Calcium Binding Proteins Regulate Susceptibility to Damage in the Inner Ear

钙结合蛋白调节内耳损伤的易感性

• 批准号: 10202072
• 负责人: DWAYNE D SIMMONS
• 金额: $ 42万
• 依托单位: BAYLOR UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10202072
• 关键词: Acquired Deafness; Acute; Address; Affect; Age of Onset; Aging; American; Animal Model; Animals; Auditory; Auditory system; Binding Proteins; Biochemical; Biological Assay; Buffers; Calcium; Calcium Signaling; Calcium-Binding Proteins; Cell Culture Techniques; Cell Death; Cell Line; Cell Nucleus; Cell Survival; Cell physiology; Cells; Cellular Stress; Cochlea; Collection; Complex; Confocal Microscopy; Cues; Cultured Cells; Cytoplasm; Data Analyses; Defect; Development; Ear; Electron Microscopy; Environmental Risk Factor; Exposure to; Frequencies; Genetic; Hair; Hair Cells; Hearing; High Prevalence; High-Frequency Hearing Loss; Hyperacusis; Imaging Techniques; Injury; Kinetics; Labyrinth; Lead; Location; Loudness; Mammalian Cell; Mediating; Methods; Mitochondria; Molecular; Morphology; Mus; Mutant Strains Mice; Noise; Nuclear Translocation; Outer Hair Cells; Oxidative Stress; Play; Predisposition; Preparation; Presbycusis; Process; Proteins; Public Health; Quantitative Reverse Transcriptase PCR; Regulation; Research; Research Personnel; Research Project Grants; Resolution; Role; Sentinel; Shapes; Signal Transduction; Source; Stress; Structural defect; Synapses; Testing; Time; Tweens; Western Blotting; Wild Type Mouse; age effect; age related; aging population; biological adaptation to stress; cell injury; cholinergic; comparative; genetic strain; graduate student; hearing impairment; immunocytochemistry; in vivo; inhibitor/antagonist; light microscopy; mutant; nerve supply; oncomodulin; programs; repaired; response; sound; tool; undergraduate student

PROJECT SUMMARY/ABSTRACT Approximately 15% of Americans have high frequency hearing loss caused by exposure to loud sounds and 50% of Americans over 75 years old are affected by presbycusis. Although substantial progress has been made in determining the genetic and cellular functions disrupted by acquired hearing loss, comparatively little is known about the endogenous cellular and molecular mechanisms used to protect cochlear hair cells from the damaging effects of aging and noise. The long-term objective of this research is to investigate the role of mobile Ca2+ buffers in the inner ear especially during development and aging. It is our contention that understanding maturational processes that occur during development may provide important cues to understanding attempts to repair damage during aging. In the cochlea, outer hair cells (OHCs) act as sentinels of cochlear injury. Calcium regulation is fundamentally important to OHC development, function and aging. This proposal focuses on the development and age-related role of oncomodulin (OCM), a major Ca2+-binding protein preferentially expressed in OHCs. We hypothesize that OCM is necessary for the maturation of calcium signaling in OHCs and protects OHCs from the damaging effects of cellular stress. Specific Aim 1 determines the role OCM has in regulating Ca2+ signaling in pre-hearing and post-hearing OHCs. We will use Ca2+ imaging techniques and organotypic and cell culture methods to address the following hypotheses: 1. OCM shortens Ca2+ signaling kinetics and magnitudes in pre- and post-hearing OHCs; 2. OCM modulates the expression of other proteins involved in OHC Ca2+ signaling; 3. OCM Ca2+ signaling depends on the Ca2+ source. Specific Aim 2 tests whether OCM mediates sensitivity to aging and noise. Using in vivo functional assays assessing cochlear thresholds (ABRs and DPOAEs) and light, confocal and electron microscopy in Ocm mutant mice, we will investigate the following hypotheses: 1. targeted deletion of Ocm accelerates ARHL independent of genetic strain leading to decreased suprathreshold responses, loss of OHC cholinergic efferent synapses, and cell death; 2. OCM deficiency makes OHCs more susceptible to damage after cochlear injury. Specific Aim 3 tests whether OCM modulates Ca2+-mediated cellular stress and promotes cell survival. Using qRT-PCR, western blots, and immunocytochemistry in wild-type and mutant ears and in transfected cell lines, we will investigate the following hypotheses: 1. In response to stress, OCM translocates from cytoplasm to the nucleus ; 2. OCM modulates cellular responses to mitochondrial stress; and 3. OCM intracellular location and modulation of cell stress promote overall cell survival. In summary, these studies on OCM provide new tools that should significantly enhance our understanding of the role of Ca2+ regulation in protecting auditory function. Undergraduates will play significant roles roles in the collection and analysis of data of each aim.

