Non-apoptotic functions of caspase-3 in neural development

Caspase-3在神经发育中的非凋亡功能

• 批准号: 10862033
• 负责人: Karina S Cramer
• 金额: $ 47.33万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10862033
• 关键词: Acceleration; Acoustic Nerve; Apoptosis; Apoptotic; Appearance; Auditory; Auditory Perception; Auditory system; Axon; Brain; Brain Stem; CASP3 gene; CASP6 gene; CASP8 gene; CASP9 gene; Caspase; Categories; Cell Death; Cell Death Signaling Process; Cell Membrane Permeability; Cell Nucleus; Cells; Cessation of life; Chick Embryo; Cochlea; Communication; Cytoprotection; Data; Defect; Dendrites; Development; Developmental Process; Electroporation; Embryo; Embryonic Development; Ganglia; Genes; Goals; Growth Associated Protein 43; Hearing Tests; Language; Mediating; Mitochondria; Modeling; Molecular; NCAM1 gene; Nerve; Neuron-Glia Cell Adhesion Molecules; Neurons; Neurosciences; Ontology; Outer Mitochondrial Membrane; Pathway interactions; Peptide Hydrolases; Permeability; Plasmids; Process; Proteins; Proteolysis; Proteome; Proteomics; Regulation; Research; Role; Sampling; Site; Source; Structural defect; Synapses; Systems Development; Testing; Time; Transfection; Transplantation; Work; auditory pathway; axon growth; axon guidance; cytotoxic; exosome; extracellular vesicles; neural circuit; neurodevelopment; receptor; sound; tandem mass spectrometry; vesicular release; x-linked inhibitor of apoptosis protein

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.

项目概要 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 听觉神经科学领域面临的挑战是了解这些神经回路在 发展。我们之前的工作表明 caspase-3（一种最好的蛋白酶）具有重要功能 以其在细胞凋亡中的作用而闻名。剪切的（活性的）caspase-3 存在于发育中的听觉中 程序性细胞死亡之前的脑干。在胚胎发育过程中，首先 见于听神经轴突，然后见于大细胞核中这些轴突的突触目标 (NM)，然后在 NM 的突触目标中，在层状核 (NL) 树突中。 Caspase-3 抑制 发育过程中会导致 NM 轴突靶向和结构异常出现重大错误 在荷兰。我们建议研究发育过程中 caspase-3 激活的调节。我们将 通过听觉通路和测试检查 caspase-3 激活进展的基础 假设听觉轴突中剪切的 caspase-3 对于激活 caspase-3 是必需的 他们的突触目标。细胞凋亡过程中 Caspase-3 的激活是由细胞死亡信号激活的 线粒体透化。我们假设在听觉发育过程中，caspase-3 通过非规范途径激活，该途径可保护细胞中含有裂解 caspase-3 的细胞 轴突。我们将测试上游分子的功能并确定它们是否影响 NM-NL 途径的发展。我们已经开始研究通过哪些分子 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基材 介导轴突生长。我们将使用这些蛋白质的半胱天冬酶不可切割形式来测试它们的 发育过程中依赖 caspase 的功能。基因本体分析表明，最 caspase-3 底物丰富的细胞定位类别是外泌体/细胞外囊泡 （电动汽车）。这一发现提出了一个总体模型，其中 caspase-3 影响 EV 反过来又提供了细胞间本地通信的有效手段 发展。我们将使用串联质谱法检查富集的 EV 样品，以确定 EV 中存在哪些 caspase-3 底物。我们将使用 EV 嫁接策略来研究 EV 是否可以挽救 caspase-3 抑制的宿主胚胎的发育缺陷。在一起，这些 研究将增进我们对发育中听觉神经回路组装的理解 脑干。