Jeffrey T. Corwin - US grants

[unreadable] DESCRIPTION (provided by applicant): Millions of Americans are affected by permanent hearing deficits and balance dysfunctions that result from losses of sensory hair cells. In mammalian ears, when hair cells die they are not effectively replaced, but the case is quite different in non-mammalian vertebrates. In those species hair cell loss leads to cell proliferation and the subsequent differentiation of new replacement hair cells, which then become innervated. These regenerative events lead to structural healing of damaged ears and can restore hearing and balance a few weeks after non-mammalian vertebrates have experienced damage that would result in permanent deficits for humans. Research has shown that human and rodent ear tissues can activate important elements of the biological machinery that underlies regeneration, but the regenerative responses in mammalian ears are normally limited. The research proposed seeks knowledge that may provide the means to eventually overcome those limits. This request for renewal of a project in its 24th year proposes to continue investigations that focus on identifying and understanding the signaling mechanisms that control the production of cells and the processes that lead to their specialization as sensory hair cells in embryonic and postembryonic ears. The mechanisms that limit self-repair in the ears of mammals will be investigated and treatments that may overcome those limits will be tested. The information sought may identify targets for the development of therapeutic approaches to stimulate self-repair in the ears of mammals. In addition, this project will seek to scale up the in vitro production of hair cells from lines of passaged cells. The availability of specialized cells that are produced in vitro has provided the basis for many examples of significant gains in cancer cell biology, neurology, and other fields. These cell lines and the knowledge gained in their further development and utilization holds the potential for contributing to improved treatments for diseases of the ear. The goals of this research directly pertain to possible recovery from sensorineural hearing loss and balance dysfunctions that contribute to prevalent communication disorders and to falls by elderly individuals. They also are likely to lead to improved understanding of the development of normal and abnormal auditory and vestibular function in human ears. With the proposed investigations, we seek to contribute to better understanding of the cellular lineages and the cellular mechanisms that contribute to the development, regenerative replacement, and in vitro production of inner ear hair cells. Hair cell loss is the leading cause of permanent hearing impairment as well as a contributing cause of balance impairments that can lead to falls in older individuals. The knowledge we seek holds the potential to ultimately contribute to the development of treatments that may lead to partial or complete recovery from forms of hearing or balance impairment that are now permanent. [unreadable] [unreadable] [unreadable]

Destruction of hair cells in the human ear results in irreversible loss of equilibrium or hearing sensitivity since our ears do not produce hair cells after birth. Recently I demonstrated hair cell production occurring perpetually in the ears of sharks, skates, and amphibians. In some species this results in new cells equal to more than 50 times the number in the human cochlea and a 500-fold increase in physiological sensitivity. Neurons here also grow continually expanding their arbors to innervate new hair cells, and the populations of supporting cells in these epithelia turnover rapidly as demonstrated by labeling of DNA. In these ears, due to the persistence of hair cell production, the effects of hair cell damage may be reversible through regeneration. Here a plan is presented to continue investigating persistent sensory epithelium growth, to evaluate the regenerative capabilities of perpetually growing ears, and to test hypotheses concerning mechanisms that may control growth in hair cell sensory epithelia. The ultrastructure of the hair cell precursors in persistent germinative zones will be investigated by transmission electron microscopy. Aminoglycoside antibiotics and cryogenic surgery will be used to damage hair cells, then scanning electron microscopy will be used to evaluate the ear's ability to generate new hair cells in toads that normally continue to produce these cells. Rates of supporting cell turnover will be measured in the ears of adult toads and in the vestibular epithelia of mice through autoradiography. The regeneration of the processes of peripheral statoacoustic neurons and their guidance will be studied by denervating epithelia and in neuronal cell cultures. Experimental surgery in salamanders and transplantation of altered embryonic chick otocysts into host embyros and into organ culture will be used to examine trophic and inductive interactions between neurons and hair cells. The characteristics of growing hair cell epithelia are common to many forms including the embryonic ears of birds and mammals and the perpetually growing ears of anamniotes. The information sought here is essential for understanding the limits and capacities of regeneration and self repair in hair cell epithelia. It pertains directly to possible recovery from sensorineural hearing loss and balance disorders and to the development of normal and abnormal function in human ears.

