Jochen Schacht, Ph.D. - US grants

The aim of these interdisciplinary studies is the elucidation of biochemical mechanisms underlying aminoglycoside-induced hearing loss. Specifically, we wish to describe the molecular events leading to inner ear damage; to establish structure-toxicity relationships and develop in-vitro tests for ototoxicity; and to define conditions of reversibility or amelioration of toxic effects. The work is based on previously obtained results and begins with the hypothesis that these antibiotics bind to polyphosphoinositide lipids, displace calcium, and disturb membrane structure and function. The studies will combine biochemical, physicochemical, electrophysiological, and morphological techniques. The interaction of aminoglycosides with phosphatidylinositol bisphosphate is quantitated by physicochemical measurements, e.g., with monomolecular lipid films, differential scanning calorimetry or permeability changes of liposomes. Structurally modified drugs are tested as to their lipid binding and their effect on cochlear microphonics and action potentials in perilymphatic perfusions in order to determine those sites on the antibiotic that are involved in its ototoxic action. Drug/lipid interactions in monolayers will also be investigated for their usefulness as an in-vitro correlate of drug ototoxicity. Pharmacokinetic studies will determine the distribution of preferentially cochleotoxic and vestibulotoxic aminoglycosides in the inner ear and this will be compared to phosphoinositide content in these structures. Reversibility of gentamicin-induced hearing loss, previously established in cochlear perfusions, will be explored after systemic injection of the drug. The proposed studies should expand our understanding of the biochemical mechanisms underlying aminoglycoside ototoxicity and provide guidance for drug modification and amelioration of toxic effects.

The sensory cells of the inner ear are called hair cells, and they are responsible for hearing and balance. With modern tools, there has been a new sophistication in analyzing how these receptors work at the cellular level. This conference will bring together biophysicists, biochemists, pharmacologists, physiol- ogists and anatomists to focus on three major topics: hair cell motility, information processing, and intercellular connections among hair cells and nerve cells. The emphasis will be on reaching some consensus on these three topics, since they have recently emerged as leading issues, as the technology to address them has advanced in the last decade. The impact of this meeting is likely to be a new formulation of the role of interactions between the peripheral sensory system and the central nervous system, and will be important to understanding sensory processing in general, as well as understanding auditory and vestibular function in particular.

Determination of the nature of the transduction, encoding, processing, and perception of acoustic signals is the major objective of this proposal. An interdisciplinary approach to such a problem is essential. Investigators trained in different disciplines combine their expertise and collaborative on four closely integrated projects to define and analyze the major events from acoustic activation of inner ear receptors to perception. The proposal's objectives are accomplished by experiments on: (1) the molecular and structural substrate underlying auditory processing and the active biochemical mechanisms in the cochlea; (2) the biochemical characteristics and control mechanisms that determine the molecular events involved in auditory transduction and its regulation; (3) electrophysiological features of hair cells and central auditory cells from cochlear nucleus to auditory cortex that determine encoding and processing; and (4) behavioral responses that reflect perceptual capacities of the auditory system for the identification, discrimination, and classification of these complex signals. These four projects are designed to yield information on the processing and perception of auditory signals at four levels of analysis, and data from each level of analysis impacts on the definition of experimental questions in the other projects. The Kresge Hearing Research Institute provides an ideal facility for this research with its multidisciplinary staff, appropriate laboratories, technical and support capabilities. The research team includes specialized personnel in anatomy, biochemistry, immunology, neuropharmacology, electrophysiology, bioengineering, and animal behavior. This group has a successful history of collaboration and provides a critical mass for the conceptualization and design of the proposed studies. This timely program continues to represent the core focus of the Institute and its faculty. The results will eventually give us a comprehensive view of the characteristics of the auditory system that underlie perception of biological signals. Clear and important clinical relevance of this program lies in the definition of specific events in auditory transduction, which will aid our understanding of possible causes of hearing loss. The analysis of the processing of complex signals will lead to improved designs for speech processing for cochlear prosthesis and hearing aids, and a clearer understanding of the basis and course of speech discrimination deficits with sensorineural hearing loss.

