微管结合蛋白STOP参与孤独症发病的机制研究

Autism is a highly disabling neurodevelopmental disorder, however the pathogenesis of the disorder is still unknown. Increasing evidence suggests that abnormalities in synaptic function and myelination may contribute to phenotypic deficits. The microtubule-associated proteins (MAPs) are proteins that interact with the microtubules of the cellular cytoskeleton and participate in determining axon guidance and synaptic plasticity in nervous sysytem. Previous research showed that stable tubule only polypeptide (STOP, also known as MAP6) protein was significantly reduced in the plasma of autistic subjects and the brain from BTBR mice, a mouse model that displays an autism-like phenotype. Further, the evoked glutamate release and the levels of myelin-related proteins in the cerebral cortex of BTBR mice were significantly lower than that in B6 mice with high level sociability. The mechanism underlying STOP protein involved in the mediation of autism-like behaviors through regulating synaptic function and myelination is poorly understood. In this project, using the BTBR mouse model of autism, the STOP protein will be locally elevated by intracerebroventricular injection of STOP lentivirus. The behavioral tests including social behaviors, ultrasonic vocalizations and repetitive behaviors will be carried out to analyze whether the autism-like behaviors are modulated. The magnetic resonance spectroscopy, diffusion tensor imaging, immunofluorescence and electrophysiological techniques will be employed to explore the effects of STOP protein in the structure and function of excitatory synapse in neurons and myelination in oligodendrocytes in vivo and in vitro. The goal of this study is to investigate the possible mechanism of microtubule-associated protein STOP in the pathogenesis of autism.

孤独症是一种严重的神经发育障碍性疾病，其发病机制尚不明确。近来的研究表明，突触发育和髓鞘化异常是孤独症发病的重要因素。微管结合蛋白是神经细胞骨架的关键成分，参与轴突导向及突触发育。项目组前期的研究发现，微管结合蛋白STOP在孤独症患者外周血和动物模型BTBR小鼠大脑皮层显著低表达；BTBR小鼠兴奋性递质谷氨酸的释放及髓鞘相关蛋白的表达明显减少，但两者间的作用机制仍不清楚。本项目拟利用BTBR小鼠作为研究对象，通过增强脑内STOP蛋白的表达，考察其对小鼠孤独症相关行为学的效应，进一步证实其在孤独症发病中的作用；运用磁共振波谱和扩散张量成像技术从整体层面，采用形态学、免疫学和电生理学等方法从细胞分子层面，体内和体外研究STOP蛋白对神经元兴奋性突触结构和传递功能以及少突胶质细胞髓鞘化的影响，探讨微管结合蛋白STOP参与孤独症发病的作用机制，以期为治疗孤独症提供新的理论基础和实验依据。

孤独症是一种严重的神经发育障碍性疾病，其发病机制尚不明确。近来的研究表明，突触发育和髓鞘化异常是孤独症发病的重要因素。微管结合蛋白是神经细胞骨架的关键成分，参与轴突导向及突触发育。本项目的主要内容是以孤独症模型BTBR小鼠作为研究对象，增强脑内STOP蛋白的表达，考察其对BTBR小鼠孤独症相关行为学的效应，探讨微管结合蛋白STOP参与孤独症发病的作用机制。在本研究项目的资助下，研究以孤独症模型BTBR小鼠为对象，项目的主要研究发现包括：⑴通过侧脑室局部注射导入STOP基因和腹腔注射微管稳定剂Epothilone D两种方法，增强了脑内STOP蛋白的表达；⑵侧脑室局部注射STOP基因慢病毒后，孤独症BTBR小鼠的自我理毛修饰行为有减少的趋势，大脑皮层神经元突触蛋白Shank3和髓鞘相关蛋白MBP的表达没有明显差异，磁共振波谱结果显示谷氨酸代谢没有明显改变；⑶腹腔注射Epothilone D后，孤独症BTBR小鼠的自我理毛修饰行为明显减少，社交行为能力有增加的趋势；BTBR小鼠大脑皮层神经元兴奋性突触的数目、突触蛋白的表达以及突触后致密物厚度明显增加；髓鞘相关蛋白MAG、MBP的表达增加；⑷构建了BTBR胎鼠大脑皮层神经元和新生鼠少突胶质前体细胞的原代培养体系；⑸建立了BTBR小鼠大脑皮层miRNA与mRNA表达谱，分析了差异表达miRNA与mRNA的负调控网络；⑹检测了孤独症BTBR小鼠大脑皮层新型蛋白质翻译后修饰的表达，BTBR小鼠大脑皮层的蛋白巴豆酰化、琥珀酰化表达水平升高。本项目的研究结果进一步充实了微管结合蛋白调节微管功能影响脑发育中突触和髓鞘功能进而参与孤独症发病的研究理论。