Bruce Tempel - US grants

Potassium currents are important for controlling the membrane potential and, therefore, the level of electrical activity in neurons, muscles and various other cell types. Although the physiological properties of K+ channels have been studied for a number of years, lack of either a rich tissue source or a good ligand has prevented the biochemical purification of any K+ channel. Using the genetic approach available in Drosophila, the first complete sequence for a K+ channel was recently reported from the Shaker locus of Drosophila. A homologous gene expressed in the mouse brain was subsequently cloned. Here we propose to extend our studies on the probable mouse-brain K+ channel gene, called MBK1. We will address the following specific aims: 1) To express physiologically the MBK1 cDNA, thereby confirming that it encodes a K+ channel component. 2) To study the function of residues that are conserved between Shaker and MBK1 using in vitro mutagenesis. 3) To characterize the MBK1 protein, including the channel of which it is likely to be a part. 4) To identify the tissues that express MBK1 and, within the brain, to localize MBK1 expression by in situ hybridization and the MBK1 protein using immunocytochemistry. 5) To characterize the MBK1 gene, its structure and regulatory elements. 6) To identify the chromosomal location of MBK1 and to determine if the MBK1 locus correlates to any behavioral mutants in mice. Answers from these studies may help to explain, at the molecular level, how membrane excitability is controlled and, in diseases like epilepsy, periodic paralysis or diabetes, how uncontrolled membrane excitability may come about.

Work in our lab is aimed at elucidating the molecular structure and function of potassium (K+) channel genes as well as attempting to understand the role(s) these genes might play in mammalian behavior. Here we propose to study voltage-gated K+ channel genes in the central nervous system (CNS) and auditory system of normal, mutant and transgenic mice. During the previous funding period, we cloned and mapped 6 different murine voltage-gated K+ channel genes to their chromosomal locations and found that the expression of one of these, MK1, is reduced approximately 50% in brains and cochlea of a deaf mutant mouse, deafwaddler. Here we propose to extend these findings by determining the cellular and subcellular distribution of these K+ channels in both the central nervous system and in the cochlea of normal and deaf mice. We will also explore the regulation of these genes and construct transgenic mice that express normal and mutant K+ channels. These studies will provide molecular insights into the distribution, gene expression and function of K+ channels in the mammalian brain and auditory system. The studies on dfw mutant animals provide the exciting possibility of linking K+ channel function with a neurological phenotype.

Work in our lab is aimed at elucidating the molecular structure and function of potassium (K+) channel genes as well as attempting to understand the role(s) these genes might play in mammalian behavior. Here we propose to study voltage-gated K+ channel genes in the central nervous system (CNS) and auditory system of normal, mutant and transgenic mice. During the previous funding period, we cloned and mapped 6 different murine voltage-gated K+ channel genes to their chromosomal locations and found that the expression of one of these, MK1, is reduced approximately 50% in brains and cochlea of a deaf mutant mouse, deafwaddler. Here we propose to extend these findings by determining the cellular and subcellular distribution of these K+ channels in both the central nervous system and in the cochlea of normal and deaf mice. We will also explore the regulation of these genes and construct transgenic mice that express normal and mutant K+ channels. These studies will provide molecular insights into the distribution, gene expression and function of K+ channels in the mammalian brain and auditory system. The studies on dfw mutant animals provide the exciting possibility of linking K+ channel function with a neurological phenotype.

