Elucidating the synaptic interactome of the high risk autism gene ANK2

项目总结 网络基因分析表明，高置信度自闭症谱系障碍(HcASD)基因通过 主要支持突触功能的会聚机制，而突触功能反过来又可以决定解剖结构 和正常的大脑连接。已在ASD患者中发现ANK2的几个变异，该基因 一直被认为是hcasd的最大风险。ANK2编码Ankyrin-B(AnkB)，在轴突中起作用 生物学可能是ASD病理生理学的一个潜在因素。然而，我们最近的发现表明，锚定蛋白- B在皮质兴奋性突触后也起着重要作用。此外，两种主要的AnkB亚型 在神经元中(220 kDa和440 kDa AnkB)在树突中大量表达，它们可能在那里执行 与独特的伴侣相互作用，包括由其他hsASD基因编码的蛋白质。因此， 重要的是要确定AnkB在突触后的异构体特定的角色和伙伴。要实现这些目标 目标我们将1)使用体内邻近依赖的生物素鉴定(IBioID)结合串联质量 光谱(MS)通过利用我们的新技术来捕获和识别突触后AnkB交互伙伴 生物素连接酶BIRA标记的条件性AnkB(AnkB-BioID)敲入小鼠及异构体特异的AnkB基因敲除 小鼠；2)结合活的和超分辨率的显微镜和细胞分析来验证引人注目的AnkB 3)明确新型AnkB PSD复合体对突触发育和功能的意义。

PROJECT SUMMARY Network gene analysis suggests that high confidence autism spectrum disorder (hcASD) genes operate through convergent mechanisms that predominantly support synaptic function, which, in turn, can determine anatomical and functional brain connectivity. Several variants in ANK2 have been identified in ASD patients and this gene has been consistently considered a top hcASD risk. ANK2 encodes ankyrin-B (AnkB), which roles in axonal biology may be an underlying factor in ASD pathophysiology. However, our recent findings indicate that ankyrin- B also plays important roles at the cortical excitatory postsynapse. Moreover, both predominant AnkB isoforms in neurons (220 kDa and 440 kDa AnkB) are abundantly expressed in dendrites, where they likely perform isoform-specific roles and interact with unique partners, including proteins encoded by other hsASD genes. Thus, it is important to define the isoform-specific roles and partners of AnkB at the postsynapse. To accomplish these goal we will 1) use in vivo proximity-dependent biotin identification (iBioID) combined with in tandem mass spectrometry (MS) to capture and identify AnkB interacting partners in the postsynapse by leveraging our novel biotin ligase BirA-tagged conditional AnkB (AnkB-BioID) knock-in mouse and isoform-specific AnkB knockout mice; 2) combine live and super-resolution microscopies and cellular assays to validate compelling AnkB interactors; and 3) define the significance of novel AnkB PSD complexes for synaptic development and function.