Neil Segil - US grants

Injury or loss of the mechano-sensory hair cells of the inner ear is considered to be the major cause of hearing and balance impairment. Because of the intimate relationship between hair cells and the sensory neurons that innervate them, substantial secondary loss of sensory neurons can occur following hair cell damage. Survival of the sensory neurons is a requirement for the effective use of cochlear prosthesis which depend on these cells to communicate electrical information from a speech processor to the auditory regions of the central nervous system. Sensory neuron survival is also likely to be important for restoring function following hair cell regeneration that reportedly can occur in the mammalian vestibular system. In the mammalian inner ear, the normal development and survival of these neurons is dependent on the correct expression of the growth factors known as neurotrophins. Two of the neurotrophins, the Brain Derived Neurotrophic factor (BDNF) and Neurotrophic Factor-3 (NT-3) are produced by cells of the sensory epithelium and required for the normal growth and survival of the cochlear and vestibular sensory neurons. The best evidence for this requirement comes from targeted deletion of the genes that code for BDNF and NT-3 which causes a total loss of cochlear and vestibular sensory neurons. The experiments proposed here are designed to identify the gene promoter sequences that control the spatial, temporal and physiological regulation of BDNF and NT-3 gene transcription in the inner ear. However, difficulties associated with the introduction of foreign DNA (transfection) into post-mitotic cells such ass the hair cells, support cells, and the sensory neurons of the inner ear have so far precluded a detailed analysis of the mechanisms of gene regulation in this tissue. To overcome these problems we have begun applying newly developed transfection techniques to study of gene expression in organ cultures made from the sensory tissue of the inner ear of mice. We propose to: 1) Develop reliable and consistent means to transiently introduce foreign DNA into the cells of the inner ear in organ cultures in order to provide a test system for analyzing specific promoter regulatory elements; 2) Analyze the promoters of BDNF and NT-3 genes by transient transfection in order to identify specific sequences needed to maintain the normal pattern of gene expression during development and in the adult. BDNF and NT-3 MRNA levels respond to a variety of physiological and environmental stimuli such as ototoxic shock. The development of new techniques for analyzing gene expression in the inner ear is crucial for understanding these transcriptional mechanisms and is a first step towards designing means of manipulating the expression of potentially clinically important proteins such as neurotrophins.

DESCRIPTION: (Adapted from the Investigator's Abstract) Most hearing loss and balance disorders are due to the death of sensory hair cells located in the auditory and vestibular systems of the inner ear. In lower vertebrates these cells are able to regenerate following damage, but in mammals this capacity is either absent or extremely limited. Regeneration, like embryonic development, requires coordination between the biochemical machinery that governs cell proliferation with that which governs cell differentiation and morphogenesis of structures like the sensory epithelium of the inner ear. The long-term objective of this proposal is to understand the signal transduction pathways mediating this coordination. These pathways are likely to be good targets for future efforts to manipulate the process of regeneration. This laboratory has recently discovered that p27kip1, a cyclin-dependent kinase inhibitor that plays an important role in regulating cell proliferation during development, is a key element in establishing normal cell numbers and morphology of the organ of Corti. In mutant mice lacking the gene for p27kip1 the sensory hair cells and supporting cells are over-produced during embryogenesis bringing about morphological abnormalities. The mutant mice are severely deaf. Abnormal cell proliferation continues postnatally in the organ of Corti, consist with a role for p27kip1 in maintaining the normally quiescent state and impeding regeneration following damage. The goal of this proposal is to test the hypothesis that p27kip1 regulates cell number and organ of Corti morphogenesis during development of the inner ear, and to investigate the mechanism of p27kip1 regulation in vivo. This hypothesis will be tested in knockout and transgenic mice, by correlating p27kip1 developmental expression with the cessation of cell division and with the morphological consequences of our genetic manipulations (Aim 1). Next, we will study the restricted pattern of p27kip1 expression during development, first by correlating temporal and spatial expression of the p27kip1 mRNA and protein levels during embryogenesis and, subsequently, by using transgenic mice to test the significance of these regulatory mechanisms for organ of Corti development (Aim 2). Finally, the role of p27kip1 in regeneration will be investigated, using the p27kip1 knockout mice for a comparison of regenerative responses between mutant and wild type animals. In addition, we will compare the regulation of p27kip1 expression in mice and in chickens, which are able to undergo proliferative proliferation in response to hair cell loss (Aim 3).

