Elucidating the role of the E3 ubiquitin ligase UBE3B in neuronal activity

项目摘要 自闭症谱系障碍(Asd)是一种神经发育障碍，每54个儿童中就有一个受到影响。 显著的表型和遗传异质性。编码E3泛素连接酶的UBE3B发生突变， 已经在表现为智力残疾、缺乏语言和自闭症的患者中被发现。具体的 UBE3B的破坏以及随后其底物的失调导致 神经发育异常是未知的。我们的小组之前已经证明了UBE3B的本构成分 基因敲除小鼠模型表现出完全丧失发声和筑巢缺陷，以及减少 树突的复杂性、长度和棘突密度。在这项提议中，我将研究UBE3B的神经元功能 通过表征UBE3B缺失后神经元活性的变化并确定其神经元底物。我 将应用免费的电生理和生化方法，使用特定于大脑的条件 UBE3B基因敲除小鼠模型(CKOnstin)。我将描述大脑皮层的电生理特性 CKOnstin小鼠的神经元，通过评估它们的基本性质，包括内在兴奋性和流变性 电流，以及短期和长期突触可塑性。此外，我将确定神经元的底物 通过分析蛋白质水平、蛋白质之间的相互作用和泛素化状态来分析UBE3B。我也会 评估UBE3B介导的底物修饰，包括泛素化位置和泛素 链拓扑。成功完成拟议的目标将为深入了解 泛素信号和神经发育，以及提高我们对特定病原体的知识 神经发育障碍的潜在机制。 得克萨斯大学西南医学中心的卓越研究环境，与集体 专业的导师团队，将提供卓越的培训。这次团契培训的目标包括 精通相关文献，并熟练掌握实验程序和 向不同的受众传播科学信息。通过这些目标获得的技能将建立 为一名独立和成功的科学研究带头人奠定基础。

Project Summary Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects 1 in 54 children and exhibits significant phenotypic and genetic heterogeneity. Mutations in UBE3B, which encodes an E3 ubiquitin ligase, have been identified in patients presenting with intellectual disability, lack of speech, and ASD. The specific mechanism through which disruption of UBE3B, and subsequent dysregulation of its substrates, leads to neurodevelopmental abnormalities is unknown. Our group has previously shown that a Ube3b constitutive knockout mouse model exhibits a complete loss of vocalization and defects in nest building, as well as reduced dendritic complexity, length, and spine density. In this proposal, I will investigate the neuronal function of UBE3B by characterizing changes in neuronal activity following loss of UBE3B and identifying its neuronal substrates. I will apply complimentary electrophysiological and biochemical approaches using a brain-specific conditional Ube3b knockout mouse model (cKOnestin). I will characterize the electrophysiological properties of cortical neurons from cKOnestin mice, by evaluating their basal properties, including intrinsic excitability and rheobase current, and both short-term and long-term synaptic plasticity. Furthermore, I will identify the neuronal substrates of UBE3B through analysis of protein levels, protein-protein interactions, and ubiquitination status. I will also assess the UBE3B-mediated modifications of substrates, including the site of ubiquitination and the ubiquitin chain topology. Successful completion of the proposed aims will provide new insights into the interplay of ubiquitin signaling and neurodevelopment, as well as advance our knowledge of the specific pathogenic mechanisms underlying neurodevelopmental disorders. The exceptional research environment at UT Southwestern Medical Center, combined with the collective expertise of the mentorship team, will provide excellent training. Goals for this fellowship training include becoming knowledgeable in the relevant literature and attaining proficiency in both experimental procedures and communicating scientific information to varied audiences. The skills obtained through these goals will build the foundation for an independent and successful lead investigator in scientific research.