Anthony W. Peng, Ph.D - US grants

[unreadable] DESCRIPTION (provided by applicant): The purpose of this research is to discover proteins involved in hearing transduction and to begin to understand their importance in the mechanism of hearing transduction. A shotgun proteomic screen of inner ear hair bundles has been done to identify proteins located in stereocilia, which are the structures important to hearing transduction. This proteomic screen takes advantage of multidimensional protein identification technology (MudPIT) in order to increase coverage of low abundance and membrane proteins. Analysis of the resultant dataset will determine which proteins are potentially involved in the hearing transduction mechanism, and these proteins will be further explored. Antibody labeling will be an important verification that our technique works, and the labeling will helps us elucidate the function of the protein in stereocilia. Understanding of the proteins involved in hearing transduction will help to understand what malfunctions occur in deafness, and may eventually lead to an improved treatment of deafness. For the 2 to 3 out of every 1,000 children in the United States born deaf or hard-of-hearing, communication with the hearing world is a great hardship. In order to improve treatments for these deaf people, the first step is to understand the normal function of the system. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The broad dynamic range of the auditory system is a critically important feature, allowing it to detect stimuli at the subatomic level while at the same time not saturating for stimuli several orders of magnitude larger. In part, the dynamic range of the system is related to the ability of the hair cell mechanotransduction process to shift its activation curve by hundreds of nanometers in a calcium dependent manner known as adaptation. The second messenger cyclic AMP (cAMP) also shifts the operating range of the hair bundle, but no physiological relevance or biochemical mechanism is currently known for this process. The goal of this proposal is to explore the role of cAMP in modulating the hair bundle dynamic range at both the cellular and system level. The first aim of this research is to determine the subcellular localization of adenylate cyclase isoforms in the hair cell, since these proteins generate cAMP. The second aim explores the mechanism of cAMP action with five specific experiments: 1- determine whether inner and outer hair cells respond similarly to changes in cAMP, 2- determine the effects of cAMP on hair bundle mechanics as a means to identify the target site, 3- Photolytic release of caged cAMP will determine the kinetics of cAMP action, 4- The role of PKA in the cAMP will be determined pharmacologically and also with the use of knockout animals. 5- Whether calcium is involved in the cAMP response will be directly assayed using swept field confocal calcium imaging. The third aim will determine, the role cAMP plays in cochlear processes via the analysis of mutant mice using whole animal auditory testing of auditory brainstem responses, cochlear microphonics, and distortion products in order to understand how the system modulates the properties of the hair bundle. Together these data will provide new data regarding the biochemical pathway of cAMP regulation and also the role of cAMP in modulating hearing at the systems level. PUBLIC HEALTH RELEVANCE: Hearing impairment affects 28 million Americans. Furthermore, the incidence of hearing loss increases with age;about 30% of Americans over the age of 65 and 40-50% of Americans age 75 and older suffer from hearing loss. The prevalence of hearing loss along with the ever growing population of people afflicted with this potential disability is a clear indication that we need to better understand how the auditory system functions in order to better treat patients with hearing impairments.

DESCRIPTION (provided by applicant): Sound vibrations enter the outer ear through the ear canal and are converted into pressure waves by the middle ear. Pressure waves in the inner ear are converted to an electrical signal via the mechano-electrical transduction (MET) process in the hair bundle of sensory hair cells; this electrical signal drives synaptic transmission resultin in information traveling to the brain. Failures in this process lead to hearing loss and deafness. Multiple human genetic mutations exhibit deficits in the MET process. Understanding the basic properties of MET will lead to a better understanding of genetic deafness, leading to targeted treatments and therapies. A growing body of data on mammalian cochlear hair cell MET properties is incompatible with existing molecular models of MET. Specifically, adaptation, a key process of MET universally accepted to be signaled by calcium, does not appear to be driven by calcium ion entry, thus challenging current models of adaptation. To better understand the underlying mechanisms responsible for cochlear MET, mechanical changes in the hair bundle need to be measured at rates that match the fast rates of MET processes in cochlear hair cells. In this proposal, to overcome current technological limitations, new micro-electro-mechanical systems (MEMS) devices are developed to specifically measure cochlear hair bundle mechanics. Using whole-cell voltage clamp recordings of mammalian cochlear hair cells along with new MEMS devices, kinetics and mechanics of fast cochlear MET processes will be measured. This data will be used to generate new models of cochlear MET. Myosin motors localized to the upper tip-link region have been proposed to be important to MET. New experiments in the cochlea will be performed using these novel MEMS devices to characterize mechanics of the hair bundle when modifying motor activity. From these experiments, the role of molecular motors as well as the upper tip-link region in cochlear hair cells in MET processes will be determined. During acoustic trauma, hair bundles are stressed from overstimulation resulting in stiffness changes to the hair bundle. To characterize mechanical properties of the mammalian hair bundle, this proposal aims to quantify the contribution of stereocilia links and the stereocila rootlet to passive hair bundle stiffness using drug application and genetic mouse models lacking specific structures. The experiments in this proposal will further our understanding of the molecular mechanisms of mammalian cochlear MET. Understanding the crucial components in passive hair bundle stiffness will lay groundwork for understanding the key regulation points of hair bundle properties and the effects of acoustic trauma on stereocilia. The technology developed will greatly enhance auditory research and likely have broader mechanics applications in the auditory field and beyond.

