Mechanisms of non-apoptotic caspase-3 regulation of auditory brainstem development
Mechanisms of non-apoptotic caspase-3 regulation of auditory brainstem development
批准号:
10335160
负责人:
Forrest Weghorst
金额:
$3.11万
依托单位国家:
美国
项目类别:
财政年份:
2021
资助国家:
美国
项目状态:
已结题
起止时间:
2021-04-01 至 2022-10-18
关键词:
Acoustic NerveApoptoticAspartateAuditoryAuditory systemAxonBinding ProteinsBiochemicalBrain StemC-terminalCASP3 geneCaspaseCategoriesCause of DeathCell DeathCell NucleusChickChick EmbryoComplexCytoskeletal ModelingDataData SetDefectDendritesDevelopmentDevelopmental ProcessElementsEmbryoGene DeliveryGene ExpressionGenesGeneticGenetic TranscriptionGlutamatesHearing problemKnowledgeMethodsMolecularMusNeurodevelopmental DisorderNeuronsPathway AnalysisPathway interactionsPeptide HydrolasesPeptidesPhaseProcessProteinsProteolysisProteomeRNARNA BindingRNA purificationRNA-Binding ProteinsRecombinant ProteinsRegulationRoleSchizophreniaShapesSound LocalizationStructureSynapsesTestingTimeTrainingTransfectionauditory pathwayauditory processingautism spectrum disorderaxon growthaxon guidancecrosslinkdesign and constructiondevelopmental diseaseexperienceexperimental studyextracellular vesiclesfascingenetic regulatory proteinin vivointerestmultiple omicsmyotrophinneural circuitneurodevelopmentneuroregulationnovelpreventprotein expressionprotein purificationsoundsynaptogenesistranscriptome
中文摘要
项目摘要
声音定位依赖于听觉脑干中精确神经回路的发育。异常
电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然未知。我们实验室
先前已经表明,半胱天冬酶-3活性是听觉脑干回路发育所必需的,
鸡胚在整个发育过程中,caspase-3在听性脑干的轴突和树突中表达活跃
听觉信息上行通路中的神经元:首先在听觉神经轴突中;然后在其
突触靶,大细胞核(NM);最后在NM的突触靶树突,核
laminaris(NL)。当半胱天冬酶-3存在于NM轴突中时,抑制半胱天冬酶-3活性导致NM轴突损伤。
靶向错误,即使在这段时间之后听脑干中没有发生凋亡细胞死亡,
期这些数据表明,caspase-3是负责引导NM轴突在一个非凋亡的方式。到
确定caspase-3如何影响NM轴突导向,我的目的是确定听觉脑干caspase-3
印刷受体.我筛选了半胱天冬酶-3抑制和对照脑干的肽段,
显示半胱天冬酶蛋白水解的生物化学特征(谷氨酸或天冬氨酸的C-末端裂解
残留物),并且仅在对照脑干中观察到。符合这两个标准的421种肽
来自287种不同的蛋白质,这些蛋白质富含几种功能类别,包括细胞骨架蛋白,
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是如何
这些底物类别的分裂导致正确的听觉脑干回路发育。在目标1中,
我建议用表达参与细胞凋亡的caspase-3底物的不可裂解形式的构建体来抑制NM。
细胞骨架调节:肌营养素和肌成束蛋白-1。因为我相信半胱天冬酶-3抑制会导致NM轴突
通过阻止caspase-3对细胞骨架调节的控制来靶向缺陷,我假设这些
不可裂解的底物将复制由整体胱天蛋白酶-3抑制引起的轴突靶向缺陷。在目标2中,我
将使用UV交联,然后进行正交有机相分离(OOPS)来纯化RNA结合的
蛋白质,蛋白质结合的RNA,和剩余的蛋白质组和转录组从胱天蛋白酶-3-抑制和
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,
探讨caspase-3蛋白水解RNA结合蛋白对听觉神经元基因表达的影响
脑干因此,这些目标将阐明半胱天冬酶-3在底物类别方面的作用
通过我的初步数据,有助于更全面地了解凋亡途径如何为非-
在神经发育和可塑性中的凋亡功能。
英文摘要
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.
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