Congenital, perinatal, or early onset hearing loss occurs in approximately 7 out of 1000 neonates in the United States. Especially in young people, noise-induced hearing loss is increasingly common. In the adult population, more than 50% of U.S. males age >65 years have a 53dB loss at 4 kHz, substantially impairing perception of normal speech. The primary pathohistological defect in each of these hearing impaired groups is the loss of outer (and inner) hair cells near the base of the cochlea, where high frequency sounds are transduced. We have recently identified a mutant mouse strain, called deafwaddler (dfw), in which hearing is most severely affected in the high frequency range. Anatomical studies show that outer hair cells (OHC) are absent from the basal region of the cochlea in dfw, becoming more frequent near the apex where low frequencies are heard. Inner hair cells (IHC) are sometimes missing at the base of dfw cochlea but appear intact in the mid- and apical regions. Other physiological measures (e.g. endocochlear potentials, 8th nerve conduction, and anatomical structures appear intact in dfw. Thus, the dfw mutant provides a unique genetic model for understanding the physiological changes leading to sensorineural deafness and loss of functional hair cells in the cochlea. Here we propose to identify the dfw gene by positional cloning techniques. We will: 1.) Refine dfw 's chromosomal location by analyzing inbred backcross (IB) panels between M. musculus (dfw) and M. castaneus, scoring the mutant phenotype relative to molecular microsatellite markers. This panel will provide the high resolution pedigree required for successful positional cloning of dfw. 2.) Clone the dfw region in YACs, establishing a physical map and developing new polymorphic markers that will be scored in IB panel for yet more accurate localization of dfw. 3.) Screen candidate genes from the region for the mutation causing dfw. 4.) Analyze the structure and function of the dfw gene product in parallel with, 5.) Further analyze the developmental and spatial changes in hair cell loss in the dfw model. These studies should provide new insight into the molecular basis of congenital auditory hair cell loss, and relate directly to hair cell loss in aging and due to noise exposure.

Hearing loss is the most frequent sensory defect in humans. Congenital, perinatal or early onset hearing loss occurs in approximately 7 out of 1000 neonates in the United States. In approximately half of the children born with severe hearing impairment, a genetic contribution is suspected. The powerful molecular and genetic techniques available in mouse combined with the functional similarities between mouse and human audition make mouse a useful model system for studying human deafness. Recently, we have shown that the plasma membrane calcium ATPase type 2 gene (Pmca2) is altered in both alleles of the deafwaddler (dfw) mouse mutant strain. We have also shown that PMCA2 (the protein) is highly concentrated in stereocilia of mouse outer hair cells and in the basolateral membrane of inner hair cells. These data, along with electrophysiological studies from other labs, suggest that PMCA2 clears calcium from hair cells, thereby allowing stereocilia to transduce auditory information. Here we propose: to analyze PMCA2 expression in the auditory and vestibular systems, to examine developmental changes in PMCA2 expression, to identify new genes that interact with deafwaddler, and, toward identifying human families with mutations analogous to deafwaddler, to develop genetic markers for the human PMCA2 gene. We have also developed high resolution genetic and physical maps for the quivering locus on mouse chromosome 7. Mutations in the quivering gene cause hyperactivity as well as deafness that arises at the level of the cochlear nucleus in the auditory brainstem. We propose to clone the gene underlying quivering and to analyze the expression and function of the quivering gene product in mice. Because seven independent alleles of quivering exist, we should be able to correlate the predicted severity of mutations in the quivering gene with the observed differences in severity in the phenotypes in different strains of quivering. Our studies on the deafwaddler and quivering mutants will identify genes critical to normal functioning of hair cells and to normal transmission of auditory information, respectively. Beyond simply identifying the gene, mice provide the additional advantage that we can use electrophysiological, developmental, and genetic techniques to more fully understand the biological role of these genes in auditory function.

