Yehoash Raphael - US grants

The injured avian auditory epithelium can regenerate by replacing lost hair cells. In contrast, loss of hair cells in the mammalian organ of Corti is irreversible. We propose to use a comparative approach to determine why acoustic trauma in mammals results in a scar, whereas in chicks new hair cells are generated. We hypothesize that early responses to acoustic trauma in the organ of Corti are similar to those in the avian basilar papilla. The first two aims will test this hypothesis, using electron microscopy, immunocytochemistry and DNA-specific tracers. We will identify changes in surface morphology, cytoskeletal organization and intercellular junctions which occur during hair cell degeneration and scar formation in noise-exposed chick basilar papillae. We will then compare these parameters in the chick with those in the mammalian organ of Corti. The third aim will test the hypothesis that generation of new hair cells in chicks is dependent on loss of hair cells and formation of scars. Preliminary evidence that chick supporting cells divide after trauma focused our interest on the responses of supporting cells and the interaction between supporting cells and damaged hair cells. Markers for DNA and proliferation will be applied together with markers for junctional and cytoskeletal proteins to identify changes associated with supporting cell proliferation. Elucidating the spatial and temporal changes which precede supporting cell divisions may help determine the point of divergence at which avian inner ears begin the regenerative process whereas mammals do not. This work pertains to biological mechanisms of tissue healing in general, and to the repair of mosaic epithelial sheets in particular. Elucidating the mechanism of scar formation in the mammalian cochlea may help develop methods to clinically reduce susceptibility to acoustic trauma. Knowledge of the spatial and temporal patterns of scar formation and regeneration in chicks and scar formation in mammals may be useful in developing clinical means to induce generation of new hair cells in humans. Future efforts to unveil a method for inducing mammalian hair cell regeneration will likely focus on the point of divergence, in time and space, where chicks regenerate new hair cells and mammals do not.

DESCRIPTION: Hair cell loss in the organ of Corti is the most common cause of permanent sensorineural deafness. In contrast to mammals, birds have been shown to recover both structurally and functionally from injury to the inner ear epithelium. Supporting cells which surround the sensory hair cells have been shown to play an important roll in maintaining the injured epithelium. In mammals these supporting cells create scars that repair the tissue and in birds they generate new hair cells. The goal of this proposal is to understand mechanisms of repair in the auditory epithelia of mammals and birds and to elucidate the differences between these repair mechanisms. Our first specific aim is to examine the response of the auditory epithelium to trauma in mammals. Specifically, we will study changes induced by trauma in tissue distribution of molecules that are involved in signaling, and localize organelles responsible for tissue organization. In parallel to these studies in mammals, our second specific aim analyzes repair and regeneration in the avian auditory epithelium, to determine the cellular and molecular regulation of avian supporting cell response to injury. specifically, we will correlate regenerative activity of supporting cells with their morphology, characterize conversion of supporting cells into hair cells, localize signaling molecules, and determine the spatial-temporal distribution of a parathyroid hormone-like protein and a GTP-binding protein called CDC42, 2 gene products that are preferentially expressed in supporting cells after injury of the basilar papilla. Our third specific aim is to influence the outcome of trauma to the mammalian organ of Corti, with the ultimate goal of reducing or reversing injury. For this purpose, we will use topical conditioned culture media and purified bio-active agents to influence epithelial repair. The long term goal of our research work is to induce regeneration in the mammalian auditory epithelium. We expect that the localization of gene products activated by trauma, the comparative analysis between birds and mammals, and the utilization of data and research tools from other biological systems should enable us to develop clinically relevant procedures for prevention and therapy of deafness.

The Morphology Core of Kresge Hearing Research Institute provides morphological processing and assessments for the entire Program Project. The Core will handle the processing and analysis of tissues with a variety of morphological methods. The major part of the work will be performed by two full time research assistants, Peter A. Finger and Annapurna Ramakrishnan. Planning and supervision of the work, quality control and assessments of the results will be performed by Dr. Raphael. Drs. Altschuler and Hawkins will be available as consultants. Dr. Raphael and Dr. Middlebrooks will share the responsibility for coordinating the distribution of the load of work between the Core and the PIs on the sub- projects. All methods proposed for use by the Core are already in routine use in our laboratory. Publication for previous years include extensive use of the majority of these methods. Close working relationship exist between the PIs on the different projects and between the PI on the Core and the technical assistants. To further promote efficient and successful Core operation, frequent meetings will be set between the Core PI and the other PIs, to discuss progress and plan next steps in each of the specific aims. Dr. Raphael will also be involved in preparing the data for presentation in meetings or peer reviewed papers.