The goals of the proposed research are to elucidate the mechanisms of aminoglycoside ototoxicity and to develop a rational preventive treatment. Aminoglycoside antibiotics have been a frontline in systemic therapy for serious Gram-negative infections for nearly five decades although their ototoxic and nephrotoxic side effects were recognized soon after the introduction of streptomycin in the 1940s. To date, the mechanisms of toxicity have not been established and the absence of protective therapeutic measures continues to complicate the clinical application of these drugs. Previous attempts to define mechanisms of action and to design antidotes were based on the belief that aminoglycosides do not undergo significant metabolism in the body. Our novel hypothesis which is based on recent experimental evidence that a toxic metabolite exists, contradicts this dogma and enables us to address questions that previous hypotheses left unanswered. Our aim is to characterize the metabolite and elucidate its contribution to the mechanisms of ototoxicity. The goals of the proposal will be accomplished by experiments on 1) chemical analysis of the metabolite and the enzymatic mechanisms of its formation; 2) mechanisms of the toxic action of the metabolite at the cellular and molecular level; 3) mechanisms of organ-specific toxicity; and 4) prevention or amelioration of aminoglycoside ototoxicity. These specific questions will be addressed with well-established biochemical, analytical and physicochemical techniques. The elucidation of the mechanism of aminoglycoside toxicity will provide a scientific rationale with far-reaching implications for the prevention or amelioration of adverse effects of a family of drugs whose primary efficacy is unquestioned.

This proposal offers an exciting interdisciplinary approach to fundamental problems of cochlear function and dysfunction. It is based on current theories of regulatory mechanisms governing cell physiology and on novel molecular genetic approaches to the identification of genes and gene products. The unifying theme of cochlear homeostatic mechanisms is addressed in five projects representing molecular biology and genetics (Projects I, II and III) and molecular cell biology and biochemistry (Projects IV and V). The proposal combines diversified and powerful steps to investigate the normal system (Project V), the system under stress (Projects III and IV) and the genetically altered system (Projects I and II). Projects IV and V propose hypothesis-driven investigations related to the role of calcium in cochlear mechanisms (Project V) and to the function of heat shock proteins in cochlear trauma (Project IV). These studies are paired with the exciting new methods that molecular biology and genetics provide. Projects I and II propose positional cloning of mouse deafness mutants ames waltzer and pirouette, respectively. Project III uses auditory-specific trauma to identify genes by differential display. The interdisciplinary approach strengthened additionally by the resource available through the Kresge Hearing Research Institute. The Histology Core is responsible for providing morphological and histochemical assessments. Likewise, the Physiology Core will provide electrophysiological characterization of mutations, trauma and normal function. These cores are essential components of the Program Project, providing anatomical and functional feedback critical to the hypotheses and specific aims of other projects. In summary, the present proposal represents a broadly based yet focused approach to the study of molecular mechanisms in cochlear homeostasis. It is particularly powerful through its interdisciplinary nature and the ability to place molecular genetics and molecular biology into a systems context. As disease or dysfunction are an aberration of homeostatic mechanisms, the current studies will yield major fundamental information and enable us to design novel ways to prevent trauma to the inner ear or to accelerate recovery from such trauma.

DESCRIPTION (provided by applicant): Aminoglycoside antibiotics remain the most commonly used antibiotics and the primary cause of preventable hearing loss worldwide. The impact of aminoglycoside ototoxicity has recently been aggravated by the global resurgence of tuberculosis and the increased occurrence of resistant bacteria necessitating multidrug regimens including aminoglycoside. Given the ten to 20% incidence of cochlear and vestibular disturbances associated with aminoglycoside treatment, this constitutes a major global health problem. The last decade has brought major advances in understanding the mechanisms of aminoglycoside action and designing interventions to prevent the ototoxic side effects. The proposed research is founded on past and recent discoveries from this laboratory that have led to the hypothesis of metal chelation and free radical formation by amino glycosides, and spurred by a first successful human trial demonstrating protection from gentamicin-induced hearing loss. The anticipated studies will follow new leads from preliminary experiments to delineate further the molecular mechanism of ototoxic action including the pathways of cell death and protection. Since we have recently established the adult mouse as a model for aminoglycoside ototoxicity we can combine biochemical and physiological investigations with the tools of molecular biology and the availability of mutant animals. In particular, our studies will 1. Characterize the potential contribution of lysosomal pathways to drug-induced hair cell death; 2. test the contribution of NF-kappaB mediated gene activation to cell survival; 3. analyze the regulation of the NF-kappaB pathway by signaling through phosphoinositide 3-kinase and Akt; 4. identify improved pharmacological protection based on an improved understanding of the mechanism of drug action. The results will define biochemical and molecular events involved in cell death and survival in aminoglycoside ototoxicity. The data may also help to understand other pathologies that are associated with oxidant stress, such as cisplatin ototoxicity, noise trauma and perhaps presbycusis. Optimized interventions to prevent aminoglycoside ototoxicity may serve as a basis for the translation of laboratory findings to the clinic. The attenuation or prevention of adverse effects of aminoglycosides will have far reaching implications for the continued but safe use of a family of drugs whose primary efficacy is unquestioned.

