Elucidating the Role of Epithelial PCP signaling in SGN Axon Guidance in the Cochlea

阐明上皮 PCP 信号传导在耳蜗 SGN 轴突引导中的作用

基本信息

批准号：
10536706
负责人：
Shaylyn Clancy
金额：
$ 3.54万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10536706
关键词：
Acoustic Trauma Adhesions Afferent Neurons Alleles Auditory Axon Behavior Binding Brain CRISPR/Cas technology Cell Adhesion Cell Adhesion Molecules Cell-Cell Adhesion Characteristics Chimeric Proteins Cochlea Cochlear nucleus Coculture Techniques Congenital Abnormality Cytoskeleton Data Development Developmental Process Disease Embryo Epithelial Fc domain Foundations Genes Goals Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Hair Hair Cells Hearing Immunoglobulins Immunohistochemistry Inner Hair Cells Intercellular Junctions Knock-out Knockout Mice Knowledge Label Labyrinth Ligands Light Mammals Mediating Membrane Molecular Morphogenesis Morphology Mus Natural regeneration Nature Nervous system structure Neurons Noise Organ Organ of Corti Outer Hair Cells Pain Pathway interactions Pattern Peripheral Pillar Cell Play Property Proteins Regulation Reporter Role Sensory Sensory Disorders Signal Transduction Stereotyping Supporting Cell Testing Tissue imaging Tissues Transgenic Organisms Work apical membrane axon guidance base cell behavior conditional knockout emx2 protein experimental study hearing impairment hearing restoration hindbrain inner ear development insight loss of function member mutant nerve supply nervous system development new therapeutic target novel novel therapeutic intervention noxacusis planar cell polarity polarized cell receptor repaired rho GTP-Binding Proteins sound spiral ganglion therapeutic target ubiquitin-protein ligase

项目摘要

Our sense of hearing is critically dependent on the spiral ganglion neurons (SGNs), which connect the sound receptors in the organ of Corti (OC) to the cochlear nuclei of the hindbrain. During development, SGNs establish stereotyped innervation patterns with specific hair cell targets in the OC. Type I SGNs innervate inner hair cells (IHCs) to transmit sound signals, while type II SGNs (SGNIIs) innervate outer hair cells (OHCs) to detect acoustic trauma. Despite their essential functions in hearing, our understanding of the molecular mechanisms that mediate wiring of the auditory periphery is still fragmentary. It has been shown recently that guidance of SGNII peripheral projections is regulated by the Planar Cell Polarity (PCP) pathway. Intercellular PCP signaling mediates polarized cell behaviors within the plane of a tissue in a plethora of developmental processes. In the wild-type OC, SGNII afferents make a characteristic 90-degree turn toward the base of the cochlea and innervate multiple OHCs. In several PCP mutants, SGNII afferents turn randomly towards either the cochlear base or the apex. Although it has been shown that PCP proteins localize asymmetrically to supporting cell (SC)-SC junctions and act in the cochlear epithelium to guide SGNII afferents, the underlying mechanisms are currently unknown. Based on a strong foundation of preliminary data, we will test the hypothesis that PCP signaling regulates multiple downstream effectors including adhesion molecules and Rho GTPases to influence cell adhesion and the cytoskeleton in SCs, which serve as intermediate targets of SGNIIs. Specifically, we found that PCP signaling regulates the localization of Nectin3, a member of the immunoglobulin superfamily of adhesion molecules, in SCs. Moreover, our preliminary data have suggested that the Rac GTPases are required in the cochlear epithelium for SGNII afferent guidance; however, their constitutive activity was insufficient to direct SGNII afferents when PCP signaling is disrupted. To test our hypothesis, Aim 1 seeks to elucidate the role of Nectin3 in SGNII afferent turning. To this end, we have generated Nectin3 knockout mice using CRISPR-Cas9. We will characterize Nectin3 mutant alleles and analyze SGNII afferent turning in Nectin3 knockout mutants. Additionally, to determine whether Nectin3-mediated heterophilic adhesion between SCs and SGNII afferents plays a role in their turning direction, we will test the effect of soluble Nectin3 ecto domain-Fc fusion proteins on SGNII axon outgrowth and turning in cochlear explants and dissociated SGN cultures. Aim 2 will further investigate mutual regulation between Rac signaling activity and asymmetric PCP protein localization, using a newly generated transgenic Rac1 activity reporter line and Rac conditional knockout mutants. Furthermore, we will also determine whether the E3 ubiquitin ligase POSH/Sh3rf1, a known Rac1 downstream effector, is involved in SGNII afferent guidance using loss-of-function perturbations. Together, the proposed experiments will provide novel insights into how PCP signaling directs polarized cell behaviors and fill our knowledge gaps about SGNII wiring mechanisms in the mammalian cochlea.

