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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：
10739288
负责人：
Shaylyn Clancy
金额：
$ 3.63万
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/10739288
关键词：
Acoustic Trauma Adhesions Afferent Neurons Alleles Apical Auditory Axon Behavior Binding Biological Assay 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 Dissociation Embryo Epithelium 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 Mammals Mediating Membrane Molecular Morphogenesis Morphology Mus Natural regeneration Nature Nervous System 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 nectin 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 tissue fixing transmission process 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）