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.

项目总结/摘要 大约15%的美国人有高频听力损失，这是由于暴露在响亮的声音中造成的， 75岁以上的美国人中有50%受到老年性耳聋的影响。虽然在这方面取得了实质性进展， 在确定后天性听力损失所破坏的遗传和细胞功能时， 是已知的内源性细胞和分子机制，用于保护耳蜗毛细胞， 老化和噪音的破坏性影响。本研究的长期目标是调查 移动的Ca 2+在内耳中的缓冲区，特别是在发育和衰老过程中。我们认为， 了解发育过程中发生的成熟过程可能会提供重要线索， 试图修复老化过程中的损伤。在耳蜗中，外毛细胞（OHC）起着哨兵的作用 耳蜗损伤钙调节对OHC的发育、功能和衰老至关重要。这 一项提案的重点是肿瘤调节蛋白（OCM）的发展和年龄相关作用，OCM是一种主要的钙结合蛋白， 蛋白质优先在OHC中表达。我们假设OCM是钙成熟所必需的 在OHC中的信号传导和保护OHC免受细胞应激的破坏作用。具体目标1确定 OCM在调节听前和听后OHC中的Ca 2+信号传导中的作用。我们将使用Ca 2 + 成像技术和器官型和细胞培养方法，以解决以下假设：1. OCM 缩短听力前和听力后OHC中的Ca 2+信号传导动力学和幅度; 2. OCM调节 参与OHC Ca 2+信号传导的其他蛋白质的表达; 3. OCM Ca 2+信号传导依赖于Ca 2 + 源头特定目标2测试OCM是否介导对老化和噪音的敏感性。使用体内 评估耳蜗阈值（ABR和DPOAE）和光、共聚焦和电子 通过在Ocm突变小鼠中进行显微镜检查，我们将研究以下假设：1. Ocm的靶向缺失 加速ARHL，不依赖于遗传应变，导致阈上反应降低，OHC丧失 胆碱能传出突触和细胞死亡; 2. OCM缺陷使OHC更容易受到损害 耳蜗损伤后具体目标3测试OCM是否调节Ca 2+介导的细胞应激， 促进细胞存活。在野生型和突变型中使用qRT-PCR、蛋白质印迹和免疫细胞化学 耳和转染细胞系中，我们将研究以下假设：1。为了应对压力，OCM 从细胞质易位到细胞核; 2. OCM调节细胞对线粒体应激的反应;以及 3. OCM细胞内定位和细胞应激的调节促进总体细胞存活。总而言之， OCM的研究提供了新的工具，应该大大提高我们对Ca 2+作用的理解 保护听觉功能的调节。本科生将在收藏中扮演重要角色， 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