Mark E. Warchol - US grants

The sensory hair cells of the inner ear are necessary for the detection of sound and head movement. Hair cells may be lost from the inner ear as a result of noise exposure, treatment with certain drugs, or as a consequence of aging. Recent evidence has shown that hair cells can regenerate in the ears of adult birds and mammals, and that supporting cells in organ cultures of ears taken from adult humans will proliferate after the death of hair cells. These findings strongly suggest that hair cell regeneration may be induced in the ears of humans. In order to develop clinical strategies that will bring about hair cell regeneration in the human ear, it is first necessary to understand how regeneration is initiated. More specifically, we must know what cellular and molecular signals are responsible for triggering the regenerative proliferation of inner ear supporting cells after the death of hair cells, and how these cells then go on to form replacement hair cells. The goal of the proposed research is to identify factors that trigger regenerative proliferation in the ears of warm-blooded vertebrates. Experiments will be carried out that test the ability of identified mitogenic growth factors to enhance the regenerative proliferation of supporting cells in organ culture preparations of inner ear sensory epithelia taken from birds and from adult humans. Additional experiments will determine whether the vestibular sensory organs of adult birds produce a diffusible mitogenic growth factor. The role of leukocytes in the process of hair cell regeneration will also be explored. Finally, experiments will be conducted that will determine whether hair cells can also regenerate by differentiating from existing supporting cells, without any accompanying cell division.

DESCRIPTION (provided by applicant): Hearing loss and vertigo are serious health problems that currently affect millions of Americans. Normal sensory function of the inner ear relies on mechanoreceptive hair cells, which convert sound vibrations and head movements into electrical signals that are conveyed to the brain. Hair cells can be injured or lost as a result of noise exposure, ototoxic medications, inner ear infections, or as part of normal aging. The inner ears of mammals (including humans) are unable to replace these cells, and the death of hair cells is the most common cause of sensorineural deafness. In contrast, the ears of nonmammalian vertebrates can quickly regenerate hair cells after injury, and it is of great interest to understand the cellular basis of this regenerative process. This proposal focuses on a novel and relatively unexplored aspect of regenerative biology. Prior research has shown that macrophages - the primary effector cells of the innate immune system - are critically involved in repair and regeneration of many types of somatic tissues. The cochlea and vestibular organs contain large numbers of macrophages, but the function of those cells within the ear is completely unknown. Our prior work has suggested that macrophages may help promote hair cell regeneration, but a direct demonstration has remained elusive. The proposed studies will address these long-standing issues, and will make use of newly-developed methods for selective elimination of hair cells and/or macrophages from the mature ear, both in vitro and in vivo. The outcome of the proposed experiments will indicate whether macrophages play a stimulatory or suppressive role in regeneration. Notably, knowledge acquired from this work will also extend beyond the realm of regenerative medicine. Many inner ear pathologies respond to immunosuppressive therapies. The cellular basis of such treatments is unknown, but these observations point to direct effects of inflammatory cells on the sensory structures of the ear. As such, completion of these projects will resolve many outstanding questions regarding the contribution of otic macrophages to sensory repair and will also provide new insights into the nature of the cellular dialogue between the innate immune system and the inner ear. PUBLIC HEALTH RELEVANCE: Hearing loss and vertigo are serious health problems that affect millions of Americans. This proposal focuses on identifying the role of macrophages and the innate immune system in the processes of repair and regeneration in the mature inner ear. Such knowledge could suggest new mechanisms for restoring sensory function.