This proposal will investigate gene transfer and cell death in the cochlea. Gene transfer has recently become an important method for tissue engineering for experimental and clinical purposes. The experimental field of inner ear biology and the otology clinic may benefit greatly from gene transfer technology. Viral vectors have become the vehicle of choice for in vivo genetic interventions in the nervous system. The cochlea may be among the organs that can benefit from genetic manipulation via viral vectors. We have demonstrated efficient infection of guinea pig spiral ganglion cells in vitro and in vivo with the Ad.RSVntlacZ adenoviral vector. Transgene expression has been detected as long as eight weeks after the inoculation. In this application we propose to continue our successful preliminary experiments to optimize variable parameters of adenoviral mediated gene transfer into cochlear cells. Specifically, we will (a) determine the optimal inoculation parameters needed for efficient infection of spiral ganglion cells, (b) characterize the immune response to the viral infection, (c) determine the duration of viral gene expression, (d) characterize the spread of infection to adjacent tissues, and (e) determine the functionality of infected spiral ganglion cells using ABR measurements. We also propose to determine the effects of gene transfer on spiral ganglion cell survival. Specifically, we will determine if it is possible to (a) rescue spiral ganglion cells by overexpressing bcl-2 and (b) induce cell death in spiral ganglion cells by viral-mediated overexpression of bcl-xs. The data we have been generating, along with the experiments we now propose will solidify the foundation of viral mediated gene transfer in the cochlea. This technology will facilitate experimental elimination of specific cell types in the cochlea, and create a powerful tool for physiological studies in this organ. As such, it will be of benefit to the other projects described in our Program Project. Moreover, gene transfer will enable to study function of specific genes in the ear. It will also help the important clinical goal of preserving spiral ganglion cells, for reducing progressive trauma and enhancing benefits from cochlear implants.

Inner ear lesions can result in permanent balance and hearing deficiencies and have significant negative impact on quality of life. Therefore it is essential to expand the fundamental knowledge of the molecular basis for inner ear deficiencies and develop clinical interventions for their prevention and therapy. This application is to test the influence of two growth factors, glial cell-derived neurotrophic factor (GDNF) and insulin-like growth factor-I (IGF-I) on protection rescue and regeneration in the inner ear. Experiments in the first specific aim of this proposal will test the hypothesis that the IGF-I family of growth factors is necessary and sufficient to enhance repair and regeneration in the sensory epithelia of the inner ear. IGF-I, IGF receptor and IGF-I binding proteins will be localized using in situ hybridization and immunocytochemistry in normal and traumatized inner ears. We will also test the hypothesis that addition of exogenous IGF-I into the perilymph will enhance hair cell regeneration in the mammalian vestibular epithelium, and provide protection in the auditory and balance systems. The second specific aim is to test the hypothesis that viral-mediated transgene expression of GDNF in the cochlear fluids can protect and rescue auditory hair cells and function from temporary and permanent threshold shifts. The third specific aim will test the hypothesis that that infusion of exogenous GDNF into the perilymph leads to GDNF binding to hair cells in the sensory epithelium of the inner ear. We expect that our work will continue to advance auditory and vestibular research towards clinical treatment of environmentally based impairments in the auditory and vestibular systems. The hypothesis driven aims of this proposal are based on recent exciting preliminary data and on innovative in vivo experimental approaches combined with traditional assays. We anticipate that the work will contribute to the understanding of the inner ear response to trauma at the molecular level, and lead to the development of means for protection, rescue, repair and regeneration in the organ of Corti and the vestibular sensory epithelia.

