肥胖主效基因SH2B1介导IGF-1R信号通路影响ASD认知和社交功能的机制研究

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders. It has been identified that obesity could aggravate the impairment of cognition and social function in ASD model mouse. Our previous work has reported that methylation levels of SH2B1 were significantly different between identical twins with or without ASD, which implied that SH2B1 gene was correlated to ASD. In the current study, ASD children and BTBR model mice with Sh2b1 knock-out (BTBR+Sh2b1-/-mice) are used as the research objects to analyze the effect of obesity and its major gene SH2B1 on cognitive and social function of ASD. We explore whether SH2B1 mediated Insulin-like growthfactor-1 receptor (IGF-1R) signal pathway to regulate neuronal function impairment and apoptosis, by the neurons derived from human induced pluripotent stem cells (hiPSCs) from ASD patients and BTBR+Sh2b1-/-mice. Furthermore, we also use path clamp technique to detect the effect of SH2B1 mediated IGF-1R signal pathway on voltage-gated potassium channel based on PI3K/Akt/mTOR signal transduction pathways, to explore the potential mechanism whether SH2B1 mediated IGF-1R signal pathway affects cognitive and social function, which could provide new insight contributed to comprehensive intervention of ASD.

孤独症谱系障碍（ASD）是一种严重的神经发育障碍性疾病。研究表明肥胖可加重ASD模型鼠的认知及社交损伤。本课题组前期针对同卵双生非共患ASD的表观遗传学研究发现，肥胖主效基因SH2B1的甲基化水平存在明显差异，提示该基因与ASD有一定关联。本项目以ASD患儿及所构建的Sh2b1缺失的BTBR孤独症模型鼠为研究对象，明确肥胖及肥胖主效基因SH2B1对ASD认知及社交行为的影响；应用ASD人源性iPSCs和BTBR+Sh2b1-/-模型鼠来源的神经元共同探讨SH2B1是否介导IGF-1R信号通路造成ASD神经元功能损伤及凋亡；此外，采用膜片钳技术，阐明SH2B1基因参与IGF-1R对神经元Kv钾通道的调控以及下游信号转导通路PI3K/AKT/mTOR在其中的作用，从细胞和分子水平揭示肥胖主效基因SH2B1介导IGF-1R信号通路影响ASD认知和社交功能的机制，为开展ASD综合干预提供新的思路。

孤独症谱系障碍（ASD）是一种严重的神经发育障碍性疾病。研究表明肥胖可加重ASD模型鼠的认知及社交损伤。基于课题组前期针对同卵双生非共患ASD的表观遗传学研究发现，肥胖主效基因SH2B1的甲基化水平存在明显差异，提示SH2B1基因可能与ASD有一定关联。本研究通过人群病例-对照研究明确ASD与正常儿童体成分及肥胖检出率及其差异，证实了ASD儿童较正常儿童肥胖发生率明显增高。对ASD儿童肥胖与其发育水平进行关联研究发现，肥胖可以加重ASD儿童的认知和社交功能损伤。对ASD患者脑组织样本数据进行分析，证实肥胖主效基因SH2B1与ASD患者认知和社交功能高度相关。此外，成功构建了SH2B1基因缺失的ASD模型BTBR鼠和B6小鼠，并证实SH2B1基因缺失后的各组鼠体脂率均升高，同时进行行为学测试，证明肥胖及肥胖主效基因SH2B1缺失会加重ASD模型鼠认知和社交损伤。IGF -1R在大脑神经发育中起关键作用，本研究发现在SH2B1鼠敲除鼠中IGF-1R表达显著降低。分别在Neuro-2a、PC12细胞两种细胞系上构建SH2B1-/-稳转细胞系，给予IGF-1干预进行神经突长度测量，发现SH2B1和IGF-1同时存在的细胞中神经突的长度明显增高，提示SH2B1基因与IGF-1R通路共同作用会影响神经元的发育。通过分子生物学，证实SH2B1缺失时BTBR鼠海马组织中CREB、CaMKⅡ蛋白的磷酸化水平明显降低，GAD1蛋白水平显著升高，凋亡相关Bcl-2/Bax水平降低、CDK5水平升高，提示肥胖会加重BTBR鼠认知社交损伤，并与神经元凋亡和认知相关蛋白功能异常相关。通过Luminex检测技术，检测各组鼠神经炎性因子及IGF-1的表达水平，发现相比于B6鼠，BTBR鼠中BAFF和IL-33水平明显降低，SH2B1缺失时B6鼠海马组织中的IL-1F2、MMP-9以及IGF-1的水平明显升高，SH2B1缺失时BTBR鼠海马组织中的IL-1F2、IL-33、BAFF以及IGF-1的水平明显升高，提示SH2B1缺失加重ASD认知社交障碍损伤可能与炎性反应相关。本项目从人群研究到动物模型的机制验证，在整体、细胞及分子水平上，综合阐明了肥胖主效基因SH2B1对ASD认知社交功能的影响及可能机制，为ASD临床靶向干预提供新思路。