Peter S. Steyger - US grants

Although clinical use of aminoglycoside antibiotics is essential against life-threatening bacterial infections, there are serious ototoxic and nephrotoxic side-effects in 4-14 percent of all aminoglycoside prescriptions (4 million annually). Ototoxicity causes sensory hair cell death, hearing loss, and vestibular disorders, leading to physical, mental, educational, and language difficulties in patients. The overall aim of our studies is to identify, and then prevent the cellular mechanisms that initiate ototoxic drug-induced hair cell death. The long- term goal of this project is to develop interventional strategies that will allow future clinicians to use aminoglycosides without serious side effects. Gentamicin and forskolin are two unrelated ototoxic drugs that depolarize mitochondria, and increase production of reactive oxygen species and calcium levels in hair cells. Each of these toxic sequelae is a powerful trigger for inducing hair cell death. These common cellular responses to toxicity suggest that unrelated ototoxic drugs trigger a common pathway that lead to hair cell death. Therefore, we will use explants of bullfrog saccular hair cells to: (1) Identify the acute effects of gentamicin and forskolin in hair cells, and determine if they trigger these toxic sequelae by similar or differing mechanisms (2) Determine if inhibitors of drug-uptake can prevent toxic sequelae in hair cells (3) Determine if effective inhibitors of drug-uptake and toxic sequelae enhance hair cell survival during ototoxic drug treatment These studies provide a direct link between the molecular and biochemical studies of ototoxic drugs and the morphological analysis of fixed tissues after drug treatment. This knowledge will advance development of clinical strategies to prevent drug- induced hair cell death, and preserve inner ear function during this critical pharmaceutical therapy.

DESCRIPTION (provided by applicant): Aminoglycosides are clinically essential for treating life-threatening Gram-negative bacterial infections, e.g., meningitis, and in preventing infection in burns and premature babies. However, there is a high incidence of ototoxicity and sensory hair cell death. Sensitive hair cell function is dependent on a structurally and physiologically intact ear. Thus, studies to determine mechanisms of ototoxicity have used intact animals, or inner ear explants excised from animals. There are a number of functional, toxicological, and pharmacological similarities between inner ear cells and cells of the kidney tubules. To exploit these similarities, we have developed cloned sub-lines of currently-available kidney cell lines to determine their validity as in vitro models to study the mechanisms of aminoglycoside transport and toxicity in the inner ear. Using the aminoglycoside gentamicin, we propose to use cloned kidney cells to study two distinct aspects of aminoglycoside ototoxicity: 1) Like hair cells, proximal tubule epithelial cells of the kidney are among the few cell types that are toxicologically sensitive to clinical doses of aminoglycosides. These cell types also share pharmacological sensitivities to several other drugs. We will use a clone of OK cells, a proximal kidney tubule line, to determine the feasibility of using proximal tubule cells as valid in vitro models of aminoglycoside uptake and toxicity by hair cells. 2) Little work has been done to determine the mechanism of drug entry into the endolymph. Research suggests that hair cell uptake of aminoglycosides is across the apical membrane that is exposed only to the highly-regulated endolymph. Thus, pathways of drug entry into the endolymph will be highly-potent sites of intervention to prevent ototoxicity during treatment. The various epithelia surrounding the endolymph are cumbersome to acquire as explants in the quantity and proper configurations to facilitate direct examination of aminoglycoside transit across these epithelia. Therefore, we have developed a kidney distal tubule cell line to use as a model for aminoglycoside transport across the various epithelia enclosing the endolymph compartment. Distal tubule cells share several characteristics with inner ear epithelia such as the marginal cells of the stria vascularis. We will use a clone of MDCK cells, a distal tubule line, to determine the feasibility of using distal tubule cells as valid in vitro models for aminoglycoside transport across the epithelia enclosing the scala media. Results obtained from both studies will periodically be compared with animal models for confirmation. Preliminary data obtained with use of our cloned kidney cell lines have already dramatically changed our understanding of aminoglycoside entry into, distribution within, and transit across epithelia. Both of these in vitro models should provide powerful tools for finding co-therapeutics to reduce or eliminate ototoxicity during aminoglycoside treatment, blocking both toxic events in the hair cells and drug access to the endolymph and, thus, the hair cells.

