Alan G. Cheng - US grants

DESCRIPTION (provided by applicant): The main goal of this award is to further my career as a surgeon-scientist by establishing a successful research program. My longstanding interest in treating hearing disorders and, in parallel, performing basic science research on the same topic has fueled my decision to become a pediatric otolaryngologist-scientist. Since my clinical practice focuses on pediatric otology, I am frequently confronted with children suffering from hearing loss. As a physician, I have noticed a major deficiency in our current treatment approach where we cannot reverse sensorineural hearing loss. As a scientist, I hope to establish a research program that will shed light on therapies that restore hearing. To build on my previous research experience, this award is designed to help me develop into an independent investigator through hands-on training and close guidance on experimental designs, techniques, and grant writing. Because Wnt signaling plays major roles in maintaining stem cell populations in other organ systems, we hypothesize that canonical Wnt pathway is involved in the maintenance of cochlear progenitor/stem cells. Likewise, we hypothesize that loss of Wnt signaling or responsiveness is responsible for the inability of the mammalian cochlea to maintain regenerative capacity throughout life. Over the course of a five-year plan, we propose to conduct a series of experiments designed to test two unexplored hypotheses: 1) Wnt responsive cells are endogenous cochlear stem/progenitor cells, and 2) Wnt activation promotes proliferation in cochlear supporting cells. As single cells isolated via flow cytometry, different cochlear cell populations, including Wnt responsive cells and various cochlear supporting cells, are assessed for their ability to self-renew by sphere assays and their ability to generate new hair cells and supporting cells in vitro. Using markers for proliferation and cell lineage analysis in organotypic cultures, we will investigate the effects of Wnt agonists, mitogens, and neomycin-induced hair cell loss on these cells. At the end of this award, we will have evaluated and reported the roles of canonical Wnt signaling in regulating the endogenous Wnt responsive cells and cochlear supporting cells, and whether the Wnt pathway is a promising therapeutic target for hearing restoration. The long-term goal of our research is to maintain or activate a reserve of endogenous stem cells capable of restoring hearing and balance disorders through hair cell/supporting cell regeneration.

DESCRIPTION (provided by applicant): Cochlear degeneration is a major cause of sensorineural hearing loss (SNHL), and the lack of spontaneous regeneration contributes to the irreversible nature of SNHL. Prior studies have examined the utility of forced differentiation of hair cells in the degenerating cochlea, yet it remains unclear whether a restoration in cell number via cell proliferation can also aid cochlear regeneration. Recent work demonstrates that activation of canonical Wnt signaling via genetic or pharmacologic manipulation induces proliferation in the neonatal cochlea. Also, the competence to proliferate in response to Wnt signals is observed in both cochlear supporting cells and tympanic border cells below the basilar membrane. The goal of this proposal is to investigate whether supplementation of Wnt signals can stimulate proliferation and/or regeneration after hair cell degeneration in both the neonatal and mature cochleae. We will initiate degeneration with aminoglycoside application or via a transgenic strategy and concurrently fate-map supporting cells and tympanic border cells in vitro and in vivo. In parallel experiments using multiple transgenic mouse strains, we will study ablation targeting sensory hair cells or supporting cell subtypes. Wnt activation is achieved by using the Cre-Lox system in transgenic mice or local application of Wnt agonists and whether they will initiate proliferation of and regeneration by supporting cells and tympanic border cells are examined. The degree of damage and possible recovery in the cochlea are examined histologically and correlated with pre- and post-treatment auditory physiology in the whole animal. To gain an unbiased insight into the genetic signature of the damaged cochlea, supporting cells and tympanic border cells from undamaged and damaged cochlea are isolated via flow cytometry and subjected to gene array analyses. All transgenic mouse strains, pharmacologic agents, expertise to manipulate and examine the cochlea in vitro and in vivo, and techniques to isolate and enrich cochlear cells are at hand. Together, our research will determine 1) whether Wnt supplementation can help initiate cochlear regeneration and 2) additional targets to enhance this regenerative process in both the neonatal and mature cochleae.

