Steven H. Green - US grants

The goal of this study is to determine the nerve growth factor (NGF) signal transduction mechanism - the molecular mechanism by which binding of NGF to its receptor is converted into an intracellular signal that initiates the characteristic program of cellular responses. NGF and other neuronotrophic factors are of central importance in the ontogeny and maintenance of neurons. These studies will therefore be relevant to basic understanding of the cellular mechanisms underlying control of cell number, survival, growth and differentiation in the nervous system. Such understanding has potentially significant application to neuronal repair and regeneration in the amelioration of genetic and other neuro-degenerative disorders, trauma, and stroke. To the extent that NGF serves as an intracellular signal outside of the nervous system these studies will yield insight into the development and physiology of these tissues as well. It is also expected that these studies will be relevant to understanding the mechanisms of action of other neuronotrophic and growth factors and therefore to neoplasia, given the associations known to exist between oncogenes and growth factors. This proposal for study of NGF signal transduction consists of four projects which make use primarily, if not exclusively, of the NGF- responsive PC12 cell line and of the previously isolated non-NGF- responsive PC12 mutants (PC12nnr lines). The first project involves studies of the NGF receptor (NGFR) itself, focussing on regulation of its affinity, internalization and intracellular transport. The other three projects are all aimed at identifying and isolating NGF transduction-related proteins, other than the NGFR itself, by three different approaches. Each approach exploits a different aspect of the nature of such proteins, based on the results of previous studies of the NGFR and the mechanism of action of NGF. These projects will identify and isolate proteins involved in NGF signal transduction and produce immunological and molecular probes for them thereby allowing future studies of their function and expression. One project will be the biochemical isolation of NGFR-associated proteins, for which previous results have suggested key functional roles, exploiting their affinity for the NGFR. Following the isolation of such proteins, antibodies recognizing them will be prepared. The second project involves identification of transduction-related proteins by introducing DNA from NGF-responsive cells into PC12nnr cells using means that facilitate molecular cloning of those introduced genes that restore to PC12nnr cells the ability to respond to NGF. Additional PC12 cell mutants defective in responses to NGF will be isolated following proviral insertion mutagenesis. This tags the mutated genes with viral sequences, facilitating molecular cloning of the genes of interest. This project will identify genes involved in transduction or in the molecular processes underlying neuronal differentiation and neurite outgrowth. The products of the genes cloned by these means are proteins required for NGF responses. Antibodies recognizing these proteins can be produced from the translation products of the cloned genes. Third, transduction-related proteins will be assumed to possess particular enzymatic activities, such as GTP binding or tyrosine kinase. The identification, the function, and the role in NGF signal transduction of neuronal proteins possessing such activities will be explored in these studies.

Depolarization is a trophic stimulus for neurons, i.e., prevents neuronal death. Spiral ganglion neurons (SGNs) die following the loss of hair cells. Electrical stimulation promotes survival of deafferented SGNs in vivo, raising the possibility of using electrical stimulation to maintain survival of SGNs in humans deaf as a result of loss of hair cell function. In particular, the efficacy of electronic cochlear implants would be greatly augmented if SGN death was prevented. To this end, we propose studies directed towards determining the mechanism by which depolarization promotes survival of SGNs. These studies take advantage of an in vitro model of the regulation of SGN survival by neurotrophic stimuli. Using this model, we find that depolarization, functioning by increasing cytosolic Ca2+, is a stronger trophic stimulus than neurotrophic factors. There are three general objectives: The first is to further characterize the in vitro model of the maintenance of SGN survival by depolarization and neurotrophic factors: (1) Molecular criteria derived from studies of neuronal apoptosis in other systems will be applied to the death of SGNs to determine if it is apoptotic. (2) Are there different subpopulations of SGNs that differ in their ability to be supported by depolarization and neurotrophic factors? (3) How long after withdrawal of trophic support do SGNs become committed to a cell death fate? The second objective is to initiate studies of the mechanism by which depolarization prevents the death of SGNs become committed to a cell death fate? The second objective is to initiate studies of the mechanism by which depolarization prevents the death of SGNs in vitro: (1) Does depolarization promote survival by inducing autotrophic mechanisms in the spiral ganglion cells? A variety of techniques will be used to detect induction of neurotrophic factors, neurotrophic factor receptors, and apoptosis-inhibiting genes by depolarization in neuronal and non-neuronal cells in the culture. (2) What intracellular signal pathway(s) does depolarization use to accomplish trophic signaling? These studies proposed here focus on signaling through MAP kinases and on signaling through CREB family transcription factors because these are known to be activated by neurotrophic factors and by cytosolic Ca2+. Molecular genetic and pharmacologic techniques will be used to activate or inhibit specific molecules in these pathways to assess their involvement in neurotrophic signaling . The third objective is to relate these studies of trophic stimulation by depolarization in vitro to the inhibition of SGN death in vivo by electrical stimulation. (1) Is the death of deafferented SGNs in vivo apoptotic? (2) Does electrical stimulation of deafferented SGNs in vivo result in the same molecular events that are induced in vivo by depolarization: The studies included in the first two objective will identify particular depolarization-induced events relevant to cell survival, e.g., induction of neurotrophic factors. Electrically stimulated cochlea from deafened rats will be examined for expression of these same events in vivo.

