Elucidating the synaptic interactome of the high risk autism gene ANK2

阐明高风险自闭症基因 ANK2 的突触相互作用组

• 批准号: 10391766
• 负责人: Damaris N Lorenzo
• 金额: $ 45.17万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-07 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10391766
• 关键词: ANK2 gene; ASD patient; Action Potentials; Affect; Anatomy; Antibodies; Architecture; Axon; Binding; Biological Assay; Biology; Biotin; Brain; Cells; Cellular Assay; Complex; Couples; Cytoskeletal Proteins; Dendrites; Dendritic Spines; Development; Exhibits; Functional disorder; Genes; Glutamates; Goals; Image; Impairment; In Vitro; Ion Channel; Knock-in Mouse; Knockout Mice; Label; Ligase; Link; Mass Spectrum Analysis; Membrane; Membrane Proteins; Membrane Transport Proteins; Microscopy; Molecular; Morphology; Motor; Mus; Neurons; Pathway Analysis; Pathway interactions; Phenocopy; Play; Positioning Attribute; Protein Isoforms; Proteins; Proteomics; Resolution; Role; Sodium Channel; Synapses; Variant; Vertebral column; Vesicle; autism spectrum disorder; base; chromatin remodeling; density; disorder risk; excitatory neuron; experimental study; falls; gene network; high risk; hippocampal pyramidal neuron; in vivo; individuals with autism spectrum disorder; nanoscale; novel; postsynaptic; postsynaptic density protein; protein complex; scaffold; synaptic function; tandem mass spectrometry; trafficking; voltage

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.

项目摘要 网络基因分析表明，高置信度自闭症谱系障碍（hcASD）基因通过 会聚机制主要支持突触功能，这反过来又可以决定解剖结构， 和功能性大脑连接。已经在ASD患者中鉴定了ANK 2的几种变体，并且该基因 一直被认为是hcASD的最高风险。ANK 2编码锚蛋白B（AnkB），其在轴突生长中起作用。 生物学可能是ASD病理生理学的潜在因素。然而，我们最近的发现表明锚蛋白- B在皮层兴奋性突触后也起重要作用。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， 亚型特异性作用并与独特的伴侣相互作用，包括其他hsASD基因编码的蛋白质。因此，在本发明中， 重要的是确定AnkB在突触后的同种型特异性作用和配偶体。完成这些 我们的目标是：1）使用体内邻位依赖性生物素鉴定（iBioID）结合串联质谱， 质谱（MS）捕获和识别AnkB相互作用的合作伙伴在突触后，利用我们的新的 生物素连接酶BirA标记的条件性AnkB（AnkB-BioID）敲入小鼠和同种型特异性AnkB敲除 小鼠; 2）联合收割机结合活体和超分辨率显微镜检查以及细胞测定来验证令人信服的AnkB 相互作用;和3）定义新的AnkB PSD复合物对突触发育和功能的意义。