Mechanisms of non-apoptotic caspase-3 regulation of auditory brainstem development

非凋亡 caspase-3 调节听觉脑干发育的机制

• 批准号: 10335160
• 负责人: Forrest Weghorst
• 金额: $ 3.11万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2022-10-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10335160
• 关键词: Acoustic Nerve; Apoptotic; Aspartate; Auditory; Auditory system; Axon; Binding Proteins; Biochemical; Brain Stem; C-terminal; CASP3 gene; Caspase; Categories; Cause of Death; Cell Death; Cell Nucleus; Chick; Chick Embryo; Complex; Cytoskeletal Modeling; Data; Data Set; Defect; Dendrites; Development; Developmental Process; Elements; Embryo; Gene Delivery; Gene Expression; Genes; Genetic; Genetic Transcription; Glutamates; Hearing problem; Knowledge; Methods; Molecular; Mus; Neurodevelopmental Disorder; Neurons; Pathway Analysis; Pathway interactions; Peptide Hydrolases; Peptides; Phase; Process; Proteins; Proteolysis; Proteome; RNA; RNA Binding; RNA purification; RNA-Binding Proteins; Recombinant Proteins; Regulation; Role; Schizophrenia; Shapes; Sound Localization; Structure; Synapses; Testing; Time; Training; Transfection; auditory pathway; auditory processing; autism spectrum disorder; axon growth; axon guidance; crosslink; design and construction; developmental disease; experience; experimental study; extracellular vesicles; fascin; genetic regulatory protein; in vivo; interest; multiple omics; myotrophin; neural circuit; neurodevelopment; neuroregulation; novel; prevent; protein expression; protein purification; sound; synaptogenesis; transcriptome

PROJECT ABSTRACT Sound localization depends on the development of precise neural circuits in the auditory brainstem. Abnormal circuit assembly can contribute to auditory dysfunction in developmental disorders. However, the molecular mechanisms responsible for correct auditory brainstem circuit development remain largely unknown. Our lab has previously shown that caspase-3 activity is necessary for development of auditory brainstem circuits in the chick embryo. Throughout development, active caspase-3 is seen in axons and dendrites of auditory brainstem neurons in the ascending pathway of auditory information: first in auditory nerve axons; then in axons of their synaptic target, nucleus magnocellularis (NM); and finally in dendrites of NM’s synaptic target, nucleus laminaris (NL). Inhibition of caspase-3 activity when caspase-3 is present in NM axons results in NM axonal targeting errors, even though no apoptotic cell death occurs in the auditory brainstem until after this time period. These data suggest that caspase-3 is responsible for guiding NM axons in a non-apoptotic manner. To determine how caspase-3 influences NM axon guidance, I aimed to identify auditory brainstem caspase-3 substrates. I screened the peptidomes of caspase-3-inhibited and control brainstems for peptides that displayed a biochemical signature of caspase proteolysis (cleavage C-terminal of glutamate or aspartate residues) and that were observed only in control brainstems. The 421 peptides that fulfilled these two criteria hailed from 287 distinct proteins, which were enriched for several functional categories, including cytoskeletal regulatory proteins and RNA-binding proteins. Here I propose several experiments to test how caspase-3 cleavage of these substrate categories brings about correct auditory brainstem circuit development. In Aim 1, I propose to transfect NM with constructs expressing uncleavable forms of caspase-3 substrates involved in cytoskeletal regulation: myotrophin and fascin-1. Because I believe that caspase-3 inhibition causes NM axon targeting defects by preventing caspase-3 control of cytoskeletal regulation, I hypothesize that these uncleavable substrates will replicate axon targeting defects caused by global caspase-3 inhibition. In Aim 2, I will use UV cross-linking followed by orthogonal organic phase separation (OOPS) to purify RNA-bound proteins, protein-bound RNAs, and the remaining proteome and transcriptome from caspase-3-inhibited and control auditory brainstems. I will then use gene co-expression network analysis of these four datasets to probe the effect of caspase-3 proteolysis of RNA-binding proteins on gene expression in the auditory brainstem. These aims will thus clarify the role of caspase-3 with regard to the substrate categories revealed by my preliminary data, contributing to a fuller understanding of how the apoptotic pathway serves non- apoptotic functions during neurodevelopment and plasticity.

项目摘要 声音定位取决于听觉脑干中精确神经元电路的发展。异常 电路组件可以导致发育障碍的听觉功能障碍。但是，分子 负责正确听觉脑干电路发育的机制在很大程度上未知。我们的实验室 以前已经表明，caspase-3活性对于开发听觉脑干电路是必不可少的 小鸡胚胎。在整个开发过程中，在听觉脑干的轴突和树突中都可以看到主动caspase-3 听觉信息的上升途径中的神经元：首先是听觉神经轴突；然后在他们的轴突中 合成靶标，核细胞核（NM）；最后，在NM合成目标的树突中 拉米纳利（NL）。 caspase-3活性抑制caspase-3存在于NM轴突中导致NM轴突 靶向错误，即使在听觉脑干中没有发生凋亡细胞死亡，直到此后 时期。这些数据表明，caspase-3负责以非凋亡方式引导NM轴突。到 确定caspase-3如何影响NM轴突指导，我旨在确定听觉脑干caspase-3 基材。我筛选了caspase-3抑制和控制脑干的petidides的petidomes 显示了胱天蛋白酶水解的生化特征（谷氨酸或天冬氨酸的裂解C末端 残留物），仅在对照脑干中观察到。满足这两个标准的421肽 来自287种不同的蛋白质，这些蛋白质富含多种功能类别，包括细胞骨架 调节蛋白和RNA结合蛋白。在这里，我提出了几个实验来测试caspase-3如何 这些底物类别的分裂带来了正确的听觉脑干电路的发展。在AIM 1中，我 提议用表达不可泄漏形式的caspase-3基板的构造来翻译NM 细胞骨架调节：肌营养蛋白和fascin-1。因为我相信caspase-3抑制会导致NM轴突 通过防止caspase-3控制细胞骨架调节，靶向缺陷，我假设这些 不可泄漏的底物将复制由全球caspase-3抑制引起的轴突靶向缺陷。在AIM 2中，我 将使用紫外线交联，然后使用正交有机相分离（OOPS）净化RNA结合 蛋白质，结合蛋白质的RNA以及抑制caspase-3抑制的其余蛋白质组和转录组 控制听觉脑干。然后，我将使用这四个数据集的基因共表达网络分析 探测RNA结合蛋白对caspase-3蛋白水解对听觉中基因表达的影响 脑干。因此，这些目标将阐明caspase-3在揭示的底物类别方面的作用 根据我的初步数据，有助于更充分了解凋亡途径如何服务 神经发育和可塑性期间的凋亡功能。