[unreadable] DESCRIPTION (provided by applicant): After loss of hair cells, the deafferented spiral ganglion neurons (SGNs) lose their peripheral process and gradually die. SGN degeneration reduces the efficacy of cochlear implants, currently the only treatment for sensorineural deafness. Electrical stimulation promotes survival of deafferented SGNs in vivo, raising the possibility of using electrical stimulation to maintain survival of SGNs in deaf individuals - in effect allowing cochlear implants to replace the trophic as well as the sensory function of hair cells. We use in vitro and in vivo approaches to determine how electrical activity prevents SGN death and apply this knowledge to prevention of SGN degeneration in vivo. We showed that SGN death in deafened rats is correlated with increased proapoptotic signaling in the JNK-Jun pathway. Early in the post-deafening period there is also decreased prosurvival signaling in SGNs, evident as decreased CREB phosphorylation. Aim 1 uses intracochlear infusion of a JNK inhibitor and JNK3-/- mice to determine whether JNK activity is necessary for SGN death in vivo and, if so, when is it necessary. We also ask the extent to which JNK inhibition or JNK3 deletion promotes degeneration of peripheral processes. Because Jun phosphorylation and SGN death occur long after hair cells have died, we ask, in Aim 2, whether other post-deafening degenerative changes in the cochlea can account for SGN death, focusing on the death of glial cells, peripheral process degeneration, and loss of NT-3 expression. We next turn to the question of how membrane electrical activity promotes SGN survival. We have developed molecular reagents to selectively activate or silence individual intracellular signaling pathways in specific subcellular compartments. Using these, we showed that CaMKII links depolarization to suppression of proapoptotic JNK signaling. We further show that CaMKII does so by recruiting nonreceptor protein-tyrosine kinases, FAK and Pyk2, and protein kinase B (PKB). This is reminiscent of the mechanism by which peptide neurotrophic factors suppress JNK signaling via their receptor protein-trosine kinases and PKB. In Aim 3, we further develop this novel signaling pathway, and parallelism with neurotrophins, by testing the role of Rac/Cdc42 small GTPases in suppression of JNK signaling by the depolarization-CaMKII-Pyk2/FAK pathway. As in our previous studies, the experimental approach using transfection into cultured SGNs of inhibitory and gain-of-function constructs targeting specific steps in the proposed pathway. Physiologi,cal activity and stimulation by cochlear implants consists of impulses of various frequencies. In Aim 4, we extend our studies of intracellular signaling to patterned electrical activity using a system for in vitro electrical stimulation (ES). We ask whether patterned ES recruits the novel signaling pathways we have identified in depolarized SGNs. We also ask in Aim 4 what is the optimal frequency for suppression of proapoptotic signaling in deafferented SGNs in vivo and whether in vivo ES also recruits FAK/Pyk2 in a CaMKII-dependent manner.Sensorineural hearing loss affects about 20,000,000 Americans and the only current means to replace the function of the lost sensory cells is the cochlear implant, which directly stimulates cochlear neurons. Our research focuses on improving the survival and function of surviving neurons in order to improve the long-term efficacy of cochlear implants, currently used by over 40,000 Americans. [unreadable] [unreadable] [unreadable]

