智力障碍相关蛋白CUL4B/Jab1和干细胞微光环境整合调控神经发生的分子机理研究

There is a limited understanding of the molecular mechanisms underlying X-linked mental retardation (XLMR or XLID) pathogenesis, intervention and treatments. Cullin 4B (CUL4B) is a scaffold protein involved in the assembly of cullin-RING ubiquitin ligase (E3) complexes. Contemporary reports have identified multiple mutations of CUL4B gene as being causally associated with XLMR. Our studies explored (1) c-Jun activation domain-binding protein (Jab1) in the COP9 signalosome complex as a novel proteolytic target for the CUL4B ubiquitin ligase complex. (2) low light-emitting diode (LED) irradiation promoted the murine neural stem/progenitor cells (NSCs) proliferation and blocked the neuronal differentiation through elevation of Jab1. These findings are discussed with regard to the known function of Jab1 as a negative regulator of p27, MEF2C and Smad7 by promoting their degradations. Based on these observations, we would conduct a series of experiments to reveal the molecular mechanism for regulation of stem cell behavior involved with the CUL4B/Jab1 axis. LED irradiation is a potentially photostimulator to manipulate stem cell microenvironment through opsin-triggered phototransduction in NSCs. Furthermore, we would reveal the blue, green and NIR (Near Infrared) LED irradiation-induced effects on neurogenesis at the molecular, cellular and system levels. Delineation these mechanisms may be important in the application of in vivo recruitment of endogenous NSCs with the use of noninvasive LED manipulation. Taken together, the proposed grant may have significant implications for the pathogenesis and intervention in mental retardation disease.

在神经系统相关罕见病中，智力障碍致病基因突变导致脑发育异常的病因机制和干预治疗策略迫切需要运用新的思路和方法去探索。前期研究发现（1）XLMR致病基因CUL4B突变导致Jab1蛋白泛素化降解异常的新通路。（2）低能级LED“微光”操控影响神经干细胞增殖分化的新现象，并初步证实Jab1蛋白在此过程中处于调控位置并蕴含重要功能。综合前期研究基础和科学发展趋势，课题拟利用Cul4bNestin-Cre小鼠模型，系统研究CUL4B/Jab1在干细胞行为及神经功能塑造中的调控机理。在此病因机制基础上，利用交叉学科技术手段，采用细胞和动物模型并重、单纯和组合光谱技术，体内和体外指标相结合的方法，从“非基因修饰光操控”的新视角解析干细胞微环境对神经发生的调控。探讨内因 (CUL4B/Jab1) 和外因 (干细胞微光环境) 整合调控神经发生的分子机理，将为病因机制和干预策略发展提供原创性的理论和实验依据。

智力障碍等发育异常疾病的神经损伤机制和干预治疗策略迫切需要从新的视角、运用新的方法去探讨。在本项目资助下，我们发现神经干细胞NSCs和间充质干细胞MSCs表达光敏感蛋白。无需病毒载体转染外源基因，特征参数蓝光（455nm，300µW/cm2）显著提升小鼠神经干细胞增殖分化行为。分子机制研究显示光感受/光传导信号通路为Melanopsin-TRPC6-Jab1，Jab1蛋白在这其中处于核心调控位置。体内实验研究显示成年小鼠深部脑区同样表达此光感受/光信号转导通路，进一步结合红外上转换光遗传操控，通过非线性效应在大脑深处诱导产生所需蓝光。研究结果显示该技术可以成功诱导内源性神经干细胞进行分化，实现了非基因修饰/非侵入式神经干细胞的体内原位动员。我们利用蓝光操控和血管内皮细胞共培养体系，发现人脐带间充质干细胞（供体细胞）分泌外泌体（MSC-Exs）可被内皮细胞（受体细胞）吸收。血管环形成和基质胶塞模型实验结果显示体外/体内血管新生显著增强。特征蓝光刺激后，miR-135-5p和miR-499-3p在MSC-Exs外泌体中富集并促进血管形成。此外，我们基于光照产生的热释电效应，通过改变水滴湿润性操控其运动变化，提出了一种新的光操控方法，对于后续计划开展的细胞原位操控提供了原理性支撑和指标参数。本项目研究结果发现了干细胞的光响应特征，加深了对脑发育异常中神经调控策略的科学理解，揭示了非基因修饰/非侵入式光调控可以实现对干细胞的动员和功能重塑。本项目结果对于全面阐释脑发育异常病因机制和有效干预治疗提供了重要的理论和实验依据。