Partial support is requested for a conference on the Mechanisms of Sensory Regeneration, to be held at the University of Virginia in May 1994. The symposium will be the second major conference on the topic of sensory regeneration held in the U.S. It will provide a forum for researchers to present findings pertaining to basic mechanisms that regulate cell proliferation, cell differentiation, neurotrophic support, neurite regeneration, regenerative repair in vertebrate limbs and peripheral nerves, and the regeneration of sensory cells and neurites in the auditory, vestibular, lateral line, olfactory, gustatory organs, and the retina. The principal focus will be on the regeneration of hair cells and their innervation. The first specific aim of the conference is to facilitate the transfer o information on the latest findings from cell biologists, biochemists, and molecular biologists, who are investigating the mechanisms that control cell growth and differentiation, to researchers in sensory biology who are addressing the application of those findings in efforts to develop regenerative therapies for sensory deficits that affect millions of Americans. The second aim is to inform scientists and their trainees who work at the cutting edge of research in the fields of cell and molecular biology about opportunities for research that may have practical applications for the treatment of sensory disorders. The third aim is to foster collaborations between those scientists and scientists in the various subdisciplines and laboratories that address sensory regeneration. The fourth aim is to provide the members of a wider audience with an up-to-date review of recent developments in research on sensory regeneration, with an assessment of the important issues that remain to be addressed, and with access to potential collaborators and consultants; so that they may more readily begin productive research on sensory regeneration. Contributed poster presentations will be solicited and abstracts will be reviewed for selection. The presentations and discussions will be videotaped and the proceedings will be published so that ideas and controversies emerging from the meeting will be widely disseminated in a timely manner. The topics addressed are important for a better understanding of the basic mechanisms of regeneration and self- repair in sensory organs and neurons, which could potentially lead to the development of treatments to reverse effects of damage and disease in the sensory organs of millions of people.

[unreadable] DESCRIPTION (provided by applicant): Millions of Americans are affected by permanent hearing deficits and balance dysfunctions that result from losses of sensory hair cells. In mammalian ears, when hair cells die they are not effectively replaced, but the case is quite different in non-mammalian vertebrates. In those species hair cell loss leads to cell proliferation and the subsequent differentiation of new replacement hair cells, which then become innervated. These regenerative events lead to structural healing of damaged ears and can restore hearing and balance within one month after non-mammalian vertebrates have experienced types damage that would cause permanent deficits in our own ears. Research has shown that human and rodent ear tissues can activate the biological machinery that underlies regeneration, but the regenerative responses in mammalian ears are normally limited. We are seeking the means to overcome those limits. In this project we shall conduct experiments that are designed to identify the mechanisms that lead to hair cell differentiation and we are seeking to produce large numbers of hair cells in vitro. The controlled expression of transcription factors will be used to induce hair cell differentiation in vitro. In addition we shall seek to identify extracellular signals that will induce embryonic stem cells to differentiate otic phenotypes in vitro. To determine whether embryonic stem cells, transfected inner ear epithelial cells, and different lines of immortalized otic cells can be induced to differentiate as hair cells we will transplant them into ears developing in vivo and then assess the expression of otic markers. We also shall seek to identify culture conditions that promote the differentiation of cells dissociated from ears of embryonic mice and we shall develop methods for clonal expansion of otocyst-derived stem cells. The availability of lines of specialized cells that can be produced in vitro has provided the basis for significant gains in understanding cellular development, function, and pharmacology in cancer cell biology, lipid metabolism, diabetes, neurology, and other fields of biomedical science. Such cell lines and the knowledge gained in the development of lines of cells differentiated from embryonic stem cells are likely to play vital roles in the development of treatments for disease. By meeting the goals of this project we hope to contribute to the translation of cell biology into advances that will be useful for understanding and treating diseases of hearing and balance. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Advanced Microscopy Facility (AMF) at the University of Virginia is a core service and user facility sponsored by the School of Medicine that has provided microscopy services to the university and surrounding community for over 30 years. An average of 85 Principal Investigators (PIs), and upwards of 200 individual researchers uses the services and instrumentation of the AMF annually. Electron microscopy has been a mainstay of the facility since its establishment in 1979. To aid PIs at UVA, many of whom are NIH supported, in their conduct of cutting edge research, the AMF strives to offer state-of-the-art instrumentation. The existing scanning electron microscope (JSM-6400) at the AMF is over 20 years old and does not meet two specific requirements of many current or potential future users: the ability to achieve very high resolution at a high magnification, and the ability to achieve nearly as high resolution in imaging delicate parts of samples at low accelerating voltages. The existing JSM-6400 is simply not capable of meeting the needs of the NIH funded projects of the current users. The purchase of the proposed JEOL JSM-7001F TTLS thermal field emission SEM would result in a large number of biomedical researchers at UVA finally being able to acquire high quality, high resolution scanning electron micrographs. Specifically, the thermal field emission gun, combined with through-the-lens optics, guarantees a resolution of 1.2 nm at 30 kV and 2.0 nm at 1 kV on the requested SEM, compared to 3.5 nm at 35 kV achievable with the existing tungsten filament SEM. Importantly, no other scanning electron microscope at the University of Virginia is set up for and available for dedicated biomedical research use. Therefore, acquisition of the JSM-7001F TTLS will allow major users of the AMF to achieve the stated aims of their funded projects and will offer a significantly improved scanning electron microscope with capabilities needed but not currently available to biomedical researchers at UVA.