Wang Xiaoming, Xu Qiong, Bey Alexandra L, Lee Yoonji, Jiang Yong-Hui
Division of Medical Genetics, Department of Pediatrics, Duke University School of Medicine, 27710 Durham, NC, USA.
Division of Medical Genetics, Department of Pediatrics, Duke University School of Medicine, 27710 Durham, NC, USA ; Department of Child Health Care, Children's Hospital of Fudan University, 201102 Shanghai, China.
Mol Autism. 2014 Apr 25;5:30. doi: 10.1186/2040-2392-5-30. eCollection 2014.
Considerable clinical heterogeneity has been well documented amongst individuals with autism spectrum disorders (ASD). However, little is known about the biological mechanisms underlying phenotypic diversity. Genetic studies have established a strong causal relationship between ASD and molecular defects in the SHANK3 gene. Individuals with various defects of SHANK3 display considerable clinical heterogeneity. Different lines of Shank3 mutant mice with deletions of different portions of coding exons have been reported recently. Variable synaptic and behavioral phenotypes have been reported in these mice, which makes the interpretations for these data complicated without the full knowledge of the complexity of the Shank3 transcript structure.
We systematically examined alternative splicing and isoform-specific expression of Shank3 across different brain regions and developmental stages by regular RT-PCR, quantitative real time RT-PCR (q-PCR), and western blot. With these techniques, we also investigated the effects of neuronal activity and epigenetic modulation on alternative splicing and isoform-specific expression of Shank3. We explored the localization and influence on dendritic spine development of different Shank3 isoforms in cultured hippocampal neurons by cellular imaging.
The Shank3 gene displayed an extensive array of mRNA and protein isoforms resulting from the combination of multiple intragenic promoters and extensive alternative splicing of coding exons in the mouse brain. The isoform-specific expression and alternative splicing of Shank3 were brain-region/cell-type specific, developmentally regulated, activity-dependent, and involved epigenetic regulation. Different subcellular distribution and differential effects on dendritic spine morphology were observed for different Shank3 isoforms.
Our results indicate a complex transcriptional regulation of Shank3 in mouse brains. Our analysis of select Shank3 isoforms in cultured neurons suggests that different Shank3 isoforms have distinct functions. Therefore, the different types of SHANK3 mutations found in patients with ASD and different exonic deletions of Shank3 in mutant mice are predicted to disrupt selective isoforms and result in distinct dysfunctions at the synapse with possible differential effects on behavior. Our comprehensive data on Shank3 transcriptional regulation thus provides an essential molecular framework to understand the phenotypic diversity in SHANK3 causing ASD and Shank3 mutant mice.
自闭症谱系障碍（ASD）患者中存在显著的临床异质性，这已得到充分记录。然而，对于表型多样性背后的生物学机制却知之甚少。基因研究已证实ASD与SHANK3基因的分子缺陷之间存在密切的因果关系。SHANK3存在各种缺陷的个体表现出显著的临床异质性。最近报道了不同品系的Shank3突变小鼠，其编码外显子的不同部分存在缺失。这些小鼠表现出可变的突触和行为表型，在不完全了解Shank3转录本结构复杂性的情况下，对这些数据的解读变得复杂。
我们通过常规逆转录聚合酶链反应（RT-PCR）、定量实时RT-PCR（q-PCR）和蛋白质免疫印迹法，系统地检测了Shank3在不同脑区和发育阶段的可变剪接及异构体特异性表达。利用这些技术，我们还研究了神经元活动和表观遗传调控对Shank3可变剪接及异构体特异性表达的影响。我们通过细胞成像技术，探究了不同Shank3异构体在培养的海马神经元中的定位及其对树突棘发育的影响。
在小鼠脑中，由于多个基因内启动子的组合以及编码外显子的广泛可变剪接，Shank3基因呈现出大量的mRNA和蛋白质异构体。Shank3的异构体特异性表达和可变剪接具有脑区/细胞类型特异性，受发育调控，依赖于活性，并涉及表观遗传调控。不同的Shank3异构体表现出不同的亚细胞分布以及对树突棘形态的不同影响。
我们的结果表明，小鼠脑中Shank3存在复杂的转录调控。我们对培养神经元中特定Shank3异构体的分析表明，不同的Shank3异构体具有不同的功能。因此，预计在ASD患者中发现的不同类型的SHANK3突变以及突变小鼠中Shank3的不同外显子缺失会破坏选择性异构体，并导致突触处不同的功能障碍，可能对行为产生不同的影响。我们关于Shank3转录调控的全面数据，为理解由SHANK3导致的ASD以及Shank3突变小鼠中的表型多样性提供了一个重要的分子框架。
