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

项目摘要 声音定位依赖于听觉脑干中精确神经回路的发展。异常 回路组装可能导致发育障碍中的听觉功能障碍。然而，分子 负责正确的听觉脑干回路发育的机制在很大程度上仍不清楚。我们的实验室 先前的研究表明，caspase-3的活性对于大脑听觉脑干回路的发育是必需的。 小鸡胚胎。在整个发育过程中，活跃的caspase-3出现在听性脑干的轴突和树突中。 听觉信息上行通路中的神经元：首先在听神经轴突中；然后在其 突触靶点大细胞核(NM)；最后在NM突触靶核的树突中 板层(Laminaris)。当NM轴突中存在caspase-3时，抑制caspase-3活性会导致NM轴突 靶向错误，即使在此之后之前，听觉脑干中没有发生细胞凋亡 句号。这些数据表明，caspase-3负责以非凋亡的方式引导NM轴突。至 确定caspase-3如何影响NM轴突引导，我的目标是识别听觉脑干caspase-3 底物。我筛选了caspase-3抑制多肽和对照多肽 显示caspase蛋白分解的生化特征(裂解谷氨酸或天冬氨酸的C末端 残留物)，并且只在对照脑组中观察到。符合这两个标准的421个多肽 来自287种不同的蛋白质，这些蛋白质富含几种功能类别，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 这些底物类别的切割带来了正确的听觉脑干回路发育。在目标1中，我 建议用表达不可切割形式的caspase-3底物的构建体转染NM 细胞骨架调节：肌营养因子和筋膜蛋白-1。因为我认为抑制caspase-3会导致NM轴突 通过阻止caspase-3控制细胞骨架调控来靶向缺陷，我假设这些 不可切割的底物将复制由caspase-3整体抑制引起的轴突靶向缺陷。在目标2中，我 将使用紫外光交联和正交有机相分离(OOPS)来纯化RNA结合 蛋白质，蛋白结合的RNA和来自caspase-3的剩余蛋白质组和转录组-抑制和 控制听觉脑电波。然后我将使用这四个数据集的基因共表达网络分析来 探讨RNA结合蛋白caspase-3蛋白降解对听觉基因表达的影响 脑干。因此，这些目标将阐明caspase-3在底物类别中的作用 根据我的初步数据，有助于更全面地理解凋亡途径是如何服务于非 细胞凋亡在神经发育和可塑性中的作用。

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.