DESCRIPTION (provided by applicant): Sensory hair cell loss is the leading cause of deafness in humans. The mammalian cochlea cannot regenerate its complement of sensory hair cells and thus at present, the only treatment for deafness due to sensory hair cell loss is the use of prosthetics such as hearing aids and cochlear implants. Strategies for hair cell repair that involve either stimulation of quiescent cells in the mature organ of Corti, or which involve transplantation of progenitor cells able to differentiate into hair cells will require markers to identify such cells, as well as a detailed understanding of hair cell precursor biology. However, progenitor cells that give rise to hair cells of the mammalian inner ear are currently uncharacterized, largely because no definitive makers are available for their identification. We propose a pilot project to develop molecular genetic markers that identify unique subpopulations of otic epithelial progenitors. To accomplish this, we will use an important new resource (The Gensat Project) to identify markers of sub-populations of cells in the developing inner ear. This is a collection of transgenic animals that harbor bacterial artificial chromosomes (BACS) as transgenes and which express Green Fluorescent Protein (GFP) under the control of a single gene contained in the BAC. In each transgenic animal, GFP is expressed in a unique sub-population of cells dictated by the expression pattern of the gene in question. We have identified current BAC Transgenic Collection, with a list of genes reported in the literature to be expressed in the inner ear has identified twenty-three genes expressed in the inner ear. To explore the usefulness of the BAC Transgenic collection, we propose to analyze four BAC transgenic animals chosen from among this group, based on their potential to add to our knowledge of sensory hair cell progenitors. In Specific Aim 1, each transgenic line will be characterized developmentally for expression of GFP in otic sub-populations each sub-populations will be purified by Fluorescence Activated Cell Sorting. In Specific Aim 2, GFP-expressing sub-populations will be assayed for their ability to differentiate into hair cells using a newly developed dissociated culture system for sensory epithelium. In the Specific Aim 3, the same purified otic epithelial sub-populations will be profiled using micro-arrays to provide initial information about gene expression networks in the developing inner ear.

DESCRIPTION (provided by applicant): This proposal is based on the novel observation that the cell cycle machinery in hair cells is activated following antibiotic administration. We hypothesize that this cell cycle reentry is intimately involved in regulating the subsequent cell death of hair cells. The importance of the cell cycle machinery in stress responses and cell death is well established. Our earlier work suggested a role for the cell cycle machinery, in particular the cyclin-dependent kinase (CDK) inhibitor p19lnk4d, in maintaining the non-dividing state of hair cells and thus in regulating the sensitivity to ototoxic stress and subsequent death. Preliminary results presented in this proposal confirm this suggestion. In response to neomycin, activation of the cell cycle is observed. In addition, inhibitors of the cell cycle machinery are shown to block cell death following administration of aminoglycoside antibiotics. To pursue these observations, we propose three specific aims incorporating a combined pharmacological and genetic approach. In Specific Aim 1, the effect of blocking specific cyclin-dependent kinase (CDK) activity on response to ototoxin will be tested. In Specific Aim 2, we will focus on the response of hair cells to ototoxins and attempt to tie this response to the activation of the cell cycle at the molecular level. Finally, in Specific Aim 3 we propose experiments to test the hypothesis that upon reentry into the cell cycle, hair cells activate the 'downstream' DNA damage checkpoint and that this is the, proximal inducer of the cell death machinery... Loss of sensory hair cells is the leading cause of deafness in humans. The mature mammalian cochlea cannot regenerate its complement of sensory hair cells and thus at present, the only treatment for deafness due to sensory hair cell loss is the use of prosthetics such as hearing aids and cochlear implants. Strategies to protect hair cells from ototoxin induced cell death would allow more aggressive use of chemotherapy agents and antibiotic treatment of infections that are now limited'by -their known ototoxic side effects.