A. Administrative structure This research center is integrating research efforts across four (departments and two colleges within the University of lowa with major contributions from the Departments of Biology and Otolaryngology and minor contributions from the Departments of Physiology and Biochemistry. This multi-departmental distribution requires appropriate supervisory structure, which will be coordinated through the administrative organization of the administrative core. The Center Director, Dr. Steve Green, will coordinate the day-to-day business of the Center. The Executive Committee (EC), which includes Dr. Steve Green, Dr. Bernd Fritzsch (Chair of Biology) and Dr. Richard Smith (Vice-chair of Otolaryngology) will meet once each month. In addition, at least once each year, the Executive Committee will hold an expanded EC meeting with all core Co-Is to review progress, goals and objectives in light of recommendations made on an annual basis by the External Advisory Committee. The implementation of new initiatives will be facilitated by the Internal Advisory Committee (lAC), which will be required to attend the expanded EC meeting. The lAC will include five nonecenter faculty selected to serve based on their scientific and administrative credentials (for example, Howard Hughes investigators, members of the National Academies or AAP, Departmental Chairs). The lAC will review and approve the Annual Report of the expanded EC meeting. The Annual Report and semi-annual progress reports will be submitted to the External Advisory Committee (EAC) for review and suggestions. Comments of the EAC will be implemented by the EC;implementation will be reviewed and approved by the lAC. This interplay between the EC, lAC and EAC will proyide a well-structured framework of meetings with defined functions and deadlines to ensure coordinated working of the various parts of the core grant. This structure also reflects the democratic culture at the University of lowa, which recognizes chairs and co-chairs in all departments involved in this grant application. This organizational structure is not only effective but allows the core center PI a high degree of autonomy while ensuring, through proper committee interactions, that this autonomy is used most effectively for the advancement of the core center agenda and research. Dr. Steve Green, the Director of this Center application, is a Full Professor in the Department of Biology with an international reputation of excellence for his ground-breaking in vitro and in vivo research on the molecular basis of neurotrophic support of the sensory neurons of the inner ear through molecular and physiological means. As a long-time resident at the University of lowa, Dr. Green has had multiple interactions with all members of the core center grant including the new members that have only recently arrived (Drs. Lee and Fritzsch). Dr. Green has initiated and participated in multiyear interdepartmental research seminar series and is an active member of the trans-departmental graduate school program in neurobiology that spans both the Carver College of Medicine and the College of Liberal Arts and Sciences, home of all departments involved in this grant application. Dr. Green is a member of the two major departments involved in this application, with a primary appointment in Biology and a secondary appointment in Otolaryngology - Head and Neck Surgery. Dr. Green is well funded, has published numerous high impact papers and has extensive experience as NIH reviewer as well as reviewer for numerous journals and other granting agencies in all matters concerning research and publications. Given his familiarity with both the infrastructure and faculty at the University of lowa, his long standing trans-departmental activities at multiple levels not limited to research alone, combined with his long-standing interactions and publications with members of the Otolaryngology Department make him the logical choice for the Center Director. In his capacity as Core Center Director, Dr. Green will oversee the daily work of all research cores and will manage, direct and supervise the administrative, scientific and training functions of the Center. His guidance will helfD integrate and coordinate the activity on the individual cores and will provide appropriate oversight to enhance and evaluate progress and trouble shoot potential problems in interaction with the Executive Committee.

DESCRIPTION (provided by applicant): The proposed Center for Molecular Auditory Neuroscience will enhance productivity, innovation, and collaborative interactions of auditory researchers at the University of Iowa. Center investigators are members of five clinical and basic science departments: Otolaryngology, Biology, Biochemistry, Physiology, and Communication Sciences and Disorders, with 15 auditory-related R01s, nine funded by NIDCD, a P50, two T32s, and other grants, for a total current year direct cost >$9M. There are extensive interactions among basic researchers, among clinical researchers, and interactions bridging clinical and basic research. The cores aim to strengthen these interactions and develop new ones. The Center consists of three research cores and will provide first-rate facilities for state-of-the-art experimental techniques crucial to molecular, cell, developmental and neurobiology of the inner ear; provide training in these techniques; make investigators aware of alternative experimental approaches and model systems that will facilitate their research; foster new collaborations that result in innovative approaches to problems in auditory research, including translation of basic research data to the clinic. The research cores are: (1) Histology and Imaging core to provide facilities and training for analysis of model organisms using light microscopy and EM: histology, including sectioning, staining, immunofluorescence, EM, and use of fluorescent dyes to label nerve fibers; confocal imaging, including multiphoton, of live or fixed tissue; (2) Genomics core for routine molecular biology techniques - sequencing, nucleotide synthesis, nucleic acid quantitation and quality - as well as analysis of gene expression; (3) Tissue/Cell Culture Core to provide facilities and training for preparation of in vitro cochlear model systems including sensory, neural and glial cells, organotypic inner ear cultures, and means for gene transfer into these cells. Individual experiments may use multiple cores and workflow will be coordinated among Core directors. The Administrative Core will manage day-to-day operation of the Center, coordinate activities of Core directors, and conduct symposia and seminars to facilitate interaction among the Center investigators.

Auditory; Auditory system; base; Biological Models; Cell Culture Techniques; Cell Line; Cell Nucleus; Coculture Techniques; Collaborations; Culture Techniques; Cultured Cells; Data; design; Development; Drosophila genus; Educational workshop; Environment; experience; Gene Deletion; gene gun; Genes; genetic manipulation; Genetic Techniques; Genetically Modified Organisms; Goals; Histology; Human Resources; Image; imaging modality; In Vitro; in vitro Model; in vivo; Individual; interest; Knockout Mice; Laboratories; Laboratory culture; Maintenance; Methods; Modeling; Molecular Genetics; Mus; Mutant Strains Mice; Neurobiology; Neurons; novel; Organ Culture Techniques; Organ of Corti structure; Physiological; Physiology; Production; Proteins; Rattus; recombinase; Research; Research Personnel; research study; response; Schwann Cells; spiral ganglion; Students; System; Tamoxifen; Techniques; Testing; Time; tissue culture; tissue/cell culture; Training; Transfection; Transgenic Mice; Trauma; Universities; Viral Vector; Work