DESCRIPTION (provided by applicant): The anti-neoplastic drug cisplatin is essential for treating a variety of epithelial and brain tumors. However, it is ototoxic, neurotoxic and nephrotoxic, causing permanent deafness and acute kidney failure in patients treated with these drugs. The cellular distribution of cisplatin has been only detected indirectly via pathological observations, by low-resolution positron emission tomography, autoradiography, or biochemically using high-performance liquid chromatography. The long-term goal of this research is to prevent ototoxicity and permanent deafness. We have developed a fluorescently-tagged cisplatin molecule that is bio-active and can be detected within individual cells in the inner ear, kidney and other organs after systemic administration. In addition, we can use this conjugation procedure to identify cytoplasmic cisplatin-binding proteins that may reveal novel intracellular mechanisms of cytotoxicity or protection. The working hypothesis is: Cisplatin trafficks to cochlear hair cells via endolymph, and binds to cytosolic proteins. The specific aims of this project are to: Aim 1: characterize the cochlear distribution of cisplatin in three rodent models, Aim 2: identify the conditions that modulate cellular uptake of cisplatin in vitro, Aim 3: verify that cisplatin enters cochlear hair cells from endolymph in vivo, Aim 4: identify cisplatin-binding proteins and their distribution in the cochlea. By determining which cells take up cisplatin, the mechanisms by which it enters cells and identifying its protein-binding partners, we can begin to develop new strategies to prevent cisplatin-induced ototoxicity. This will allow clinicians to use cisplatin and related drugs more efficaciously while preserving auditory function, especially important in pediatric patients acquiring language and educational skills. Understanding the mechanisms of how cisplatin crosses the blood-labyrinth barrier to enter the cochlea is crucial to prevent cisplatin-induced ototoxicity. The proposed research will enable the development of new strategies to prevent cochlear uptake of cisplatin and its derivates and subsequent ototoxic sequelae, particularly life-long deafness, tinnitus and vestibular deficits.

Abstract The Alexander Graham Bell association for the Deaf and hard of Hearing (AG Bell) requests financial support from NIDCD for the presentation of three research symposia in 2010, 2012 and 2014. AG Bell has a strong track record of producing high quality conferences/scientific meetings that are relevant to the scientific mission of the NIH and to public health. Since 2000, AG Bell has been coordinating conferences in conjunction with NIDCD that improve scientific education and work toward improving outcomes for those who are deaf or hard of hearing. Conferences bring together hearing health scientist with individuals and families affected by hearing loss to bridge the gap between bench research and practical applications that improve hearing outcomes. Past symposia have drawn an average of 1,000 participants composed of individuals who are deaf or hard of hearing, hearing health professionals (including audiologist, speech language pathologists, educators of the deaf, otolaryngologists and researchers in the hearing health field), as well as parents of children who are deaf or hard of hearing. Our past symposia have been highly rated and well attended at AG Bell's biennial conventions. The 2010 conference will address the reasons for loss of function in the cochlear - the part of the inner ear that is responsible for hearing - as well as cutting edge research that may translate into improved treatments for hearing loss. This topic will facilitate AG Bell's ultimate goal to improve communication between biomedical researchers and individuals who benefit from their efforts. The conference will be held in Orlando, FL in June 2010.