Project Summary Cochlear amplification is the process by which our auditory system amplifies and tunes responses to incoming sounds, bestowing us with our excellent sound level sensitivity, large dynamic range, and fine frequency discrimination. Auditory sensory cells have two processes hypothesized to contribute to cochlear amplification: somatic motility that occurs in the cell soma and active hair bundle mechanics that occurs in the apical stereocilia hair bundle. To assay the contribution of active hair bundle mechanics to cochlear amplification requires further understanding of the processes related to it. Hair cell mechanotransduction (MET), the process of converting sound stimuli into electrical signals in the hair bundle, is the driver of active hair bundle mechanics. MET adaptation is one key mechanism that is hypothesized to contribute to active hair bundle mechanics. Previous work in non-mammalian models show that adaptation is separated into fast and slow processes, both of which rely on the influx of calcium to drive the process. Data in the mammalian cochlea indicate that adaptation also consists of fast and slow components, but our work shows that the underlying biology driving the fast and slow processes in the cochlea is fundamentally different from what has been previously reported in non- mammalian hair cells. Thus, new investigations are needed to understand the molecular machinery responsible for both fast and slow adaptation, and their contributions to mammalian auditory processing. From new data about properties of cochlear MET, we hypothesize that tension is essential for adaptation mechanisms. In Aim 1 of this study, we will investigate the contribution of myosin motors to adaptation and hair bundle mechanics. We assay this using new, faster stimulation and high-speed imaging to monitor mechanical changes in the hair bundle coupled with hair cell electrophysiology and pharmacological manipulation. With numerous myosin motors known to be important for auditory function, in Aim 2 we will explore the contributions of specific myosin motors to adaptation and hair bundle mechanics using existing mouse models. For Aim 3, we developed a new mouse model using CRISPR/Cas9 technology to acutely inactivate myosin VIIa motor function, and we will assess the role of myosin VIIa in tension generation. The experiments in this proposal will further our understanding of the molecular mechanisms of mammalian cochlear adaptation and hair bundle mechanics to develop a new model of the mammalian auditory MET process. We are uniquely positioned to accomplish this with the new technologies that we have and continue to develop. Basic mechanistic knowledge of auditory MET will lead to experiments where we can interrogate the system in vivo to determine specific molecular contributions to cochlear amplification. Understanding cochlear amplification can lead to better prevention and/or restoration of hearing.

Project Summary Vestibular dysfunction becomes more prevalent with age and it is estmated that more than 80% of people over 80 years old experience dysfunction5. Furthermore, approximately 8 million adults in the US suffer from balance impairment due to damage to the peripheral vestibular system, but effective treatments for balance dysfunction are virtually non-existent. Vestibular hair cells within vestibular canal and otolith organs convert hair bundle motion into receptor potentials and sensory information is relayed to the brain by action potentials in vestibular afferent nerves. Afferents in central zones of vestibular neuroepithelia exhibit different responses to vestibular stimuli than afferents in peripheral zones. There are three types of vestibular afferents: calyx-only afferents innervate one or more type I hair cells, bouton dendrites innervate type II hair cells and dimorphic afferents contact both hair cell types. Calyx-only afferents are present only in central zones and have irregular firing patterns, whereas dimorphic afferents exist in both zones and have regular firing patterns. We will study age-related dysfunction in calyx-bearing afferents in gerbil vestibular organs using novel preparations developed in the laboratories of the principal investigators. We will use electrophysiological, hair bundle stimulation, immunohistochemical and behavioral approaches to address age-related changes in mature and aged vestibular epithelia. In Aim 1 we will determine if functional changes in vestibular hair cell mechanotransduction and/or basolateral currents occur with age. Aim 2 will test the hypotheses that synaptic degeneration of calyx terminals will manifest as morphological uncoupling of type I hair cells from their associated calyces and deficits in vestibular evoked responses and behaviors. In Aim 3 we will directly investigate changes at the type I hair cell/calyx synapse by recording spontaneous activity and responses to hair bundle stimulation in mature and aged calyx afferents. Our investigative team is uniquely positioned to carry out the proposed studies. Results from this work will provide new information on how the aging process impacts peripheral vestibular signals and may inform development of vestibular neurotherapeutics targeting afferent nerves in order to restore normal vestibular function.