DESCRIPTION (provided by applicant): This application requests support to continue the productive work of the University of Michigan Hearing Research Core Center to enhance the quality and scope of NIDCD-mission oriented research. We are excited to expand this core to embrace three additional regional research facilities, comprising a total of 38 projects by 29 investigators, supported by $6,886,426 annual direct cost. The application builds on experience gained from the previous P30 grant and will continue, and expand, the currently successful core services. As a major new goal, it adds a novel approach to foster interactions and collaborations among auditory and vestibular scientists at the University of Michigan, Michigan State University, Wayne State University, and University of Toledo. The Scientific Synergy Core A will bring together these scientists for discussions, workshops, and meetings of scientific affinity groups to promote new collaborations and enhance the quality and scope of research. Investigators will be supported by three scientific cores offering resources, training, and services in auditory physiology (Core B), cell and molecular biology (Core C), and substance delivery to the inner ear (Core D). A technical Core E will design and manufacture specialized electronic and mechanical instruments, as well as consult in computing-related issues. Cores will be supervised by expert core directors and staffed with experienced personnel. The overall responsibility for the direction and operation of the Center will rest with its director. The day-to-day planning and operation of each core will be the responsibility of the core directors, with standard procedures for scheduling, prioritization of services, and quality control. The Core Center director will be assisted by an administrative staff for the coordination of all activities, monitoring of funds, personnel issues, and reporting requirements to the granting agency. Within the intellectual environment generated by the Scientific Synergy Core and the availability of these resources, this Center will promote interactions and multidisciplinary research by providing novel methodologies, standardized analytical tools, and the exchange of technological experience. Regular meetings of the core users, the affinity groups, and the entire Center research community will assure the best possible implementation of collaborations and effective use of research support.

Cellular responses to a traumatic insult include the activation of multiple pathways that lead either to survival (homeostatic pathways) or cell death (apoptotic or necrotic pathways). The control of these pathways requires the concerted efforts of second messengers, protein kinases, and transcription factors that will ultimately change the patterns of gene expression in the cell. Depending on the intensity of the stress, different genes will be activated, eventually shifting the balance from homeostatic pathways (at low stress) to cell death pathways (at high stress). This project intends to unravel some of these pathways and their contributions to noise-induced hearing loss. It is the underlying hypothesis - based on solid preliminary evidence - that an initial event in noise trauma is the formation of reactive oxygen species leading to the activation of transcription factors and upregulation of oxidant stress response genes. Specifically, the following aims will be addressed in a mouse model of noise-induced hearing loss: (1) the formation and distribution of reactive oxygen species and the activation of the transcription factors AP-1 and NF-kappaB; (2) the hypothesis that the phosphoinositide pathway (phosphoinositide-3-OH kinase; PI 3-kinase) and NF-kappaB integrate noise-induced signaling by mediating the effects of ROS and neurotrophic factor; (3) the expression of specific antioxidant genes and the localization of their protein products. The project is coordinated with the other projects and core facilities in this application both by the common theme and by shared coordinated design and shared animals. The joint analysis of the data will yield a wide-ranging and integrated model of cochlear stress responses which, in turn, will provide a rational basis for designing pharmacological interventions to prevent noise-induced hearing loss.

[unreadable] DESCRIPTION (provided by applicant): Presbycusis, age-related hearing loss, is a major reason for social isolation and loss of quality of life in the elderly but we have neither a clear understanding of the mechanisms involved nor of strategies to prevent it. This program project brings together laboratories with long-standing interests in the biochemistry and molecular biology of stress pathways in the inner ear and a group of scientists with expertise in genetic studies in mice, for a series of interrelated studies of the fundamental cell biology and genetics of presbycusis. Project 1 (Antioxidant and homeostatic pathways) will test the hypothesis that antioxidant defenses and redox-sensitive signaling pathways decline with age rendering the inner ear more prone to cell injury and death. Project 2 (Stress pathways in the aging cochlea) extends this hypothesis to age-related changes in the classic stress response ("heat shock proteins"), by analyzing induced responses to stress during aging, and will generate new mouse models to examine this pathway in the cochlea. Project 3 (Genetic analysis of stress resistance and loss of hearing) focuses on the genetic controls and physiological regulators that modulate hearing loss in a population of genetically heterogeneous mice. Genotyping will provide a genomic map of loci modulating hearing acuity, its change over age, and its resistance to noise-induced damage. Specifically, the project will test the hypothesis that modulation of stress-resistance will modulate presbycusis. The projects will be supported by three cores. Core A (Administration) will coordinate reporting and evaluation activities and facilitate interactions among the projects. Core B (Physiology) will evaluate auditory function, and Core C (Otopathology/Histology) will assess morphological changes and provide histological assays. The three projects are taking distinct but complementary approaches to delineate the molecular and genetic basis of age-related hearing loss. The results will answer questions on the relationships among two well-characterized stress pathways in the aging cochlea, and identify new genetic loci associated with age-related hearing loss. [unreadable] [unreadable]