DESCRIPTION (provided by applicant): Aminoglycoside antibiotics are essential for treating life-threatening bacterial sepsis, yet induce acute nephrotoxicity and permanent deafness/balance disorders. These noxious side-effects affect as many as 120,000 individuals each year in the US. The goal of this proposal is to identify the molecular mechanisms that traffic aminoglycosides across the blood-labyrinth barrier into the cochlear fluids and sensory hair cells to induce cytotoxicity and hearing loss. The long-term goal is to protect the cochlear sensory hair cells from drug-induced ototoxicity, and maintain life-long hearing function. Our published data indicate that systemically-delivered gentamicin is trafficked from the strial vasculature, across the stria vascularis, into endolymph prior to hair-cell uptake. We have identified a candidate aminoglycoside transporter, and inhibition of this transporter reduces cochlear uptake of fluorescently-tagged aminoglycosides. Aminoglycoside uptake by hair cells is also potentiated by prior noise trauma, implicating the involvement of additional gentamicin-permeant cation channels besides the mechanoelectrical transduction channel. The specific aims of this project are: First, to determine if the candidate aminoglycoside transporter is required for gentamicin uptake, cochlear trafficking and induction of ototoxicity in vivo (Aim 1). Second, to test if other non-selective cation channels (besides the MET channel) expressed by hair cells are gentamicin-permeant and induce cytotoxicity (Aim 2). And, third, since noise trauma and aminoglycosides trigger oxidative stress in hair cells, we will determine if oxidative stress activates gentamicin-permeant cation channels and enhances hair cell uptake of gentamicin in vivo (Aim 3). Identifying the molecular mechanisms of aminoglycoside trafficking across the BLB and entry into hair cells is crucial to rational development of new clinical strategies that protect cochlear function during life-saving aminoglycoside therapy. For example, the genes involved in these mechanisms can then be screened for single nuclear polymorphisms (SNP) that induce gain-of-function activity in identified gentamicin-permeant channels and transporters. This will allow clinicians to use aminoglycosides more judiciously and personalize gentamicin therapy for individual cases prior to treatment for life-threatening bacterial sepsis, tuberculosis and for prophylaxis in premature babies, and casualties with severe burns and blast injuries.

DESCRIPTION (provided by applicant): Aminoglycoside antibiotics are essential for treating life-threatening bacterial sepsis, yet induce acute nephrotoxicity and permanent deafness/balance disorders. These noxious side-effects affect as many as 120,000 individuals each year in the US, particularly infants and premature babies. The long-term goal of this research is to prevent the cochlear uptake of aminoglycosides and subsequent ototoxicity, thus preserving life-long auditory function. Aminoglycosides like gentamicin cross the cochlear blood-labyrinth barrier into the cochlear fluids and enter sensory hair cells, inducing toxicity and hearing loss. Our recent data suggest that systemically-delivered gentamicin is trafficked from the strial vasculature, across the stria vascularis, into endolymph prior to hair-cell uptake. We have identified a candidate aminoglycoside transporter. Pharmacological inhibition of this transporter reduces cochlear, strial and hair-cell uptake of gentamicin. The specific aims of this project are: First, to determine if other aminoglycosides are predominantly trafficked to cochlear hair cells via the stria vascularis and endolymph in vivo (Aim 1). Second, to test whether inhibitors of aminoglycoside trafficking preserves auditory function by preventing hair cell toxicity, and maintain the bactericidal efficacy of aminoglycosides. We will also determine if deletion of the candidate aminoglycoside transporter ameliorates aminoglycoside ototoxicity (Aim 2). And, third, investigate whether structural modification of aminoglycosides prevents cochlear trafficking, hair cell uptake and hair cell toxicity (Aim 3). Identifying the routes and molecular mechanisms of aminoglycoside trafficking across the BLB and into hair cells is crucial to develop new pharmacotherapeutic strategies that preserve auditory function during aminoglycoside therapy. Clinically-relevant inhibitors of aminoglycoside trafficking should not affect the bactericidal efficacy of aminoglycosides. Preventing cochlear trafficking and hair cell uptake of aminoglycosides will allow clinicians to use aminoglycosides more aggressively for treating life-threatening bacterial sepsis, specific agents of bioterrorism, and for prophylaxis in combat, severe burns and blast injuries.

PROJECT SUMMARY (See instructions): Although most investigators incorporate tissue imaging in their studies, these imaging methods are conducted with varying degrees of technical sophistication, anatomical detail, tissue quality, and technical expertise. In nearly all cases, there is potential for technical refinement when assisted by experienced laboratory personnel. To provide this critical technical support and improved application of digital imaging in ongoing, pending, and new studies, we will continue to operate the Imaging Core. The Imaging Core supports 14 Core Center investigators, funded by the NIDCD and other institutes, with strong focus and a substantial record of accomplishments in imaging the peripheral and central auditory, vestibular, and olfactory sensory pathways. We will continue to centralize expertise in light, confocal and electron-microscopic imaging to enhance presently-funded research projects, permit development of new projects, and stimulate collaborations between participating investigators. The Imaging Core will: 1. Provide administrative and technical oversight to improve facility function, 2. Provide personnel who will assist investigators with current and new studies, 3. Provide access to well-maintained equipment in a more cost-efficient and effective manner, and 4. Provide training to investigators and their staff in data acquisition and interpretation. The Imaging Core will be co-directed by Dennis Trune and Peter Steyger. By providing services not available through existing grants, the Imaging Core will enhance current, planned and future new research projects carried out by Core-facility users and stimulate productive collaborations between P30 investigators, as well as with non-P30 investigators.

