Calcium Binding Proteins Regulate Susceptibility to Damage in the Inner Ear

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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.