? DESCRIPTION (provided by applicant): Aminoglycosides are a widely used broad spectrum antibiotic. A major side effect of these compounds is oto- (and vestibulo) and nephrotoxicity. Data obtained during an R21 award demonstrates that these compounds enter sensory hair cells of the inner ear via a specialized mechanosensitive ion channel in vitro. Using our unique knowledge of the biophysical properties of the mechanotransduction channel coupled with insights provided by crystallography data that identifies aminoglycoside interacting sites on the bacterial ribosome, we have developed novel compounds that greatly reduced oto- and nephrotoxicity in vivo, though at some cost to the breadth of antimicrobial activity. These proof-of-principal experiments serve as the basis for iteratively designing new compounds. The design component has three phases, first to use our initial screen as a starting point where we will alter the substituted group on sisomicin to try to better separate antimicrobial activity and the toxic side effects. Second we will synthesize and test a novel modification site based on new data arising from experiments on gentamicin derivatives. Finally, we will change the parent compound to better target specific microbes such as Pseudomonas organisms. We have developed the ability to test for purity and specificity of each compound. We can probe activity in terms of antimicrobial function as well as oto- and nephrotoxicity in in vivo and in vitro models. We can also test for sensitivity to the development of resistance and for vestibular pathologies. Together this iterative ability based on a novel hypothesis should yield a new class of nontoxic antibiotics.

? DESCRIPTION (provided by applicant): Basic science, translational, and clinical research in Otolaryngology - Head and Neck Surgery is making remarkable advances that affect the way we treat patients. A critical component of nourishing and expanding such improvements in patient care is to facilitate the partnership of clinicians and basic scientists. One way to achiev this is by training more clinician-scientists. Herein, we propose to implement a research training program designed to cultivate clinician-scientists who are interested in studying inner ear development, regeneration, function, and physiology. Our research training program is designed to provide residents and post-residency graduates with intense research experiences, a structured didactic program, and close mentorship and guidance in how to integrate clinical and research activities. Trainees will be ingrained with the philosophy that research is intrinsic to a academic surgeon's career and that they should build their career by sustaining excellence in both research and clinical care. If our training program is successful, our graduates will become independent NIDCD-funded investigators in faculty positions in academic departments. The ultimate long-term goal, of course, is for them to improve human health by advancing our field via scientific discovery that is translated to clinical care.

Abstract: Sensory hair cells are required for balance function. Vestibular hair cell degeneration causes balance dysfunction/hypofunction manifested as dizziness and vertigo. While the mammalian cochlea lacks the ability to regenerate lost hair cells, a limited degree of spontaneous regeneration occurs in the utricle, a vestibular organ detecting linear acceleration. Recent studies using fate-mapping techniques have pinpointed supporting cells as precursors of regenerated hair cells. However, it is not clear whether regenerated hair cells are fully functional and if organ function recovers. In preliminary experiments we have characterized hair cell degeneration and regeneration in the mature mouse utricle and also a loss followed by recovery of vestibular evoked potentials (VsEP) in vivo. The first aim of this proposal is to determine if increasing hair cell regeneration improves the recovery of vestibular function. Specifically, regenerated hair cells labeled via fate-mapping are probed via histology and electrophysiology to assess bundle morphology, mechanosensitvity, basolateral currents, and synaptic properties including vesicle release. In parallel, VsEP responses are measured and compared to histologic and electrophysiological measures. Next, by overexpressing Atoh1 via a transgenic approach, we will study the histology and electrophysiology of Atoh1-overexpressing hair cells and also the overall VsEP responses. In the second aim, we will determine if Atoh1 deletion prevents hair cell regeneration and the recovery of VsEP responses. In parallel, fate-mapped, surviving hair cells will be examined for possible repair via histology and electrophysiology. To gain an unbiased insight into the genetic signature of hair cell progenitors and surviving hair cells, the third aim is designed to examine the damaged mature mouse utricle using single cell RNA sequencing technologies. Here the first goal is to discover the genetic landscape of hair cell progenitors and surviving hair cells in the damaged utricle. Secondly, we will examine the gene expression of the undamaged and damaged utricle after Atoh1 overexpression. Lastly, we will use bioinformatic approaches to delineate the trajectory of the spontaneous and Atoh1-enhaced supporting cell-hair cell transition and validate this histologically. In summary, we will apply state-of-the art technologies (vestibular physiology, hair cell physiology, single cell RNA-seq, bioinformatic strategies) to study vestibular hair cell regeneration in transgenic mouse models. We have assembled a team of experts who have worked together to collect promising preliminary data. At the end of this 5-year proposal, we will have 1) determined the relationship between hair cell regeneration and functional recovery and 2) revealed and temporally ordered novel genes during mammalian hair cell regeneration.