SUMMARY Congenital and acquired hearing loss during infancy has lifelong, debilitating consequences. Early identification of hearing loss improves the efficacy of auditory (re)habilitation, communication outcomes and quality of life for these individuals. Systemic infections are a major cause of morbidity and mortality in neonates admitted to the neonatal intensive care unit (NICU). Bacterial infections (i.e., sepsis) are treated empirically with antibiotics, including the life-saving aminoglycosides, like gentamicin. In preclinical models, aminoglycoside treatment induces dose-dependent and frequency-selective sensorineural hearing and balance deficits (i.e., ototoxicity), as well as acute kidney damage. Systemic inflammation induced by bacterial ligands potentiates this drug-induced hearing loss. Infants with (suspected) sepsis require urgent gentamicin treatment, and appear to have a greater risk of hearing loss in pilot studies. Our long-term goal is to reduce the incidence, and extent, of drug-induced hearing loss among infants discharged from the NICU (graduates). We propose a non- interventional translational study of this vulnerable population to: Aim 1: Identify if gentamicin dose-dependently increases hearing loss in infants There is little rigorous data showing the dose-dependency and frequency-selectivity of aminoglycoside- induced hearing loss in humans. We will test the hypothesis that greater cumulative gentamicin dosing increases the degree of hearing loss in NICU graduates. Aim 2: Verify if (suspected) sepsis potentiates gentamicin-induced hearing loss in infants Pilot data suggest that NICU subjects with (suspected) sepsis and ?5 days of gentamicin therapy have a greater risk of hearing loss compared to their age-matched peers. We will verify these pilot data by testing the hypothesis that (suspected) sepsis increases the risk, and extent, of gentamicin-induced hearing loss in NICU graduates. If gentamicin-induced hearing loss in NICU graduates is (i) dose-dependent, and/or (ii) potentiated by (suspected) sepsis, these data will predicate the need for ototoxicity monitoring prior to, and following, discharge from the NICU. If implemented, this will (i) ensure earlier detection of hearing loss, (ii) improve the efficacy of auditory (re)habilitation strategies. In addition, identifying the incidence and dose-dependency of gentamicin- induced hearing loss will facilitate subsequent studies to determine if (i) reducing ototoxic aminoglycoside dosing, and/or (ii) alternative antibiotic or otoprotective strategies, better preserve lifelong hearing in humans. These strategies will enable NICU graduates to better meet their peers? listening and spoken language skills to fulfill their educational potential and lifelong contributions to society.

PROJECT SUMMARY/ABSTRACT Administrative Core ? Core A There are few academic centers that focus on translating novel therapeutics to preserve or restore hearing and vestibular function. In Phase I of this COBRE, the goal of the Administrative Core is to provide the thematic direction and scientific infrastructure for the Translational Hearing Center to establish a sustainable multidisciplinary research program at Creighton University with national and international recognition. We will integrate faculty development with strong mentoring and an excellent and innovative pilot project program, along with strategic planning and comprehensive evaluation, into a robust administrative structure. The Specific Aims of the Administrative Core are to: 1) Provide fiscal and administrative management of the Center including management of fiscal and personnel matters, coordination of meetings, accountability, enhanced online capabilities, and dissemination of results. 2) Develop strong Center investigators that obtain peer-reviewed grants to launch long-term research careers validating and translating novel therapeutics that preserve or restore hearing and vestibular function. We will provide intensive mentoring and access to Research Cores with opportunities for collaboration, and build a critical mass of Center-focused investigators. 3) Establish an excellent Pilot Project Program to attract new investigators to the Center, and provide access to our Research Cores, to empower them to generate sound pilot data to obtain extramural research grants. 4) Provide leadership for success and sustainability through four sub-aims to: a) recruit excellent new investigators and trainees to the Center; b) strengthen research funding of the Center, c) promote the Center?s financial independence by developing fee structures for Research Core services to enhance Center sustainability, and d) guide strategic planning for long-term sustainability of the Center; and 5) Develop an evaluation program to assess performance and outcomes of Center activities to identify and implement improvements to strengthen the impact of the Translational Hearing Center. A dedicated Administrative Core to create a supportive scientific environment will enable the Center to sustainably recruit, mentor and retain a critical mass of investigators to translate candidate ototherapeutics that preserve or restore hearing and vestibular function towards clinical studies.