我们的听觉严重依赖于连接声音的螺旋神经节神经元 (SGN) 柯蒂氏器 (OC) 中的受体连接到后脑的耳蜗核。在开发过程中，SGN 建立 OC 中具有特定毛细胞目标的刻板神经支配模式。 I 型 SGN 支配内毛细胞 (IHC) 传输声音信号，而 II 型 SGN (SGNII) 支配外毛细胞 (OHC) 来检测声音创伤。尽管它们在听力方面具有重要功能，但我们对分子机制的理解听觉外围的中介线路仍然不完整。最近的研究表明，SGNII 外围投射的引导是由平面细胞调节的极性（PCP）途径。细胞间 PCP 信号传导介导组织平面内的极化细胞行为在众多的发展过程中。在野生型 OC 中，SGNII 传入神经形成特征性的 90 度角转向耳蜗基部并支配多个 OHC。在一些 PCP 突变体中，SGNII 传入神经转变为随机朝向耳蜗基部或耳蜗尖部。尽管已经表明 PCP 蛋白定位于与支持细胞 (SC)-SC 连接不对称，并在耳蜗上皮中起作用以引导 SGNII 传入，目前尚不清楚其根本机制。基于坚实的初步数据基础，我们将检验 PCP 信号传导调节多个下游效应器（包括粘附分子）的假设和 Rho GTPases 影响 SC 中的细胞粘附和细胞骨架，作为中间靶标 SGNII 的数量。具体来说，我们发现 PCP 信号传导调节 Nectin3 的定位，Nectin3 是 Nectin3 的成员。 SC 中粘附分子的免疫球蛋白超家族。此外，我们的初步数据表明耳蜗上皮需要 Rac GTPases 来进行 SGNII 传入引导；然而，他们的构成当 PCP 信号传导被破坏时，活性不足以引导 SGNII 传入。为了检验我们的假设，目标图 1 旨在阐明 Nectin3 在 SGNII 传入转折中的作用。为此，我们生成了Nectin3 使用 CRISPR-Cas9 敲除小鼠。我们将表征 Nectin3 突变等位基因并分析 SGNII 传入转化 Nectin3 敲除突变体。此外，为了确定 Nectin3 介导的异嗜性粘附是否 SCs 和 SGNII 传入神经之间在其转向方向上发挥作用，我们将测试可溶性 Nectin3 的效果胞外域-Fc 融合蛋白对 SGNII 轴突生长和耳蜗外植体和解离 SGN 转动的影响文化。目标2将进一步研究Rac信号活动与不对称PCP之间的相互调节蛋白质定位，使用新生成的转基因 Rac1 活性报告系和 Rac 条件敲除突变体。此外，我们还将确定 E3 泛素连接酶 POSH/Sh3rf1（已知的 Rac1）是否下游效应器，参与使用功能丧失扰动的 SGNII 传入指导。在一起，拟议的实验将为 PCP 信号传导如何指导极化细胞行为和填充提供新的见解我们对哺乳动物耳蜗中 SGNII 接线机制的了解存在差距。