DESCRIPTION (provided by applicant): Cisplatin is a widely-used chemotherapeutic agent that is highly effective against ovarian, testicular, bladder, and head and neck tumors. One consequence of cisplatin treatment is ototoxicity, which occurs in about 30% of treated individuals and can result in permanent hearing loss. Unfortunately, there are often no suitable alternatives for cisplatin treatment, so cisplatin-induced hearing loss is a serious clinical problem. Little is known about the cellular mechanisms of cisplatin ototoxicity, and most prior studies have focused on injury to hair cells. Recently, however, we have found that cisplatin treatment also has direct and long-lasting effects on supporting cells of the avian ear. Moreover, our studies show that the avian inner ear is completely unable to regenerate after cisplatin ototoxicity in vitro. An enhanced understanding of these phenomena should reveal new insights into both the actions of cisplatin on the inner ear and the mechanisms of sensory regeneration in the nonmammalian ear. In the proposed studies, we will first determine whether cisplatin causes the formation of 'platinum adducts' in nuclear DNA of avian supporting cells and whether such DNA damage is correlated with the lack of regenerative ability. Next, we will further characterize the effects of cisplatin on the avian cochlea and determine whether regeneration is impaired after cisplatin ototoxicity in vivo. A third set of studies will focus on the mammalian ear. We will determine whether cisplatin: (1) damages nuclear DNA within the mouse organ of Corti and (2) causes structural changes in cochlear supporting cells. Since healthy supporting cells will be essential for future regenerative therapies for hearing loss, it is essential that we understand how those cells are affected by exposure to ototoxic drugs

PROJECT SUMMARY (See instructions): The sensory organs of the inner ear are among the most complex and exquisitely-patterned structures contained within the vertebrate body. Research on the biological mechanisms of hearing and balance requires the ability to obtain high-resolution images of the cochlea and vestibular organs, as well as their associated tissues. Modern optical and electron microscopy can provide excellent imaging data from processed samples and live tissues, but the cost of such instrumentation is typically beyond the reach of individual labs. The most cost-effective method for providing advanced microscopy services to a number of labs is for those labs to form a consortium and share imaging facilities. The present proposal is for the continuation of a P30 project that is directed toward this goal. Specifically, the Molecular and Digital Imaging (MDI) core will provide access to modern optical and electron microscopy to a group of associated labs whose research is consistent with the mission aims of the NIDCD. This section of the proposal has two Specific Aims: First, we propose to provide facilities for optical microscopy (confocal and conventional epifluorescence) to our core group of researchers, along with complete training in the operation of those instruments. The second Specific Aim is to provide access to both scanning and transmission electron microscopy services to this same group of researchers, along with equipment for sample preparation and training in all facets of EM technique. Both optical and EM imaging are heavily reliant on digital image processing, so we will also provide complete computational resources to users of our shared facility. Our core research group is large and diverse, but we are united by many common goals. In addition, we foster numerous collaborative projects, many of which have been initiated and facilitated by the MDI core resources. The MDI core has established an excellent track record of utilization and research productivity. During the previous funding cycle, we have provided imaging instrumentation and training to a total of 37 labs, and data obtained from MDI core facilities has appeared in 56 peer-reviewed publications. In light of this high level of productivity, along with our newly upgraded confocal facility and strong research group, we seek to continue to promote the research of our member labs, and increase existing knowledge of the biological basis of hearing and balance and associated pathologies.

PROJECT SUMMARY (See instructions): The sensory organs of the inner ear are among the most complex and exquisitely-patterned structures contained within the vertebrate body. Research on the biological mechanisms of hearing and balance requires the ability to obtain high-resolution images of the cochlea and vestibular organs, as well as their associated tissues. Modern optical and electron microscopy can provide excellent imaging data from processed samples and live tissues, but the cost of such instrumentation is typically beyond the reach of individual labs. The most cost-effective method for providing advanced microscopy services to a number of labs is for those labs to form a consortium and share imaging facilities. The present proposal is for the continuation of a P30 project that is directed toward this goal. Specifically, the Molecular and Digital Imaging (MDI) core will provide access to modern optical and electron microscopy to a group of associated labs whose research is consistent with the mission aims of the NIDCD. This section of the proposal has two Specific Aims: First, we propose to provide facilities for optical microscopy (confocal and conventional epifluorescence) to our core group of researchers, along with complete training in the operation of those instruments. The second Specific Aim is to provide access to both scanning and transmission electron microscopy services to this same group of researchers, along with equipment for sample preparation and training in all facets of EM technique. Both optical and EM imaging are heavily reliant on digital image processing, so we will also provide complete computational resources to users of our shared facility. Our core research group is large and diverse, but we are united by many common goals. In addition, we foster numerous collaborative projects, many of which have been initiated and facilitated by the MDI core resources. The MDI core has established an excellent track record of utilization and research productivity. During the previous funding cycle, we have provided imaging instrumentation and training to a total of 37 labs, and data obtained from MDI core facilities has appeared in 56 peer-reviewed publications. In light of this high level of productivity, along with our newly upgraded confocal facility and strong research group, we seek to continue to promote the research of our member labs, and increase existing knowledge of the biological basis of hearing and balance and associated pathologies.