ALTERATION & RENOVATION Project Summary/Abstract The Translational Hearing Center (THC) has established two functional cores: The Drug Discovery & Delivery Core (DDDC) and Auditory & Vestibular Technology Core (AVTC) to promote and enhance translational research on therapeutics for hearing loss. Currently all core facilities and equipment are located in close proximity to or within the Creighton University Core Facilities in the Criss I/II/III building complex, with additional satellite AVTCs located in neighboring institutions: University of Nebraska Medical Center (UNMC) and Boys Town National Research Hospital (BTNRH). To further empower our services for the Research Project Leaders (RPLs) and Center Investigators, we plan to align our facilities with our strength by creating new laboratory spaces through renovation of existing laboratories within first year of the COBRE award. To facilitate efficient chemical synthesis and analysis and facilitate new advanced imaging, we seek to renovate medicinal chemistry laboratory Criss II 326 and a new space for auditory hearing test laboratory Criss I 304. With these renovation projects completed, THC will have new, centralized laboratories for each core. These can be easily further expanded and integrated into the Creighton University Core Facilities. These will significantly enhance our services outlined in our proposal.

PROJECT SUMMARY/ABSTRACT Overall Plan for the Translational Hearing Center This Center of Biomedical Research Excellence (COBRE) application is to establish the Translational Hearing Center, administered by centrally-located Creighton University, with Boys Town National Research Hospital (BTNRH) and the University of Nebraska Medical Center (UNMC), as institutional partners. Our overall goal is to build a critical mass of academic translational researchers developing therapeutic interventions to preserve or restore hearing and vestibular function from a wide range of etiologies that cause hearing loss and vestibular deficits. Hearing loss in infants and children results in delayed acquisition of listening and spoken language skills critical for academic achievement and maximal career trajectories of affected individuals. In the aging population, hearing loss and vestibular deficits without appropriate rehabilitation accelerates aging and cognitive decline. Aim 1: Develop the infrastructure and expertise base for translational auditory and vestibular research. COBRE funding will enable an Administrative Core within the Center to provide a unique, transformational research environment for junior investigators to translate their basic science discoveries into therapeutic strategies that preserve or restore hearing and vestibular function. This will establish a broader nonclinical research program. The Administrative Core will coordinate interactions between project leaders with their Mentors, and an External Advisory Committee. The Administrative Core will develop a Drug Discovery and Delivery Core that will coordinate necessary drug screen assays and production of derivatives of lead compounds and their delivery to the inner ear and associated central neural pathways, as well as an Auditory Vestibular Technology Core to validate the efficacy of lead candidate ototherapeutics hits. Aim 2: Build a critical mass of funded investigators leading translational auditory and vestibular research. We will examine both peripheral and central mechanisms of hearing loss and vestibular dysfunction, and identify pharmacotherapeutic strategies preserve or restore hearing and vestibular function, with multiple levels of research funding for investigators. We also have an outstanding Mentoring Plan for project leaders, complementing their expertise with senior investigations as Internal Mentors and biostatistical support, as well as outside investigators with translational and clinical expertise as External Mentors. Additional mentoring is provided by Research Core staff, grantsmanship classes and Mock Study Sections of proposals prior to review. Evaluations of research progress, and all other Center activities, are also key to optimize Center success. The Center will also benefit from the burgeoning translational research environment in Omaha, Nebraska. Future plans call for continued expansion of the Center to include submission of Investigational New Drug applications, safety and efficacy studies and clinical trials in partnership with patient populations served by Creighton University?s academic medical center, Catholic Health Initiatives (CHI) Health system, BTNRH and UNMC.