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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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要