DESCRIPTION (provided by applicant): Inner ear lesions result in permanent deficiencies in balance and hearing. It is therefore important to reduce or eliminate the lesions that are accompanied by a variety of insults to the inner ear. Neurotrophic factors have been shown to protect inner ear structure and function against acoustic and ototoxic trauma. One neurotrophic factor, the Glial cell-line derived neurotrophic factor (GDNF) demonstrated a robust protective effect against trauma in the inner ear of the guinea pig. The experiments in this grant are designed to continue to characterize the protective effect of GDNF in the auditory and vestibular systems, and to extend the data to a different animal model, the mouse. The experiments will use gene transfer technology for GDNF transgene over-expression in the inner ear. Specifically, we propose to (a) determine the protective effect of GDNF on the mouse auditory and vestibular epithelia using electrophysiological, behavioral and morphological analyses, (b) determine the cells types that bind GDNF in normal and traumatized inner ear tissues and (c) shed light on the genes that are involved in the downstream signaling cascade of GDNF. The data we propose to generate will enhance our understanding of the mechanisms of action of GDNF in normal and injured inner ear epithelia, knowledge that may eventually lead to better clinical treatments

DESCRIPTION (provided by applicant): Sensorineural hearing loss is most commonly a result of hair cell degeneration. Hair cells in the organ of Corti cannot be replaced once lost, and therefore, sensorineural hearing loss is permanent. The bHLH transcription factor Math 1 has been shown to specify the embryonic development of hair cells and to induce growth of extranumerary hair cells in explants of rat inner ears. Using in vivo inoculation of an adenovirus vector with the Mathl gene insert into the endolymph of the mature pre-deafened guinea pig cochlea, we were able to generate new cochlear hair cells in the organ of Corti. We have shown that non-sensory cells in the mature cochlea retain the competence to generate new hair cells upon over-expression of Mathl in vivo, and that Mathl is sufficient to direct hair cell differentiation in these mature non-sensory cells. We have also demonstrated that new cochlear hair cells attract auditory neurons and that ABR-measured auditory thresholds significantly improve due to the Mathl treatment in deaf animals. In this application we propose to further characterize the structural and functional outcome of Mathl -induced hair cell regeneration in the mature deaf cochlea. Our first Aim is to determine the extent of hair cell regeneration following Mathl over expression in cochleae deafened with severe ototoxic insults, and to characterize the new hair cells and the reorganization of the auditory epithelium following the treatment. Our second Aim is characterize the response of inner ear neurons to the presence of new hair cells. Our third Aim is to test the hypothesis that transduction channels at the tips of stereocilia of new hair cells are functional, and determine the influence of Mathl gene therapy on hearing thresholds in deaf guinea pigs. The novel and feasible experiments we propose will help to characterize and enhance hair cell regeneration induced by the Mathl transgene in the mature guinea pig cochlea. The work we describe will bring the innovative field of hair cell regeneration research closer to clinical applicability.

DESCRIPTION (provided by applicant): Perception of sound with cochlear implants (CIs) is currently accomplished, in most cases, by stimulating spiral ganglion neuron (SGN) bodies in Rosenthal's canal, since cochleae without hair cells typically lack auditory nerve fibers in the basilar membrane area (BMA). If auditory nerve fibers could be induced to regenerate back into the BMA, stimulation of such fibers could potentially lower the amount of current required for stimulus detection, increase dynamic range, improve temporal response properties and decrease channel interaction, thereby enhancing the perception of CI stimulation. Using recently developed methods, we generated preliminary data demonstrating the feasibility for long-term over-expression of a neurotrophin which targeted to the BMA of deaf ears, leading to robust regrowth of auditory nerve fibers into this tissue. This nerve regeneration was accomplished using non-toxic long-acting adeno- associated viral vectors that delivered into the cochlea using clinically feasible routes. Our proposed experiments will test the global hypothesis that presence of neurotrophins secreted by cells in and around the auditory epithelium will attract and maintain auditory nerve fibers, leading to improvement in measurable parameters of the functional (psychophysical and electrophysiological) responses to CI stimulation. Experiments in Aim 1 will compare the efficiency of BDNF versus NTF3 in attracting neurons to the deaf BMA and characterize the source of the neurons and their position in the tissue. Work in Aim 2 will compare detection threshold levels, dynamic ranges, temporal integration properties, spatial selectivity, and amplitude growth functions using established animal psychophysics procedures and electrically-induced auditory brainstem responses (EABR) in deaf reinnervated cochleae and deaf non-reinnervated cochleae. Aim 3 will determine if auditory nerve fiber regeneration into the BMA improves SGN survival and central connections. Aim 4 will determine how the combined effects of long-term chronic electrical stimulation and neurotrophin over-expression influence SGN cell bodies and projections. These experiments will set the groundwork for clinical methods to induce nerve regeneration that could enhance the outcome of cochlear implant procedures in patients with severe or profound sensorineural hearing loss. PUBLIC HEALTH RELEVANCE: The need to develop methods for inducing, directing and maintaining auditory nerve regeneration has been a critical barrier to the field. Our recent break-through in the application of gene transfer now provides the ability to target cells in the deaf inner ear, and insert genes into these cells for secreting a protein of choice (neurotrophin) leading to stable, long-term nerve regeneration in these ears. Auditory nerve fibers will grow into an area where they will be in close proximity to the cochlear implant electrode. Our proposed studies can, for the first time, test the long-term influence of auditory nerve regeneration on the psychophysical responses to electrical stimulation provided by the cochlear implant. We will further correlate these responses with survival and condition of the neurons and their peripheral and central processes. The outcome of the proposed work may improve communication for thousands of people who have severe or profound hearing impairments and use cochlear implants.

