UBE3A gain-of-function and parent-of-origin influence on neurodevelopmental phenotypes

项目总结 UBE3A是一种E3泛素连接酶，以自身和其他底物为靶标进行蛋白酶体降解。在 发育中的大脑、神经前体细胞和未成熟神经元以双等位方式表达Ube3a，但作为神经元 成熟后，Ube3a的表达将限制在母系遗传的等位基因上。提升的突变 母亲或父亲的Ube3a与自闭症风险有关，但UBE3A过量到底是如何损害的 神经发育尚不清楚。最近，我们发现苏氨酸485(T485)的磷酸化抑制 UBE3A泛素连接酶活性。UBE3A T485磷酸化从胚胎开始，在出生时达到顶峰， 提示在皮质发育的早期，磷酸化可能保护性地限制UBE3A的活性。 此外，我们发现UBE3A(T485A)中与自闭症相关的从头突变破坏了这种磷酸化 站点，有效锁定UBE3A Always-On。我们设计了一只可以精确模拟人类UBE3A的小鼠 T485A突变，使我们能够评估这种新的功能获得突变如何影响大脑和行为 母系或父系遗传的表型。在初步研究中，我们发现皮质厚度 出生时，所有三种Ube3a T485A基因型(父亲、母亲、 纯合子)。其他与自闭症相关的基因突变也会增加大脑重量。这些 研究结果表明，UBE3A在大脑发育中具有一种新的、以前未被认识的产前功能。全 三个Ube3a T485A突变基因也具有与神经发育一致的行为表型 精神错乱。由于对UBE3A在任何年龄段如何损害大脑功能知之甚少，我们进行了公正的研究 蛋白质组学来确定与大脑相关的底物。我们的初步蛋白质组学数据将UBE3A直接链接到 蛋白酶体，一种可以影响细胞周期和对大脑重要的信号通路的结构 发展。这些数据和其他数据让我们假设UBE3A T485A改变了细胞的平衡 在大脑发育过程中的增殖和分化，部分是通过损害蛋白酶体功能， 并在以后的生活中导致与自闭症相关的表型。这项提案中的实验将 严格证明(1)UBE3A T485A改变了祖细胞增殖和分化的平衡 在大脑皮层，(2)Ube3a T485A的父母遗传影响自闭症相关的大脑和 行为表型；(3)UBE3A T485A与蛋白酶体相互作用，损害蛋白酶体功能 在大脑里。无偏见的蛋白质组学实验将确定UBE3A的大脑相关底物，并扩大 我们对哪些分子通路受到功能获得突变的影响的理解 UBE3A活动。

PROJECT SUMMARY UBE3A is an E3 ubiquitin ligase that targets itself and other substrates for proteasomal degradation. In the developing brain, neuronal progenitors and immature neurons biallelically express Ube3a, but as neurons mature, Ube3a expression becomes restricted to the maternally-inherited allele. Mutations that elevate maternal or paternal Ube3a are linked to autism risk, but precisely how UBE3A excess impairs neurodevelopment is unclear. Recently, we found that phosphorylation of threonine 485 (T485) inhibits UBE3A ubiquitin ligase activity. UBE3A T485 phosphorylation initiates embryonically and peaks at birth, suggesting that phosphorylation might protectively limit UBE3A activity during early cortical development. Additionally, we found that an autism-linked de novo mutation in UBE3A (T485A) disrupts this phosphorylation site, effectively locking UBE3A always-on. We engineered a mouse that precisely models this human UBE3A T485A mutation, allowing us to evaluate how this novel gain-of-function mutation affects brain and behavioral phenotypes when inherited maternally or paternally. In preliminary studies, we found that cortical thickness and brain weight were significantly increased at birth in all three Ube3a T485A genotypes (paternal, maternal, homozygous). Mutations in other autism-linked genes increase brain weight to a similar extent. These findings suggest a novel and previously unrecognized prenatal function for UBE3A in brain development. All three Ube3a T485A mutant genotypes also had behavioral phenotypes consistent with neurodevelopmental disorders. Since little is known about how UBE3A impairs brain function at any age, we performed unbiased proteomics to identify brain-relevant substrates. Our preliminary proteomics data link UBE3A directly to the proteasome, a structure that can influence the cell cycle and signaling pathways important for brain development. These and other data lead us to hypothesize that UBE3A T485A alters the balance of cell proliferation and differentiation during brain development, in part by impairing proteasome function, and contributes to autism-associated phenotypes later in life. The experiments in this proposal will rigorously demonstrate that (1) UBE3A T485A alters the balance of progenitor proliferation and differentiation in the cerebral cortex, (2) parent-of-origin inheritance of Ube3a T485A influences autism-related brain and behavioral phenotypes, and (3) UBE3A T485A interacts with the proteasome and impairs proteasome function in the brain. Unbiased proteomics experiments will identify brain-relevant substrates of UBE3A, and broaden our understanding of which molecular pathways are affected by gain-of-function mutations that enhance UBE3A activity.