DESCRIPTION:(from applicant's summary) A major challenge confronting neurobiology is to define how specific voltage-gated potassium (Kv) channel genes influence the timing, duration and frequency of the neuronal signals that encode and transmit information. Neurons in the auditory system have the unique advantages of relatively simple circuitry, well defined functional roles (involving precise signal fidelity) and strong expression of Kv currents. The goal of this proposal is to examine the functional roles of Kv channel genes in three types of auditory neuron-bushy neurons and octopus cells of the cochlear nucleus. and neurons of the medial nucleus of the trapezoid body -each performing related but distinct information processing tasks. Using molecular and irnmunocytochemical techniques, the applicant will determine the complement of Kv channel subunits expressed in these neurons and examine their subcellular localizations. Using electrophysiological techniques, the applicant will characterize Kv currents in these auditory neurons in brainstem slices from wildtype mice and from hearing impaired mice that lack the Kv1.1 channel subunit gene (i.e. Kvl.l knockout mice). These data should reveal rules governing Kv channel assembly and localization in parts of the neuron specialized for either encoding or transmission of information, and elucidate specialized roles in auditory information processing for different subunits, or subsets of subunits within a subfamily. Our thorough characterization of the functional role of Kv channels at the cellular level will also help to explain at the organismal level the hearing loss, movement abnormalities and seizures observed in Kvl.1 knockout mice. Using both anatomical and electrophysiological data, the applicant will develop computer models to assess the relevance of Kv channels/currents in auditory information processing. The model will be used to predict the effects of removing other Kv genes strongly expressed in auditory neurons, such as Kv 1.2 for which the applicant's predictions will be tested directly by examining Kvl.2 knockout mice. Episodic ataxia myokymia is caused by mutations in the Kvl.1 (KCNA1) gene in humans. Clinical reports on these patients often include tinnitus, vertigo and sometimes profound hearing loss. The proposed studies and models based on the Kvl.1 knockout mouse mutants should also be informative regarding the neuronal dysfunction that underlies this human disease.

Hearing loss is the most frequent sensory defect in humans. Congenital, perinatal or early onset hearing loss occurs in approximately 7 out of 1000 neonates in the United States. In 50% of these cases, a genetic contribution is known or suspected, the powerful molecular and genetic techniques available in mouse and zebra fish are useful model systems for studying the biology of hearing. The functional similarities between mouse and human audition make mouse a particularly attractive model for hearing loss observed clinically. In support of Users of mice to study auditory system function, Core D will provide expert mouse husbandry (receiving, setting up crosses, weaning, fostering, background transfers, etc), genotyping, and notification/delivery of requested mice to Users. Per diem charges for mouse care will be the responsibility of the User, provided that they have a funded project that includes support for mouse cage costs. To encourage innovative and new collaborative projects, we will provide per diem support for common control animals and for approved projects in funding transition. All services provided by Core D will be reviewed periodically to ensure quality and accessibility. The Genetics Core will provide services for a variety of Users. Efficiency of mouse care will be increased greatly by having centralized, expert mouse husbandry and genotyping. Users not proficient in mouse care will gain access the vast number of identified gene knockout mice and defined spontaneous mutants to further their research. Experienced mouse users will benefit from the familiarity of the Core staff with mouse behavior and care, providing better health and higher fecundity for their strains. Training in husbandry, genotyping, and behavioral characterization will be available to all Users and their lab personnel. To encourage interactions and new collaborations, information about the strains, techniques, and projects being pursued by all Users of the Core will be disseminated online and in Genetics Core User meetings.