[unreadable] DESCRIPTION (provided by applicant): Loss of sensory hair cells in mammals results in permanent deafness because regeneration does not occur. The loss of regenerative ability is tied to the inability of the specialized supporting cells within the organ of Corti to begin dividing in response to hair cell death. We have taken a developmental approach to this problem. Our hope is that by thoroughly understanding the process by which the cells of the organ of Corti stop dividing during embryogenesis, we will gain insight into why regeneration does not occur. In doing so, we hope to provide tools and targets for therapeutic intervention into the problem of deafness. During development of the organ of Corti, control of cell proliferation is tightly coordinated with the process of cell differentiation and patterning (Ruben, 1968). We have shown that the cyclin-dependent kinase inhibitor p27Kip1 is required for timing this coordination. In p27Kip1 mutant mice, cell cycle exit is delayed, leading to supernumerary cells, a disruption of the orderly pattern of hair cell organization, and deafness (Chen and Segil, 1999). Although p27Kip1 abundance is widely believed to be regulated at the post-transcriptional level through control of protein turnover, our results indicate that transcriptional regulation of p27Kip1 is largely, though not entirely, responsible for the determining the number of cells in the mature organ. Additional preliminary data indicates that Notch pathway signaling may be a key player in regulating p27 transcription during organ of Corti formation. In Specific Aim 1, we analyze the role of Notch signaling in the spatial and temporal regulation of p27Kip1 transcription during embryogenesis of the organ of Corti. In spite of the importance of p27Klp1 transcriptional regulation, we have observed that in Skp2 mutant mice, there is also a defect in cell cycle exit and organ of Corti structure. Skp2 is part of the SCF-ubiquitin ligase complex that is involved in regulating p27Kip1 protein turnover. In Specific Aim 2, we address the role of post-transcriptional mechanisms in the regulation of p27Kip1 Finally, in Specific Aims 3 and 4, we address the problem of regeneration directly, by studying p27Kip1 regulation in postnatal supporting cells. We have recently developed techniques that allow us to purify postnatal supporting cells and grow them in vitro. In doing so, we have discovered that perinatal supporting cells retain the capacity to reenter the cell cycle and divide, while supporting cells from P14 mice are unable to do so. Changes in the ability of P14 supporting cells to down-regulate p27Kip1 are partly responsible for the block to cell division that results in the lack of regeneration. This specific aim investigates the molecular basis for the age-dependent change in p27 regulation that we hypothesize underlies the lack of regeneration in the mammalian inner ear. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The University of Southern California (USC) and the House Research Institute (HRI) together have established a research training program for predoctoral, postdoctoral, and physician-scientist scholars: the USC/HRI Hearing and Communication Neuroscience (HCN) Training Program. The program brings together a broad spectrum of scientists to enhance inter-disciplinary communication, and offers the advantage of providing research training opportunities that bridge basic science with translational research and clinical hearing applications. The program serves to reinforce research and training interactions between scientists at HRI with scientists in the Dornsife College of Letters Arts & Sciences, the Keck School of Medicine, and the Viterbi School of Engineering at USC. The program combines the strengths of an outstanding group of researchers focused on basic aspects of hearing and vocal communication at both USC and HRI, the resources of USC graduate programs in Neuroscience, Psychology, and Linguistics, and the expertise in clinical otologic excellence of HRI. The rationale of this proposal is to engage predoctoral, postdoctoral, and physician- scientist trainees in a highly interactive and multi-disciplinary training experienc ranging from cell biology to cognitive neuroscience and linguistics that is unfettered by conventional departmental barriers, and actively facilitates their development as independent scientists. We have successfully filled all positions with predoctoral and postdoctoral scholars every year during the first four years of the grant. One of the postdoctoral scholars was a physician-scientist who also completed the House Neurotology Clinical Fellowship Program. Predoctoral trainees typically join the program during the second year of their graduate training, whereas the level of seniority of post-doctoral trainees participating in the program varies. All trainees receive multi-disciplinary training in all aspects of hearing and communication neuroscience, as well as practical skills that will prepare them for careers in independently-funded research, education, and industry. The ability to expose trainees directly to both cutting-edge research in basic science as well as ongoing clinical research and applications is a major strength of the program.