Age-related hearing loss is undoubtedly multifactorial, combining genetic predispositions with a plethora of lifetime insults to the auditory organ. Therefore, no single model may encompass all facets of presbycusis and research has to select appropriate approaches to selected aspects that may contribute to this pathology. The proposed project explores a major hypothesis of aging namely that reactive oxygen species (ROS) are major contributors to age-related tissue dysfunction. The evidence behind this hypothesis is compelling and preliminary data are highly suggestive that both endogenous antioxidant defenses and redox-sensitive signaling pathways decline in the aging cochlea leaving cochlear tissues and cells prone to oxidative injury and death. The hypothesis will be addressed at several levels in four specific aims. The first aim will assess cochlear pathology in aging CBA mice and measure the intrinsic antioxidants defense systems (glutathione, antioxidant enzymes) that maintain the cellular redox status. The second aim will determine the activity of redox-regulated signaling pathways that restore redox homeostasis. The control of these pathways requires the concerted efforts of second messengers, protein kinases, and transcription factors that will ultimately change the pattern of gene expression in the cell. Consequently, the third aim will analyze the expression of redox-related genes in aging CBA mice and compare this pattern to gene expression in progeny of four-way crosses (bred in Project 0003) with accelerated hearing loss. Finally, the fourth aim proposes to supplement nutritional antioxidants in an effort to restore redox homeostasis in aging animals and attenuate an oxidative stress-based cochlear failure. The results of this study, together with the results from projects 0002 and 0003, will allow us to formulate a hypothesis of stress-related injuries to the aging cochlea. Such knowledge and the potential attenuation of hearing loss by antioxidants may have far reaching consequences for the treatment of presbycusis.

Age-related hearing loss is undoubtedly multifactorial, combining genetic predispositions with a plethora of lifetime insults to the auditory organ. Therefore, no single model may encompass all facets of presbycusis and research has to select appropriate approaches to selected aspects that may contribute to this pathology. The proposed project explores a major hypothesis of aging namely that reactive oxygen species (ROS) are major contributors to age-related tissue dysfunction. The evidence behind this hypothesis is compelling and preliminary data are highly suggestive that both endogenous antioxidant defenses and redox-sensitive signaling pathways decline in the aging cochlea leaving cochlear tissues and cells prone to oxidative injury and death. The hypothesis will be addressed at several levels in four specific aims. The first aim will assess cochlear pathology in aging CBA mice and measure the intrinsic antioxidants defense systems (glutathione, antioxidant enzymes) that maintain the cellular redox status. The second aim will determine the activity of redox-regulated signaling pathways that restore redox homeostasis. The control of these pathways requires the concerted efforts of second messengers, protein kinases, and transcription factors that will ultimately change the pattern of gene expression in the cell. Consequently, the third aim will analyze the expression of redox-related genes in aging CBA mice and compare this pattern to gene expression in progeny of four-way crosses (bred in Project 0003) with accelerated hearing loss. Finally, the fourth aim proposes to supplement nutritional antioxidants in an effort to restore redox homeostasis in aging animals and attenuate an oxidative stress-based cochlear failure. The results of this study, together with the results from projects 0002 and 0003, will allow us to formulate a hypothesis of stress-related injuries to the aging cochlea. Such knowledge and the potential attenuation of hearing loss by antioxidants may have far reaching consequences for the treatment of presbycusis.

