Usher proteins in the inner ear structure and function

Usher syndrome (USH) is the most prevalent form of inherited deafness associated with retinitis pigmentosa (RP) and vestibular areflexia. In the United States, 1 per 6000 individuals is affected with USH. To date, nine USH genes have been identified. Certain mutations in some of these USH genes also cause nonsyndromic deafness (DFNB) or nonsyndromic RP. However, mutations in these USH genes do not account for all of the known cases of Usher syndrome. Therefore, proposed studies of USH proteins will improve the molecular epidemiology of USH and aid in genetic diagnosis and counseling. Besides that, studies of USH proteins have been extremely fruitful for elucidating molecular components of stereocilia bundle and transduction machinery. Growing evidence suggests that USH proteins constitute stereocilia tip link complex (cadherin 23, protocadherin 15, SANS), affect the attachments of tip links at the upper (harmonin) and lower (whirlin) ends, and influence mechanoelectrical transduction (MET). This study will elucidate the function of a novel USH protein, also improving our understanding of the basic stereocilia structure and function. The long-term goal of this project is to understand how USH proteins are involved in the biological processes of the inner ear. The objective here is to determine the mechanisms underlying hearing impairment caused by mutations of newly identified USH1J/DFNB48 gene, CIB2. Using positional cloning strategies, we have identified four pathogenic mutations in CIB2 gene responsible for USH1J/DFNB48 phenotypes in one Turkish and 57 Pakistani families. CIB2 encodes a calcium and integrin binding protein that is localized in the stereocilia as well as at the cuticular plate region of both inner (IHC) and outer hair cells (OHC). Additionally, our ex vivo studies revealed that DFNB48 mutations affect Ca2+-buffering properties of CIB2 and that CIB2 interacts with whirlin, a known Usher protein. However the exact function of CIB2 in the inner ear as well as the mechanism of hearing impairment requires animal models. Recently, we have generated these mouse models. Our central hypothesis is that CIB2 is a Ca2+ buffering protein that is required to maintain calcium homeostasis in the mechanosensory stereocilia of the inner ear hair cells. Loss of CIB2 function may affect both the structure and mechanosensitivity of stereocilia bundle. To test this hypothesis, we have already generated two mutant mouse strains, including a Cib2F91S knock-in, a model of the most common DFNB48 mutation in humans, and an exon 4 floxed allele, Cib2flox-ex4. In this project, our skills in molecular and cellular biology and genetics are complemented by Dr. Frolenkov’s expertise in the inner ear function and structure and Dr. Jones’ expertise in recording the vestibular evoked potentials in rodents, thus building a multidisciplinary team to investigate the role of CIB2. Specific aims: Aim 1: Elucidate the underlying cause of deafness in Cib2F91S mice: Our working hypothesis is that p.F91S mutation affects Ca2+ buffering ability of CIB2 and/or interaction of CIB2 with its protein partners, resulting in impaired MET function and hearing loss. To test this hypothesis, we will use Cib2F91S/F91S mouse model to a) Determine the cochlear abnormalities resulting in hearing loss in Cib2F91S/F91S mice. b) Elucidate any changes in the morphology of the inner ear due to the p.F91S mutation. c) Determine the effect of p.F91S on the MET function of inner ear hair cells. Aim 2: Determine the role of CIB2 in development and assembly of tip link-MET complex. Based on our preliminary studies in humans and zebrafish, a complete elimination of CIB2 in the inner ear is expected to have more profound abnormalities (profound deafness and vestibular dysfunction). To test this, we have generated a floxed allele (Cib2flox-ex4) of Cib2 exon 4, deletion of which will generate a Cib2 null. a) These conditional knockout mice (Cib2Äex4) will be evaluated to determine more comprehensively the functions of CIB2 in the inner ear hair cells. Aim 3: Elucidate potential interactions between CIB2 and known proteins associated with tip link-MET complex: We propose that CIB2 interaction with whirlin is required for the proper localization of CIB2 in the hair cells’ stereocilia. Furthermore, intracellular Ca2+ may regulate CIB2 interaction with whirlin and other USH protein. To test these hypotheses, we will a) Determine the localization of CIB2 in the whirlin mutant (whirler) and myosin XVa mutant (shaker-2) mice. b) Determine the effect of Ca2+ in regulating the CIB2-whirlin interaction. c) Define the relationship between CIB2 and known tip link-MET complex proteins. Our expected outcomes are to identify a) the mechanism of deafness caused by loss of CIB2 function; and b) the physiological role of CIB2 in tip link-MET complex assembly and function. Such results are expected to have an important positive impact, because deciphering the function of proteins encoded by “USH genes” is a prerequisite for the eventual development of therapies to treat and/or prevent this dual neurosensory disorder.