DESCRIPTION (provided by applicant): Noise-induced hearing loss (NIHL) and age-related hearing loss (AHL or presbycusis) are major health problems. They are common, their consequences are permanent, and their impacts on human communication and quality of life are significant. Although important advances have been made in characterizing the structural changes in the ear that are associated with NIHL or AHL, the mechanisms underlying these changes are poorly understood. In humans, hearing loss secondary to noise exposure is highly variable between individuals: some people have "tough" ears, while others have "tender" ears. In contrast to humans, laboratory mice show significantly less variability in NIHL among individuals within an inbred strain while there are striking differences in NIHL sensitivity between different inbred strains. Our long-term goal is to exploit these strain differences in mouse models to study the genetic factors influencing resistance and susceptibility to NIHL Here we propose to focus on the remarkable NIHL resistance observed in the inbred mouse strain 12986/SvEvTac (129S6). We will address the following Specific Aims: SA 1. Refine and confirm our preliminary Quantitative Trait Locus (QTL) map for NIHL resistance in 129S6. Develop a second NIHL-resistance QTL map in a different mouse strain, MOLF/Ei for comparison. SA 2: Generate congenic strains using both phenotype-driven selection for NIHL resistance and genotype-driven, marker-assisted selection for QTL regions. Isolated QTL regions will be tested for epistatic interactions. SA 3. Identify candidate NIHL resistance genes using DNA microarrays to study changes in gene expression after noise exposure. Genes differentially regulated between strains and mapping within QTL regions will be sequenced in both strains and compared for variations. SA 4. Strong candidate genes will be tested in genetic crosses to determine whether they interact functionally with the NIHL-resistant QTL. Nucleotide differences in genes suspected to account for the QTL will be tested using gene targeting knock-in techniques to see if they are sufficient to transfer NIHL resistance to another strain. The characterization of genes influencing NIHL resistance will provide fundamental insight into the cellular and molecular processes underlying noise-induced cochlear damage. In turn, these insights will be key to devising effective strategies to preserve hearing in human populations.

PROJECT SUMMARY (See instructions): Hearing loss is the most frequent sensory defect in humans. Congenital, perinatal or eariy onset hearing loss occurs in approximately 7 out of 1000 neonates in the United States. In approximately half of the children born with severe hearing impairment, a genetic contribution is suspected. The powerful molecular and genetic techniques available in zebrafish and mouse combined with the functional similarities between mouse and human audition make these organism useful model systems for studying deafness. Core D supports users of genetic models to study genes affecting auditory, vestibular and olfactory system function. We provide technical support in the form of expert mouse husbandry (receiving, setting up crosses, weaning, fostering, background transfers, etc), genotyping, and notification/delivery of requested mice to the users. Core D also supports several facilities for behavioral and physiological analyses of mutants including auditory brainstem responses (ABR), simple motor tests, rotarod testing, and vestibulo-ocular reflex (VOR) analysis. Other services include access to lentiviral transfection technologies and resources for genomic and proteomic data analysis. Advice and training is available to all users from a knowledgeable and helpful Core staff. The Genetics Core will provide greater efficiency for researchers and promote new collaborations between investigators. Quarteriy user meetings provide information on Core resources, discussion of protocols, and talks by Core users on their own research projects currently being pursued. Our goal is to facilitate the use of genetic models to broaden our knowledge of communicafive disorders toward improving human health.

DESCRIPTION (provided by applicant): The primary mission of the Research Training Program at the University of Washington Department of Otolaryngology-Head and Neck Surgery (OtoHNS) is to educate residents who have the prerequisite research training, commitment, and experience to develop and support research programs that will enhance the treatment of patients with diseases of communication, the special senses of hearing, balance and olfaction, airway regulation and cancer of the head and neck. This program creates a research culture, facilitating investigative activity throughout the residency period, and beyond. This continuation of an Institutional Training Grant (T32) that began in 1984 supports full-time research training for every OtoHNS Resident for one or two years near the beginning of residency. Residents are required to continue research productivity during each year of the Residency Program after the T32-supported research period, and this research productivity is facilitated by additional department-supported research periods free of daily clinical responsibility. In addition to four positions for Resident Research Trainees, we request one position for a Post-Residency Clinical Scholar to obtain full-time research training in conjunction with a subspecialty clinical fellowship, and one position for a Predoctoral Medical Student Research Scholar who wishes to acquire a full year of intensive research training. Trainee positions not filled by physicians in one of the programs noted above are awarded on a competitive basis for one-year PhD Postdoctoral Fellows recruited by OtoHNS Program Faculty, facilitating the collection of data enhancing successful applications for Individual NRSA F32 Award applications. Concerted effort is made to recruit and train under-represented minority candidates. The Research Training Program is continuously evaluated and altered to fit the changing needs of the research trainees. Funded investigators in biomedical science throughout the University of Washington system are available as potential Primary Research Mentors, and a group of 16 investigators with primary appointments in the OtoHNS Department or experience mentoring our T32-supported research trainees are named in addition to the Director (PI) and two Co-Directors (Co-Investigators). Analysis of the results of this program over the past 15 years reveals that >60% of the physician trainees that are out of training have joined full-time academic institutions.