Spiral ganglion neurons transmit auditory information from cochlear hair cells to the neurons of the cochlear nucleus. Thus, spiral ganglion and cochlear nucleus neurons are essential for normal hearing and for restoration of hearing via cochlear or cochlear nucleus implants in deaf individuals. However, in some circumstances these neurons may degenerate or die after deafening, limiting the potential efficacy of these devices. The reasons for this neurodegeneration and its variable nature after hair cell death remain unclear. Recent findings from our labs have revealed that elements of the innate immune system are recruited to the spiral ganglion and cochlear nucleus after deafening and suggest that the activation status of immune cells is an important determinant of neuronal survival in both structures. We also show that these elements of the innate immune system have profound effects on the survival of the auditory neurons, in some cases being cytotoxic, in others possibly neuroprotective. In at least one deafness model, the immune response, remarkably, may be a principal cause of spiral ganglion neuronal death after deafening. To resolve these complex and disparate effects of the innate immune system on auditory neuronal survival ? with the long-term goal of developing immunotherapies for neuroprotection ? we propose to systematically delete specific components of the innate immune system involving Natural Killer (NK) cells, macrophages, or microglia to determine their effect on neuronal survival. The experiments will use transgenic mice and, in some cases, inhibitory antibodies. Both macrophages and NK cells are recruited into the spiral ganglion in response to hair cell injury. The proposed experiments will determine whether macrophages and NK cells are neurotoxic or neuroprotective in the injured cochlea and the roles of specific cytokines and chemokines in stimulation and potential neurotoxicity of these immune cells. A parallel series of studies will focus on neuroimmune interactions in the cochlear nucleus, in which extensive research by one of the co-PI's has shown that neuronal survival depends on afferent input during a `critical period' in early postnatal maturation. In contrast, mature cochlear nucleus neurons survive deafferentation. Preliminary data suggest that this may be due to neuroprotection by microglia (the resident immune cells of the CNS.) The proposed experiments will test this hypothesis. Together, these studies will test fundamentally new hypotheses implicating specific components of the innate immune system as critical, if not optimal, targets for neuroprotective therapies to promote survival of cochlea and auditory brainstem neurons after cochlear pathology.

Clinical studies have suggested a link between the loss of sensory function in the inner ear and the development of Alzheimer?s disease. The biological basis of this association is not clear. Neurodegeneration that occurs in Alzheimer?s disease (AD) appears to be caused by aberrant processing and clustering of protein fragments derived from amyloid precursor protein (APP) and the microtubule-associated protein Tau. Consistent with this notion, certain identified mutations in APP and Tau are known to greatly increase the risk of developing AD. The projects outlined here are partly motivated by our observation that high levels of APP and Tau are also present in the hair cells and afferent neurons of the cochlea and vestibular organs, raising the possibility that these proteins might contribute to degeneration in the aging inner ear. However, it is equally possible that inner ear dysfunction is linked to AD because the loss of sensory input from the aging inner ear increases the likelihood of CNS degeneration and dementia. Experiments proposed here will use established mouse models of Alzheimer?s disease and engineered deafness to address these issues. In Aim 1, we will determine whether b-amyloid and Tau deposition and subsequent degeneration occurs in the inner ear. Experiments in Aim 2 will test whether disruption of inner ear function leads to increased CNS neuropathology in a mouse model of Alzheimer?s disease. To our knowledge, these will be the first studies to directly examine the potential relationship between inner ear function and amyloid-related CNS pathology. The outcomes may reveal a role for APP in degeneration of the inner ear, or indicate that input from the ear affects amyloid deposition in the CNS. A more complete understanding of the link between inner ear function and Alzheimer?s disease may also lead to novel diagnostics for the detection of early stage AD in humans.