The Scientific Synergy Core (Core A) is the intellectual fulcrum of this research core center. It represents a low-cost high impact core which will dramatically strengthen auditory and vestibular sciences at the University of Michigan and in the research community in the Michigan - Ohio region. Specifically, the core will promote scientific information exchange and scholarly discussions through regular contacts, further technology transfer to enhance research, aid principal investigators in the training of students, fellows and staff, and encourage new and increased collaboration through the formation of special interest groups. Within the P30 research cores the regular contacts between the principal investigators and key personnel of the cores will remain the customary avenue of communication. Going beyond these interactions, however, all students, fellows, and laboratory staff are included in the meetings of the interest groups of Core A and the three annual meetings that we are proposing. Despite its ambitions of creating a multi-institutional synergy core, Core A does not add significantly to the P30 budget because of the proximity of all participating laboratories, which eliminates the necessity for any overnight stays and associated costs for meetings, discussions, or service training. While the rules of the P30 restricts its use to programs with appropriate grant support, we feel that the intellectual environment created by Core A must not be limited to those with currently funded grants. Core A will serve the spirit of the P30 and the mission of the NIDCD by including young investigators trying to establish themselves and research groups between funding. In fact, we argue that the inclusion of these scientists may become one of the most important aspects of this core in strengthening and promoting NIDCD-mission research. The intellectual interactions will build mentoring relationships, encourage collaborations, and through discussion, enhance the quality of of our research, our grant applications and our publications. These aims will aid the growth of young groups and make established ones more competitive in their attempts to obtain grant funding.

Administrative Structure The scientific aspects of the Core Center, specifically the promotion of information exchange, scientific interactions, and the fostering of collaborations will be coordinated by the newly created Scientific Synergy Core (Core A). The charge to the administrative core therefore is purely organizational to 1. integrate and supervise the research core activities; 2. facilitate the operation of the cores by resolving issues of space and facilities; 3. assume administrative tasks for all cores such as personnel management and financial operations; 4. carry out or facilitate contacts with University of Michigan administration and granting agencies; 5. coordinate the submission of progress reports and renewal grant applications. Dr. Jochen Schacht is the Director of the proposed University of Michigan Hearing Research Core Center and will be responsible for both the overall goals and the administration of this grant. The research and user base comprises investigators from four institutions: The University of Michigan, Michigan State University, Wayne State University, and University of Toledo;however, all core facilities and activities will be located at the University of Michigan. Dr. Schacht has been on the faculty of the Kresge Hearing Research Institute since 1972 and holds the position of Professor of Biological Chemistry in Otolaryngology and Director of the Kresge Hearing Research Institute. He is PI on the current P30 and has extensive experience in the coordination of similar multi-investigator projects. He was the PI on a NIDCD-funded training grant "Sensory Mechanisms and Disorders" from 1983 to 1996 (continued by Dr. Pfingst), on a NIDCD-supported Program Project grant "Perception and Processing of Complex Signals" from 1991 to 1996 (continued by Dr. Middlebrooks) and on a NIDCD-sponsored Program Project grant "Molecular Mechanisms of Cochlear Function and Dysfunction" from 1996 to December 2001 when P01s were discontinued by NIDCD. Currently, Dr. Schacht is PI on an interdepartmental Program Project Grant on age-related hearing loss funded by NIA. Dr. Schacht will be supported in all aspects of research core organization by the directors of the individual research service cores, Drs. Altschuler, Dolan, Middlebrooks, and Raphael. Each research core has its own structure designed to meet the needs of the investigators and to maximize its efficiency. While the Research Core directors report to Dr. Schacht on core activities and progress, they determine the day-to-day operations of their cores as spelled out in the description of the Research Cores. In the administrative aspects of the program, Dr. Schacht is assisted by a team consisting of Ms. Jackie Blake, an experienced administrator, Mr. Gary Dootz, Administrative Assistant and grants manager, and Ms. Sue Ann Kelch, Finance Coordinator. The team will oversee all administrative services provided for the cores including fiscal planning and management, financial oversight and analysis, compilation of progress reports to NIH, personnel services, facilities and equipment needs, and resource allocations. Ms. Blake will primarily be responsible for the paperwork and recordkeeping for Core Center personnel and payroll and act as the liaison between the co-investigators of this Center and The University of Michigan's central services (The Medical School, Personnel, Plant and Maintenance Departments). Mr. Dootz is responsible for contacts with the Division of Research Development and Administration as well as with NIH. The part-time Finance Coordinator will provide the direct financial services required for the Center, including financial recordkeeping, monthly reconciliation of statements of accounts, and assistance with budget planning and review. Dr. Schacht will receive formal monthly reports on the current and planned expenses of each core and review these reports with the Research Core Directors.