Non-apoptotic functions of caspase-3 in neural development

项目总结 听觉功能依赖于高度专业化和精确的神经元连接。一个重要的 听神经科学领域面临的挑战是了解这些神经回路是如何在 发展。我们之前的工作表明了caspase-3的一个重要功能，caspase-3是一种最好的蛋白酶 以其在细胞凋亡中的作用而闻名。被切割的(活性的)caspase-3存在于发育中的听觉 在细胞程序性死亡之前的脑干。在胚胎发育期间，它是第一个 见于听神经轴突，然后见于这些轴突的突触靶点的大细胞核 (NM)，然后在NM的突触靶，在板状核(NL)树突中。Caspase-3抑制作用 在发育过程中导致NM轴突靶向和结构异常的重大错误 在内华达州。我们建议研究caspase-3在发育过程中的激活调节。我们会 通过听觉通路检测caspase-3激活过程的基础 Caspase-3在听神经轴突中被切割是激活caspase-3所必需的假说 它们的突触目标。Caspase-3在细胞凋亡过程中的激活由细胞死亡信号和 线粒体通透性。我们推测，在听觉发育过程中，caspase-3 通过一条非规范的途径激活，该途径对切割了caspase-3的细胞具有保护作用。 轴突。我们将测试上游分子的功能，并确定它们是否会影响 NM-NL途径的发展。我们已经开始研究通过这些分子 Caspase-3影响听觉发育。我们的蛋白质组学研究揭示了数百种蛋白质 在发育中的听觉脑干中被caspase-3裂解。我们已经确定了几种底物 调节轴突生长的物质。我们将使用这些蛋白质的caspase不可切割形式来测试它们的 Caspase在发育过程中的依赖功能。基因本体论分析显示，最 Caspase-3底物丰富的细胞定位类型是外切体/胞外小泡 (电动汽车)。这一发现提出了一个重要的模型，在该模型中，caspase-3影响 EVS，这反过来又提供了细胞之间的本地通信的有效手段 发展。我们将使用串联质谱仪检测浓缩的EV样本，以确定 EVS中存在哪些caspase-3底物。我们将使用电动汽车嫁接策略来调查 EVS能否挽救caspase-3抑制的宿主胚胎的发育缺陷。加在一起，这些 研究将促进我们对听觉发育过程中神经回路组装的理解 脑干。

PROJECT SUMMARY Auditory function relies on highly specialized and precise neuronal connectivity. A significant challenge for the field of auditory neuroscience is to understand how these neural circuits form during development. Our previous work suggests an important function for caspase-3, a protease best known for its role in apoptosis. Cleaved (active) caspase-3 is present in the developing auditory brainstem prior to the period of programmed cell death. During embryonic development, it is first seen in auditory nerve axons, then in the synaptic target of these axons in nucleus magnocellularis (NM), then in the synaptic target of NM, in nucleus laminaris (NL) dendrites. Caspase-3 inhibition during development results in substantial errors in NM axon targeting and in structural abnormalities in NL. We propose to investigate the regulation of caspase-3 activation during development. We will examine the basis for the progression of caspase-3 activation through the auditory pathway and test the hypothesis that cleaved caspase-3 is necessary in auditory axons for activation of caspase-3 in their synaptic targets. Caspase-3 activation during apoptosis is activated by cell death signals and mitochondrial permeabilization. We hypothesize that during auditory development, caspase-3 is activated through a non-canonical pathway that is protective for cells with cleaved caspase-3 in their axons. We will test the function of upstream molecules and determine whether they influence development of the NM-NL pathway. We have begun to investigate the molecules through which caspase-3 influences auditory development. Our proteomics study revealed hundreds of proteins that are cleaved by caspase-3 in the developing auditory brainstem. We have identified several substrates that mediate axon growth. We will use caspase-uncleavable forms of these proteins to test their caspase-dependent functions in development. Gene ontology analysis revealed that the most abundant cellular localization category for caspase-3 substrates was exosomes/extracellular vesicles (EVs). This finding suggests an overarching model in which caspase-3 influences the composition of EVs, which in turn provide an effective means of local communication between cells during development. We will examine enriched EV samples using tandem mass spectrometry to determine which caspase-3 substrates are present in EVs. We will use an EV grafting strategy to investigate whether EVs can rescue developmental deficits in caspase-3 inhibited host embryos. Together, these studies will advance our understanding of neural circuit assembly in the developing auditory brainstem.