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

• 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
• 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

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.

我们的听觉在很大程度上依赖于连接声音的螺旋神经节神经元 后脑耳蜗核的Corti器官受体(OC)。在开发过程中，SGN建立 OC中有特定毛细胞靶点的固有神经支配模式。I型SGN支配内毛细胞 (IHC)传递声音信号，而II型SGN(SGNII)支配外毛细胞(OHC)检测声音 精神创伤。尽管它们在听力中具有重要功能，但我们对它们的分子机制的理解 听觉外周的中间连接仍然是支离破碎的。 最近的研究表明，SGNII外周投射的引导受平面细胞的调节 极性(PCP)途径。细胞间PCP信号调节组织平面内极化的细胞行为 在过多的发育过程中。在野生型OC中，SGNII传入神经元以90度为特征 转向耳蜗底，神经支配多个内耳毛细胞。在几个PCP突变体中，SGNII传入细胞 随机地朝向耳蜗基底部或尖端。尽管已经证明PCP蛋白定位于 不对称至支持细胞(SC)-SC连接，并作用于耳蜗管上皮细胞以引导SGNII传入， 其潜在机制目前尚不清楚。基于雄厚的初步数据基础，我们将 测试PCP信号调节包括黏附分子在内的多个下游效应器的假设 和Rho GTP酶影响SCs的细胞黏附和细胞骨架，作为中间靶点 是SGNII的。具体地说，我们发现PCP信号调节Nectin3的定位，Nectin3是 免疫球蛋白超家族黏附分子，在干细胞中。此外，我们的初步数据表明， Rac GTP酶在耳蜗管上皮细胞中是SGNII传入引导所必需的；然而，它们的构成 当PCP信号被干扰时，活动不足以直接SGNII传入。为了检验我们的假设，目标是 1试图阐明Nectin 3在SGNII传入转折中的作用。为此，我们生成了Nectin 3 使用CRISPR-Cas9基因敲除小鼠。我们将鉴定Nectin 3突变等位基因并分析SGNII传入 转入Nectin 3基因敲除突变体。此外，为了确定Nectin3介导的异嗜性黏附 在SCs和SGNII传入之间起作用的是它们的转向，我们将测试可溶性Nectin 3的作用 ECTO结构域-Fc融合蛋白对耳蜗外植体和游离SGN中SGNII轴突生长和转归的影响 文化。目标2将进一步研究RAC信号活性和不对称PCP之间的相互调节 使用新产生的转基因rac1活性报告系和RAC条件敲除进行蛋白质定位 变种人。此外，我们还将确定E3泛素连接酶POSH/Sh3rf1，一个已知的rac1 下游效应器参与使用功能损失摄动的SGNII传入制导。团结在一起， 拟议的实验将为PCP信号如何引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连接机制的了解存在差距。