DESCRIPTION (provided by applicant): Research on derivation of stem cells and their differentiation into desirable inner ear phenotypes is rapidly progressing. However, practical use of cell replacement therapy for hearing loss will depend on the ability to insert these cells into the inner ear and incorporate them into the native tissues. Inserting cells into the layer of cells making the auditory epithelium is complex because the epithelium forms a confluent and extremely tight barrier. Here we propose novel strategies for starting to design methods for integrating exogenous cells in the auditory epithelium of the deaf ear, in vivo. The challenges include keeping the therapeutic cells alive until they are inserted, opening gaps in the tissue to allow insertion, and maintaining essential native cells intact. Our overall hypothesis is that the deaf ear can be manipulated to receive and incorporate stem cells. To test this hypothesis, we will investigate and optimize means for transient decoupling of inter-cellular junctions in order to facilitate insertion of exogenous cells (Aim 1), test means to transiently reduce potassium concentration in the scala media in order to promote survival of implanted cells and to protect sensitive and indispensable components of the cochlea while junctions are open (Aim 2), and test extent of integration of cells inserted into the endolymph while potassium levels are low and intercellular junctions are open (Aim 3). The work we propose will improve our understanding of the biology of the severely deaf cochleae, and facilitate development of future therapies for deaf ears needing cell replacement therapy. While the ideas and goals of the proposed work are innovative and never before attempted, the expertise and reagents are available, promising high feasibility for successful experiments. It is therefore expected that the results of this study will ultimately benefit a large proportion of patients with severe sensorineural hearing loss from environmental and genetic etiologies.

? DESCRIPTION (provided by applicant): Sensorineural hearing loss is caused by the death of hair cells in the organ of Corti, and once lost, cochlear hair cells in humans and other mammals do not regenerate. In contrast, non-mammalian vertebrates can functionally recover from deafening injury by mobilizing supporting cells to divide and differentiate to replace lost hair cells. Over the last 10 years, the consensus from many studies is that supporting cells in the embryonic and neonatal organ of Corti retain a limited capacity to divide and differentiate into hair cells under certain conditions, but that this ability declines precipitously prior to the onse of hearing. One facet of such an age-dependent decline in regenerative potential is the function of the transcription factor Atoh1. Ectopic expression of Atoh1 in embryonic or neonatal cochlear tissue can transform supporting cells or adjacent non-sensory into hair cells - but this ability appears to be severely diminished after the onset of hearing in mice. The goal of this proposal is to understand why the ability of Atoh1 to drive cochlear hair cell regeneration declines with age. Although there are many possible mechanisms for this age-dependent decline, we will test just two in the current proposal. First, we hypothesize that at least some of the transcriptional target of Atoh1 become epigenetically modified in supporting cells with age, rendering them unavailable for transcription. Our second hypothesis, which is not mutually exclusive with the first, is that Atoh1 requires transcriptional co-factors that are not present in the mature cochlea Studies from Drosophila and our preliminary data have identified the zinc finger transcription factor Gfi1 as a good candidate to potentiate the activity of Atoh1 during hair cell formation.