项目摘要/摘要 大约15％的美国人因暴露于大声的声音和 75岁以上的美国人中有50％受到长老会的影响。尽管取得了很大的进步 在确定因获得性听力损失而破坏的遗传和细胞功能时制造的，相对较少 关于用于保护耳蜗细胞免受的内源性细胞和分子机制已知 衰老和噪声的破坏性影响。这项研究的长期目标是调查 内耳的移动CA2+缓冲区，尤其是在开发和衰老过程中。我们的论点是 了解开发过程中发生的成熟过​​程可能会为 了解在衰老过程中修复损害的尝试。在耳蜗中，外毛细胞（OHC）充当哨兵 耳蜗受伤。钙调节对OHC的开发，功能和衰老根本重要。这 提案重点介绍了oncomodulin（OCM）的发展和年龄相关的作用，这是主要的Ca2+结合 最好在OHC中表达蛋白质。我们假设OCM对于钙的成熟是必需的 OHC中的信号传导并保护OHC免受细胞应激的破坏作用。特定目标1确定 OCM在调节Ca2+信号传导中的作用在听证前和听证后OHC中的作用。我们将使用Ca2+ 成像技术以及有机和细胞培养方法以解决以下假设：1。OCM 在听证前和听证后OHC中缩短Ca2+信号动力学和磁化； 2。OCM调节 参与OHC Ca2+信号传导的其他蛋白质的表达； 3。OCMCA2+信号取决于Ca2+ 来源。特定目标2测试OCM是否介导对衰老和噪声的敏感性。使用体内 功能评估评估人耳蜗阈值（ABR和DPOAES）以及光，共聚焦和电子 OCM突变小鼠中的显微镜检查，我们将研究以下假设：1。靶向OCM的缺失 加速ARHL独立于遗传菌株，导致上股权降低，OHC的丧失 胆碱能有效的突触和细胞死亡； 2。OCM缺乏使OHC更容易受到损坏 人工耳蜗受伤。特定目标3测试OCM是否调节Ca2+介导的细胞应激和 促进细胞存活。在野生型和突变体中使用QRT-PCR，蛋白质印迹和免疫细胞化学 耳朵和翻译的细胞系中，我们将研究以下假设：1。响应压力，OCM 从细胞质转移到细胞核； 2。OCM调节细胞对线粒体应激的反应；和 3。OCM细胞内位置和细胞应激的调节促进总体细胞存活。总而言之，这些 OCM的研究提供了新工具，应显着增强我们对CA2+作用的理解 保护听觉功能方面的调节。本科生将在集合中扮演重要角色， 分析每个目标的数据。

项目成果

Oncomodulin regulates spontaneous calcium signalling and maturation of afferent innervation in cochlear outer hair cells.

• DOI: 10.1113/jp284690
• 发表时间: 2023-10
• 期刊: JOURNAL OF PHYSIOLOGY-LONDON
• 影响因子: 5.5
• 作者: Yang, Yang;Murtha, Kaitlin;Climer, Leslie K.;Ceriani, Federico;Thompson, Pierce;Hornak, Aubrey J.;Marcotti, Walter;Simmons, Dwayne D.
• 通讯作者: Simmons, Dwayne D.

Oncomodulin (OCM) uniquely regulates calcium signaling in neonatal cochlear outer hair cells.

• DOI: 10.1016/j.ceca.2022.102613
• 发表时间: 2022-07
• 期刊: CELL CALCIUM
• 影响因子: 4
• 作者: Murtha, Kaitlin E.;Yang, Yang;Ceriani, Federico;Jeng, Jing-Yi;Climer, Leslie K.;Jones, Forrest;Charles, Jack;Devana, Sai K.;Hornak, Aubrey J.;Marcotti, Walter;Simmons, Dwayne
• 通讯作者: Simmons, Dwayne