Hearing Loss and Calcium Regulation

听力损失和钙调节

• 批准号: 8823760
• 负责人: DWAYNE D SIMMONS
• 金额: $ 15.8万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8823760
• 关键词: Affect; Aging; Alleles; Apoptosis; Auditory; Backcrossings; Binding Proteins; Buffers; Calcium; Calcium Binding; Calmodulin; Cell physiology; Cells; Cochlear Microphonic Potentials; Cytoskeleton; Data; Defect; Development; Ear; Electric Capacitance; Environment; Feedback; Foundations; Frequencies; Funding; Future; Genes; Genetic Transcription; Growth; Hair; Hair Cells; Health; Hearing; Homeostasis; Image; In Vitro; Injury; Investigation; Knock-out; Laboratories; Labyrinth; Lead; Length; Link; Measurement; Measures; Mediating; Mentors; Mus; Muscle; Muscle Contraction; Noise; Noise-Induced Hearing Loss; Organ of Corti; Outer Hair Cells; Parvalbumins; Phosphorylation; Physiologic pulse; Play; Presbycusis; Regulation; Relaxation; Reporting; Research; Research Proposals; Research Training; Role; Signal Pathway; Signal Transduction; Synapses; System; Techniques; Testing; Time; Training; Transducers; base; cell motility; deafness; extracellular; hearing impairment; immunoreactivity; in vivo; interest; mutant; oncomodulin; overexpression; protective effect; rat Pres protein; response; tool; voltage; voltage clamp; voltage gated channel

DESCRIPTION (provided by applicant): This mentored research training proposal provides the necessary time and training to augment and enhance new research into how defective Ca2+ homeostasis and signaling may be involved in hearing loss. Mentored research training will occur in the in vivo assessment of cochlear function within the laboratory of Dr. M. Charles Liberman (Aim 1); in the measurement of hair cell motility within the laboratory of Dr. David He (Aim 2); and in the measurement of hair cell cytosolic Ca2+ levels within the laboratory of Dr. Fabio Mammano (Aim 3). Our preliminary data suggest that targeted disruption of oncomodulin (Ocm), a major Ca2+ binding protein mostly in outer hair cells (OHCs), leads to progressive hearing loss. We hypothesize that Ocm regulates cyto- solic Ca2+ levels necessary to protect OHCs from noise damage and aging defects. Specifically, this proposal investigates mechanisms that in the absence of Ocm might make the ear more vulnerable to noise and aging. Specific Aim 1 tests the hypothesis that Ca2+ buffering by Ocm alters efferent-mediated responses. First, we will test in vivo OHC function in Ocm mutants by measuring DPOAE thresholds and growth curves and measuring cochlear potentials, with a focus on the cochlear microphonic (CM) potential. DPOAEs provide a window into cochlear amplification. Although dependent on frequency and SPL, the CM response can provide a measure of the transducer capability of OHCs and is an indication of hair bundle functionality. Second, we will test if a lac of Ocm alters efferent-mediated responses. Efferent stimulation normally decreases the OHC contribution to cochlear amplification, thereby eliciting fast suppression of DPOAE amplitudes. Specific Aim 2 tests the hypothesis that Ca2+ buffering by Ocm alters Ca2+-dependent motile responses of OHCs.0Increases in cytosolic Ca2+ levels lead to OHC elongation mediated by Ca2+-dependent phosphoryla- tion. However, the mechanisms that induce OHC shortening or a decrease in axial stiffness are little understood. We will investigate OHC motility and electrophysiological responses. We will measure length changes of OHCs to voltage steps in whole cell, voltage clamp conditions. To assess whether targeted deletion of Ocm affects voltage-gated channels, we will also measure OHC current-voltage relations and capacitance. Specific Aim 3 tests the hypothesis that Ca2+ buffering by Ocm alters the size of Ca2+ transients. Defects in Ca2+ regulation could lead either to a broadening of Ca2+ transients or increases in the amplitude of Ca2+ signals. We will investigate induced [Ca2+]i flux using fluorescent-based measurements. We will also compare influx of extracellular Ca2+ to internal (store) Ca2+ in Ocm mutants. In summary, the laboratories chosen for research training will provide the PI with new strategies and tools to employ within his own laboratory to further understand the role of Ca2+ regulation in deafness and enhance competitiveness for future funding.

描述（由申请人提供）：本指导性研究培训计划提供了必要的时间和培训，以增强和加强有关 Ca2+ 稳态和信号传导缺陷如何与听力损失相关的新研究。指导研究培训将在 M. Charles Liberman 博士的实验室内进行耳蜗功能的体内评估（目标 1）；在 David He 博士的实验室内测量毛细胞运动性（目标 2）；以及 Fabio Mammano 博士实验室内毛细胞胞浆 Ca2+ 水平的测量（目标 3）。我们的初步数据表明，靶向破坏癌调节蛋白 (Ocm)（一种主要存在于外毛细胞 (OHC) 中的主要 Ca2+ 结合蛋白）会导致进行性听力损失。我们假设 Ocm 调节细胞质 Ca2+ 水平，以保护 OHC 免受噪声损伤和老化缺陷。具体来说，该提案研究了在没有 Ocm 的情况下可能使耳朵更容易受到噪音和老化影响的机制。具体目标 1 检验以下假设：Ocm 的 Ca2+ 缓冲会改变传出介导的反应。首先，我们将通过测量 DPOAE 阈值和生长曲线以及测量耳蜗电位来测试 Ocm 突变体的体内 OHC 功能，重点是耳蜗微音 (CM) 电位。 DPOAE 提供了了解耳蜗放大的窗口。尽管依赖于频率和 SPL，CM 响应可以提供 OHC 传感器能力的测量，并且是发束功能的指示。其次，我们将测试 Ocm 的 lac 是否会改变传出介导的反应。传出刺激通常会降低 OHC 对耳蜗放大的贡献，从而引起 DPOAE 振幅的快速抑制。具体目标 2 检验了以下假设：Ocm 的 Ca2+ 缓冲改变了 OHC 的 Ca2+ 依赖性运动反应。0胞质 Ca2+ 水平的增加导致由 Ca2+ 依赖性磷酸化介导的 OHC 伸长。然而，引起 OHC 缩短或轴向刚度降低的机制尚不清楚。我们将研究 OHC 运动性和电生理反应。我们将测量 OHC 的长度随整个电池电压阶跃、电压钳条件的变化。为了评估 Ocm 的定向删除是否影响电压门控通道，我们还将测量 OHC 电流-电压关系和电容。具体目标 3 测试了以下假设：Ocm 的 Ca2+ 缓冲会改变 Ca2+ 瞬变的大小。 Ca2+ 调节缺陷可能导致 Ca2+ 瞬变变宽或 Ca2+ 信号幅度增加。我们将使用基于荧光的测量来研究诱导的 [Ca2+]i 通量。我们还将比较 Ocm 突变体中细胞外 Ca2+ 与内部（储存）Ca2+ 的流入。总之，选择用于研究培训的实验室将为 PI 提供新的策略和工具，供其在自己的实验室中使用，以进一步了解 Ca2+ 调节在耳聋中的作用，并增强未来资助的竞争力。