DESCRIPTION (provided by applicant): To experimentally investigate reinnervation and synaptogenesis in excitotoxically-damaged cochleae, we developed an organotypic cochlear explant in which a portion of the organ of Corti and corresponding portion of the spiral ganglion are removed intact, maintaining normal morphology and synaptic interactions. Briefly treating the explant with high levels of glutamate agonists results in excitotoxic degeneration of inner hair cell (IHC) - type I spiral ganglion neuron (SGN) synapses but does not affect hair cell or SGN viability. The synapses regenerate but the restored innervation is aberrant: the number of synapses is reduced, and individual SGN axons contact multiple IHCs. In all these respects, the in vitro model mimics what has been observed following noise or glutamatergic excitotoxic damage in vivo. Exogenous neurotrophins - BDNF or NT-3 - significantly improve recovery: the number of synapses on IHCs is increased, synapse number is increased, and innervation of multiple IHCs by single axons is reduced. In Aim 1, we quantitatively compare the ability of BDNF and NT-3 to promote regeneration with an extended recovery period and seek to improve our model by extending it to older animals. Our core set of experiments in Aims 2-4 use molecular genetic approaches, including the use of transgenic mice, to test specific hypotheses, suggested by our preliminary data, regarding the function of neurotrophins in recovery and reinnervation of IHCs after excitotoxic trauma. In Aim 2 we test whether NT-3, the endogenous neurotrophin, acts in a highly spatially restricted manner to maintain synapses on individual IHCs. We will delete NT-3 from a small number of IHCs or inhibit TrkC function in a small number of SGNs and quantitatively compare these with their unmodified neighbors. In Aim 3, we replace NT-3 with BDNF to test the hypothesis that NT-3 has a distinctive function in maintaining IHCSGN synapses and BDNF can't substitute. Finally, in Aim 4, we use p75NTR knockout mice to test the hypothesis that the neurotrophin receptor p75NTR promotes reinnervation after excitotoxic trauma. We will also assay post-trauma expression of p75NTR and putative ligands and test a specific mechanism: whether p75NTR promotes reinnervation by upregulating NT-3.

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.

Most spiral ganglion neurons (SGNs) make afferent synapses on the auditory sensory cells, the inner hair cells (IHCs), and convey auditory information to the brain. Noise damages cochlear afferent synapses even at sound levels too low to destroy hair cells. Noise-induced cochlear ?synaptopathy? (NICS) is detectable by histological examination and counting of synapses and is also evident, noninvasively, as reduced auditory brainstem response (ABR) wave I amplitude. While synaptopathy does not detectably affect auditory thresholds, it may cause hearing impairments such as poorer speech-in-noise performance or tinnitus. In the course of investigating means to prevent NICS, we observed that female mice are significantly less susceptible than are males to NICS. Remarkably, female susceptibility varies with estrous cycle phase, with lowest susceptibility correlated with the estrous phase at which progesterone (P4) levels are highest (and estrogen lowest). In vitro experiments additionally show that a high level of P4 promotes rapid regeneration of synapses. These data showing sex differences in synaptopathy are the first to show that susceptibility varies through the estrous cycle and to show a protective role for P4. To follow up, our first aim is to determine whether a high level of steroid sex hormone does reduce NICS. To that end, we will experimentally manipulate levels of P4 and estrogen in male and female mice. We have further shown that, not only P4 but also the neurotrophic factor CNTF and agents that activate cyclic AMP (cAMP) signaling promote synaptic regeneration. The latter include compounds, such as rolipram, that can be administered systemically. P4, CNTF, and rolipram represent excellent reagents for investigating the role in vivo of cAMP in synapse regeneration and may also be candidate therapeutics for post-noise synapse regeneration therapy. However, cochlear synapses may lose their capacity for regeneration with time after damage and the timecourse may differ among the different agents promoting regeneration. Our second aim will determine how long after noise these agents, P4, CNTF, or cAMP, may be administered and still promote regeneration. Unlike the case for peptide neurotrophic factors, the molecular and cellular mechanism(s) by which progesterone or cAMP promote synapse regeneration remain obscure. Our third aim asks whether these factors function via genomic actions or via cytoplasmic targets or plasma membrane receptors ? a necessary preliminary step for future detailed mechanistic studies of signaling pathways and possible transcriptome changes involved. For cAMP, the question is whether cAMP- dependent protein kinase enters the nucleus or remains a cytoplasmic signal, a question we successfully answered previously with respect to survival signaling. For progesterone, our preliminary studies suggest that a nuclear receptor is not involved so our focus will be on plasma membrane progesterone receptors.