内皮细胞和平滑肌细胞增殖和迁移增加，为CUL4B基因功能研究做了很好的补充。项目研究结果为进一步探讨智力障碍的发病机理和大脑正常发育进程的分子机制，探索有价值的干预策略提供了一定的指导意义。