Abstract: The most common form of autosomal recessive hereditary deafness is due to loss of the Connexin 26 (Cx26) protein (encoded by the human GJB2 gene). In mice, loss of Cx26 (Gjb2) leads to impaired glucose transport in the placenta and early embryonic death, whereas conditional loss restricted to the inner ear results in hearing impairment and death of epithelial and neuronal cells in the cochlea. Mouse models for reduced Cx26 exhibit many of the features found in humans with Gjb2 mutations, but the models display a more severe phenotype than humans. Specifically, onset of pathology in mice is earlier, hearing loss is progressive and more severe, and spiral ganglion neuron (SGN) degeneration occurs. The underlying mechanisms for these differences in phenotypes are unclear, but may be related to developmental or cell type-specific expression and/or functions of Cx26. The pan-otocyst deletions selected for some mouse models may also be responsible for the extremely severe mouse phenotypes. Here, we propose to use a mouse model we have recently generated (Sox10Cre-Gjb2) in which Cre, driven by the promoter of the supporting cell gene Sox10, is used for deleting Gjb2. Sox10Cre-Gjb2 mice exhibit hearing loss and degeneration of the cochlear epithelium, and SGNs which appear less severe than other existing models. Using our mice, we will characterize critical roles for Cx26 during the development of the cochlea prior to the onset of hearing, versus its roles in function and survival of hair cells, supporting cells and neurons in the mature inner ear. Our team of investigators with a strong history of productive collaboration, will test the global hypothesis that Cx26 exhibits critical requirements for promotion of sensory epithelial and neuronal function and integrity that are temporal (i.e. embryonic vs. early postnatal vs adult) and cell type-specific, and that functional effects of loss of Cx26 can be corrected by gene replacement. We have three Specific Aims: (1) Characterize inner ear structure/function in mice with loss of Cx26 in supporting cells, (2) Test whether Cx26 exhibits temporal and cell type-specific requirements for promotion of cochlear epithelial cell and spiral ganglion cell function and integrity, and (3) Determine if Ad.CX26-GFP is sufficient to (a) restore functional gap junctions in the auditory epithelium as determined by fluorescence recovery after photobleaching (FRAP) and immunocytochemistry, (b) improve ABR thresholds and (c) rescue hair cells, supporting cells, and neurons in Gjb2 deficient mice. Results from these studies are poised to improve understanding of the pathophysiology of Gjb2 mutations in the ear, and accelerate development of specific and effective gene-based therapies for human Cx26 related deafness.

ABSTRACT Stem cell technology provides a useful research tool with basic science and clinical applications. The potential use of stem cells for therapy also holds great promise, but there remain several major obstacles. The main obstacles for cochlear applications are the lack of a coherent strategy for large-scale production of true otic cell types and of a tangible approach to introduce and integrate these cells to the cochlear duct. In this proposal, we take steps to address both of these major barriers. In Specific Aim 1, we seek to push the fate of stem cell- derived inner ear organoids toward a cochlear phenotype. Current protocols for generation of inner ear organoids produce clearly defined otic vesicles that develop into organoids with hundreds of vestibular-like sensory hair cells. In normal embryonic development, hedgehog signaling is critical for dorsal-ventral patterning of the otocyst and is essential for cochleogenesis. We will use agonists and antagonists to hedgehog signaling to influence fate specification. Next-generation sequencing will be used to assay hedgehog-dependent signaling in the stem cell-derived otic vesicles, and a combination of morphology and immunohistochemistry will be used to assay cochlear-vestibular specification in the derived organoids. In Specific Aim 2, we will systematically evaluate the survival and integration of otic cells in the cochlea. The main obstacle limiting implantation of exogenous cells into the auditory epithelium is a hostile high-potassium ionic environment and a tight-junction barrier that prevents effective integration. In this aim, we will ?condition? the cochlea by transiently lowering potassium levels and by disrupting cell-cell junctions. The survival and integration of naïve stem cells and stem cell-derived otic progenitors will be optimized and maturation examined. The results will pave the way toward a rationale stem cell therapy approach for replacing cells in the inner ear, with applicability to both environmental and hereditary hearing loss.