Bradley J. Walters, Ph.D. - US grants

Project Summary: Hearing is a vital sense for nearly all animals, including humans. In mammals, the organ of Corti transduces sound into neural signaling and is well-known for the elegant arrangement of cochlear hair cells into one row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs). This marvel of cellular architecture has appeared in nearly every biology textbook since its description in 1851, yet how IHCs and OHCs differentiate from one another during development is still poorly understood. Hearing loss resulting from inherited mutations and from the loss of hair cells with age, noise, or other insults, affects millions of Americans and countless more globally. In addition, mammalian HCs do not regenerate, and although genetic manipulations suggest that stem cells or nonsensory cells may be converted into cells that resemble hair cells, these cells remain underdeveloped and do not differentiate into mature IHCs or OHCs. Thus, understanding how IHCs and OHCs become distinct cells with complementary functions is of fundamental importance to our understanding of developmental biology and the design of potential otoprotective and regenerative therapies. In this application, we present preliminary evidence that two gene products, p27Kip1 and SIX2, are critical mediators of cell fate decisions leading to IHC and OHC fates, respectively, during cochlear maturation. To test whether p27Kip1 inhibits OHC fate, we propose to delete p27Kip1 from cochlear hair cells, in vivo, and examine the IHCs to see if they adopt OHC characteristics. Conversely, we propose to ectopically express p27 Kip1 in cochlear HCs to test whether exogenous p27Kip1 prevents OHCs from fully differentiating, or causes them to become IHC-like. We further propose to similarly manipulate the expression of Six2 in IHCs and OHCs to see whether the loss of Six2 from OHCs promotes an IHC fate, or if ectopic Six2 expression in IHCs promotes an OHC fate. The altered cell fates in each of these models will be examined by several means including confocal microscopy of fluorescent reporter proteins and immunofluorescent staining to visualize the expression of various proteins that are known to be specific to either IHCs or OHCs. Fluorescent immunostaining will also be used to examine the types of innervation and synaptic connections exhibited by the cells. Sorting of small pools of IHCs and OHCs from each of these models followed by whole transcriptome gene expression analysis (RNA-seq) will further provide an in-depth characterization of the extent of phenotypic conversion of the cells. Finally, scanning electron microscopy will be used to compare the morphological characteristics of the stereociliary bundles which typically differ between IHCs and OHCs. These experiments will allow us to determine whether p27Kip1 and/or SIX2 are critical determinants of IHC and OHC fate and positive results will strongly support future investigation of the roles played by these molecules in hearing function and the potential regeneration of functional hair cells from nonsensory cells.

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.