Project Summary/Abstract: Deafness and balance disorders resulting from the loss of sensory hair cells of the inner ear are a major cause of disability and morbidity in the US. In mammals, the cells of the various sensory epithelia of the inner ear arise embryonically and subsequently do not regenerate if damaged (Rubel et al., 2013). Hearing loss resulting from the death of hair cells in the organ of Corti is thus permanent, and treatments aimed at reversing hearing loss through stimulated regeneration of hair cells are badly needed. However, experimentation on the cells of the inner ear is difficult due to their small number and extreme inaccessibility in the adult. As a result, modern techniques of cell and molecular analysis and drug discovery have been difficult to apply, and aside from prosthetics, treatment options for sensorineural deafness remain few. To overcome these problems, and in pursuit of new treatments for hair cell loss, we have used powerful new ?direct lineage reprogramming? technologies, originally developed for neuron-specific reprogramming (Son et al., 2011; Vierbuchen et al., 2010), to generate hair cell-like cells in vitro, directly from mouse and human somatic cells (fibroblasts and inner ear supporting cells). This advance allows a new range of experimentation into the mechanisms of hair cell differentiation, the development of preclinical models of genetic hearing loss (disease modeling), high-throughput screening for drug discovery related to regeneration and ototoxicity, and the application of gene therapy approaches to the problem of hair cell regeneration. The Aims of the proposal include: 1) Development of improved reprogramming strategies to induce mouse and human hair cell-like cells. 2) Development of a drug screen for ototoxicity, and an in vitro disease model of genetic hearing loss. 3) A test of reprogramming in a preclinical model of hair cell regeneration/replacement in long-deafened mice.

Project Summary/Abstract: Sensory hair cell regeneration does not occur spontaneously in the mature mammalian organ of Corti, making hearing loss permanent. The goal of this proposal is to identify the causes and mechanisms behind the failure of hair cell regeneration, as well as ways to stimulate regeneration in surviving populations of inner ear supporting cells in deafened individuals. Our primary hypothesis is that regeneration requires the re- engagement of developmental gene networks to guide supporting cells to a hair cell fate, and that during postnatal inner ear maturation, epigenetic barriers arise that block the re-activation of these gene networks. The goal is to develop methods to overcome these epigenetic barriers, and to establish new cell fates with regenerative potential within the organ of Corti. Experimentally, perinatal mice retain a latent capacity for direct supporting cell transdifferentiation to a hair cell-like fate, but this capacity is rapidly lost in the first weeks after birth. This age-dependent change in regenerative potential provides a window through which to investigate the transition from a permissive to a non-permissive state for this form of regeneration in the normally maturing organ of Corti. The work of this proposal is to elucidate the mechanism(s) of epigenetic control of transdifferentiation in perinatal supporting cells (Aim1) and to identify the machinery of maturation-related changes in epigenetic/chromatin structure in supporting cells of the inner ear. (Aim 2). We hypothesize that these changes are responsible for the failure of regeneration. Finally, to investigate the epigenetic structure of adult supporting cell chromatin in normal and deafened mice (Aim 3). We hypothesize that manipulation of the epigenetic state is an important approach for future regenerative medicine approaches to restoring lost hair cells.