Brandon C. Cox, Ph.D. - US grants

DESCRIPTION (provided by applicant): Hearing loss is primarily caused by damage to sensory hair cells (HC) in the cochlea of the inner ear. Humans and other mammals cannot replace damaged HCs;however, chicken, fish and amphibians can, by proliferation and transdifferentiation of neighboring supporting cells (SC) (Conwin and Oberholtzer, 1997). Interestingly, the tumor suppressor gene, p16INK4a, is absent in non-mammalian vertebrates that have the capacity to regenerate HCs (Kim et al., 2003;Gil and Peters, 2006). In mammals, p16INK4a acts as a cyclin-dependent kinase inhibitor that keeps cells in a quiescent state. Its expression is induced by age as well as mitogenic signaling (Zindy et al., 1997). p16INK4a is also known to play a critical role in the in vivo regenerative ability of adult cells, including neurons (Janzen et al., 2006;Molofsky et al., 2006). It is commonly thought that damaged HCs release signals that cause SCs to reenter the cell cycle. In non-mammalian vertebrates this results in HC regeneration;however, in mammals, we predict that this mitotic activity induces the expression of p16INK4a which keeps SCs in a quiescent state and thus prevents HC regeneration. Is it possible to give a mouse, the chicken's ability to regenerate HCs by inactivating p16INK4a? To achieve this goal, we propose the following central hypothesis: Inactivation of p16INK4a in mammals will allow SCs: (1) to respond to signals released from HCs damaged by antibiotics or genetic ablation, (2) to reenter the cell cycle and (3) to regenerate HCs. To test this hypothesis, we propose the following specific aims: Specific Aim 1: To determine the regenerative capacity of SCs in p161NK4a-null mice after HC damage caused by the aminoglycoside antibiotic, gentamicin. Specific Aim 2: To determine the regenerative capacity of SCs in p16INK4a-null mice after HC damage caused by acute inactivation of the retinoblastoma protein in postnatal HCs. Although mammals cannot spontaneously regenerate HCs, a process of HC regeneration similar to what occurs in non-mammalian vertebrates could be induced by genetic and/or therapeutic manipulations of cochlear SCs. This proposal is the first step toward the final goal of restoring hearing in those exposed to ototoxic drugs, loud noise or other environmental agents. In addition, HC damage induced by acute inactivation of the retinoblastoma protein in postnatal HCs is a technological breakthrough for the field.

? DESCRIPTION (provided by applicant): When auditory hair cells (HCs) are lost, they are not replaced in humans or other mature mammals, resulting in permanent hearing loss. In contrast, regeneration of auditory HCs naturally occurs in non-mammals, allowing for recovery of hearing function. We have recently observed that the neonatal mouse cochlea can spontaneously regenerate its HCs after damage; however, the majority of these regenerated cells died. In addition, many studies have shown that outer HCs (OHCs) are more susceptible than inner HCs (IHCs) to damage caused by noise or ototoxic drugs. Yet the mechanism that makes OHCs more vulnerable is not understood. There is also a lack of understanding of the pathways that regulate HC survival under normal conditions after differentiation is complete. Proposed studies will investigate HC survival during postnatal maturation and adulthood, during HC regeneration, and in stressed HCs following noise exposure. We will focus on one gene, Pou4f3, a transcription factor that is expressed in HCs beginning with differentiation. While the role of Pou4f3 in maintaining HC survival during development was discovered previously, its role in postnatal maturation, aging, regeneration, and in stressed HCs has not been explored. Preliminary data show that deletion of Pou4f3 in adult OHCs causes cell death which suggests that mature OHCs still require Pou4f3 expression to survive. In addition, POU4F3 immunostaining results show that many adult OHCs have decreased levels of POU4F3 expression, while IHCs retained strong POU4F3 expression. These data suggest that complete deletion of POU4F3 causes HCs to die, but reduced levels of POU4F3 are enough to maintain HC survival. Aim 1 will investigate this further by deleting Pou4f3 in neonatal, juvenile, and adul HCs. In addition, the majority of regenerated HCs that spontaneously form in the neonatal mouse cochlea do not express POU4F3 and we hypothesize that this causes cell death. In support of this hypothesis, another study ectopically expressed Atoh1 in supporting cells to convert them into HCs. These newly formed HCs survived at least 3 months and did express POU4F3. These data implicate Pou4f3 in the regulation of HC survival during the differentiation process and Aim 2 will rescue regenerated HCs in the neonatal mouse cochlea by ectopic expression of Pou4f3. Since Pou4f3 is known to regulate several genes involved in apoptosis, decreased levels of POU4F3 expression in OHCs of adult mice may make these cells more vulnerable under stressful conditions and account for the increased damage susceptibility of OHCs. We will test this hypothesis in Aim 3 by over-expressing Pou4f3 to protect OHCs from noise-induced damage. Collectively proposed studies will investigate Pou4f3's role in the regulation of HC survival during maturation and adulthood in the normal, undamaged cochlea, during spontaneous HC regeneration in the neonatal mouse cochlea, and in stressed HCs following noise exposure. Completion of these aims will advance our understanding of the mechanisms that regulate HC survival under multiple conditions, which could be used to develop drugs to protect HCs.