? DESCRIPTION (provided by applicant): C57BL/6J (B6) mice have a well-characterized age-related hearing loss (AHL) phenotype. A recent study has shown that this loss is only partially caused by mutations in the Cadherin 23 (Cdh23) gene which encodes the tip link protein CDH23 (Kane et al., 2011). In mice and in humans, mutations in Cdh23 are exacerbated by mutations in the plasma membrane Ca2+ ATPase 2 (PMCA2) protein, which regulates intracellular Ca2+ levels (Noben-Trauth et al., 1997 and Schultz et al., 2007). This interaction is likely due to the necessity of Ca2+ in maintaining the structural integrity of CDH23 (Sotomayor et al., 2010). There are no mutations in the B6 Atp2b2 gene, which encodes PMCA2. However, two discreet measures of gene expression show that there is down-regulation of Atp2b2 transcript in B6 compared to the good-hearing strain CBA/CaJ (CBA). Studies of the deafwaddler mutations in Atp2b2 demonstrate that the auditory system is highly sensitive to small changes in Atp2b2. These mice exhibit changes in hearing sensitivity that can be correlated to changes in regulation, function and expression of Atp2b2 (McCullough and Tempel, 2004; Watson and Tempel, 2013). All of this evidence suggests that the down-regulation of Atp2b2 in B6 is a likely contributor to the age-related hearing loss phenotype in these mice. As there are no mutations in the protein coding region of Atp2b2 but changes in transcript expression, transcriptional processes are likely involved in the down-regulation of Atp2b2 in B6. Recent experiments in the Tempel lab have confirmed the presence of a long intergenic non-coding RNA (lincRNA-83) that is in the intronic regions of the mouse Atp2b2 gene. Expression studies indicate that this gene is misregulated in the brainstem and the cochlea of B6 mice. Importantly, lincRNAs are emerging as key players in transcriptional regulation of nearby genes (Wang and Chang, 2011). The over-arching hypothesis in this proposal is that Atp2b2 misregulation in B6 contributes to AHL in this strain. We propose two aims to better understand the: 1) degree of expression differences between B6 and CBA, and 2) the extent to which non-coding RNAs regulate the Atp2b2 gene.

PROJECT SUMMARY/ABSTRACT The claudin family is composed of transmembrane proteins that are an essential part of the tight junctions forming barriers in epithelial and endothelial tissue. In the cochlea, Claudin 9 (Cldn9) is highly expressed in the tight junctions of the organ of Corti, where separation of high potassium (K+) endolymph from the low K+ perilymph fluid is necessary for protection of the outer hair cells (OHCs), as well as for maintaining the Endocochlear Potential (EP) (Nakano et al 2009, Wangemann and Schacht, 1996). Our lab has focused on identifying genes that are resistant to noise stress (Street, et al. 2014). Among the five quantitative trait loci (QTL) that appeared in the 129S6/SvEvTac (129S6) strain, the strongest QTL contains Cldn9. Surprisingly, when we started to study the noise sensitivity it became apparent that this locus also displayed an age-related hearing loss (AHL) phenotype that was non-progressive hearing loss (NPHL), and a significant mid-frequency ARHL by 12 months. To explore functional attributes of Cldn9, we have recently developed a Doxycyline (dox) regulated version of the Cldn9 gene that can be up or down regulated. We are assured that the changes that we observe after dox manipulation are due to the Cldn9 expression. Here we will examine the role of Cldn9 in an aging auditory system, including the influence noise and the question of Cldn9?s role in potassium leakage.