DataSheet4_Deficiency of autism susceptibility gene Trio in cerebellar Purkinje cells leads to delayed motor impairments.zip

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by social interaction deficits, restricted interests and repetitive behaviors. The co-occurrence of motor impairments exacerbates the severity and societal impact of ASD, but the underlying mechanism remains to be elucidated. Research on the comorbidities of ASD including motor impairments could benefit in the life quality improvement in patients with ASD. Here we aimed at investigating the motor behaviors in mice with Trio deletion in Purkinje cells (PCs), and further exploring the cellular and molecular mechanisms. The protein level of Calbindin as PCs’ marker was determined. Behaviors including spontaneous locomotion activity, rotarod, beam balance and gait were tested in mice with the ages of 12-week and 20-week. Magnetic resonance imaging (MRI) scanning with T2 and DTI sequencing was performed in 12-week old mice. Although Triofl/fl; Pcp2-Cre mice showed significant impairments of spontaneous locomotion activity in both 12-week and 20-week ages, only the 20-week but not 12-week Triofl/fl; Pcp2-Cre mice showed extra mild abnormal motor, fine motor coordination, and gait. The decreased expression of Calbindin existed in both 12-week and 20-week old mice compared with control. Differentially expressed genes analysis from RNA-Seq and Gene Co-expression Network Analysis (GCNA) showed that Syne1 and its co-expressed genes were upregulated in Triofl/fl; Pcp2-Cre mice compared to controls. In addition, abnormal ADC values suggested the long-term chronic damage in the cerebellum. Together, our findings indicate that the motor dysfunction in ASD are affected by Trio deletion in PCs with delayed in onset, accompanied with alterations in MRI, histological, and epigenetic level.

孤独症谱系障碍（Autism Spectrum Disorder, ASD）是一类以社交互动缺陷（social interaction deficits）、狭隘兴趣与重复刻板行为（restricted interests and repetitive behaviors）为核心特征的神经发育障碍（neurodevelopmental disorders）。运动障碍共病会加重ASD的病情严重程度与社会影响，但其潜在机制仍有待阐明。针对ASD合并运动障碍等共病的研究，有助于提升ASD患者的生活质量。本研究旨在探究浦肯野细胞（Purkinje Cells, PCs）特异性Trio基因敲除小鼠的运动行为表型，并进一步解析其细胞与分子层面的调控机制。

研究人员检测了作为PCs标志物的钙结合蛋白（Calbindin）的蛋白表达水平；对12周龄与20周龄小鼠开展了自发活动、旋转棒实验、平衡木实验与步态分析等行为学检测；对12周龄小鼠进行了T2加权成像与扩散张量成像（Diffusion Tensor Imaging, DTI）序列的磁共振成像（Magnetic Resonance Imaging, MRI）扫描。

实验结果显示：相较于对照组，Triofl/fl; Pcp2-Cre小鼠在12周龄与20周龄时均出现显著的自发活动能力受损；但仅20周龄的Triofl/fl; Pcp2-Cre小鼠表现出轻度异常的粗大运动、精细运动协调与步态，12周龄小鼠未出现该类表型。相较于对照组，12周龄与20周龄的Triofl/fl; Pcp2-Cre小鼠的Calbindin蛋白表达均出现下调。

通过转录组测序（RNA-Seq）的差异表达基因分析与基因共表达网络分析（Gene Co-expression Network Analysis, GCNA）发现，相较于对照组，Triofl/fl; Pcp2-Cre小鼠脑中的Syne1及其共表达基因表达上调。此外，异常的表观扩散系数（Apparent Diffusion Coefficient, ADC）值提示小鼠小脑存在长期慢性损伤。

综上，本研究结果表明，ASD相关的运动功能障碍可由浦肯野细胞中Trio基因缺失介导，且该表型存在起病延迟的特征，同时伴随磁共振成像、组织学与表观遗传层面的异常改变。