Abstract The 8th installment of the Midwest Auditory Research Conference (MARC) will take place at Southern Illinois University School of Medicine in Springfield, Illinois from July 11-13, 2019. We expect ~150 energized auditory and vestibular scientists from a broad swath of the country to visit the home town of Abe Lincoln. The goals of this meeting are to foster collaborations among basic, clinical, and translational hearing/vestibular scientists and to provide an inexpensive opportunity for graduate students and postdoctoral fellows to present their work as posters or talks. MARC 2019 focus areas conform to the mission of the NIDCD including: Inner ear development; Vestibular hair cell regeneration; Hidden hearing loss; Synaptic transmission between hair cells and neurons; Age-related hearing loss; Central auditory function/dysfunction/plasticity; Mechanisms of tinnitus; and Improving speech understanding across all populations of ages. To this end, confirmed keynote speakers include: Gabriel Corfas-University of Michigan, Judy Dubno-Medical University of South Carolina, Paul Fuchs-Johns Hopkins University School of Medicine, Andy Groves-Baylor College of Medicine, Nina Kraus-Northwestern University, Dan Polley-Harvard Medical School, Jenny Stone-University of Washington, and Thanos Tzounopoulos- University of Pittsburgh School of Medicine. MARC 2019 will provide an informal setting that encourages graduate students and postdoctoral fellows to discuss their work amongst each other, and with nationally regarded Principal Investigators. Sessions will include a broad mix of talks that focus on peripheral and central auditory neuroscience, therefore encouraging attendance across the spectrum of auditory/vestibular research. We also plan to include a Friday night event at the Abraham Lincoln Presidential Museum with an engaging keynote speaker as part of the MARC meeting. Graduate students and postdoctoral fellows will be selected for travel awards with registration waivers, based on the quality and relevance of their abstracts. Selected abstracts from trainees and early stage investigators will be given preference for talks that are slotted into the keynote appropriately themed sessions. Since we anticipate that greater than half of the attendees will be trainees, MARC 2019 will also include a career development session where strategies to improve grant writing will be discussed in a round table format.

Project Summary: It has been estimated that more than 40% of older adults suffer vestibular (i.e. balance) deficits. These losses cause numerous other problems associated with aging including cognitive decline and injurious or fatal falls. There is also a strong link between age-related vestibular dysfunction (ARVD) and Alzheimer's disease and related dementias. Despite the prevalence of these issues and the massive toll they exert on public health and associated financial costs, the underlying causes for ARVD are poorly understood. As a result, there are currently no FDA approved therapies for ARVD. While a deep understanding of mechanistic causes is lacking, it has been known for some time that a very common pathology that causes age related inner ear dysfunction is the death of sensory cells called hair cells. Exactly why these cells die with age remains a mystery. Here, we have identified a previously uncharacterized pattern in the expression of the pro-survival gene, Pou4f3, where it is normally highly expressed in inner ear hair cells, but is downregulated with age in a fashion that is correlated with hair cell death in the balance organs of the inner ear. Furthermore, preliminary data suggest that deleting Pou4f3 causes detrimental phenotypes in vestibular hair cells, exacerbates hair cell death, and leads to significant declines in vestibular function. We propose to build on these preliminary data by further examining Pou4f3 changes in expression in vestibular organs with age and in models of Alzheimer's disease. We will also more thoroughly characterize the effects of Pou4f3 deletion to better understand the effects that deletion or hypomorhpism have on balance and neurological functions. We also propose to examine genomic regulatory elements in inner ear tissues from young and aged mice to identify causal mechanisms for Pou4f3 downregulation with age as well as possibly discover other key genes involved in aging processes in the inner ear. Finally, we will test whether overexpression of Pou4f3 can prevent sensory cell death and age related vestibular declines. Our preliminary data suggest that Pou4f3 is a promising therapeutic target for preserving balance function in the aging human population. The experiments proposed will determine the validity of that overarching hypothesis and will provide a foundation from which to launch several new investigations into Pou4f3-targeted pharmacological and gene therapy approaches for the prevention of age related vestibular decline.