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Progressive Hearing Loss and Gradual Deterioration of Sensory Hair Bundles in the Ears of Mice Lacking the Actin-Binding Protein Eps8l2

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Progressive Hearing Loss and Gradual Deterioration of Sensory Hair Bundles in the Ears of Mice Lacking the Actin-Binding Protein Eps8l2 Progressive hearing loss and gradual deterioration of sensory hair bundles in the ears of mice lacking the actin-binding protein Eps8L2 David N. Furnessa,1, Stuart L. Johnsonb,1, Uri Manorc,1, Lukas Rüttigerd,1, Arianna Tocchettie,2, Nina Offenhausere, Jennifer Oltb, Richard J. Goodyearf, Sarath Vijayakumarg, Yuhai Daic, Carole M. Hackneyb, Christoph Franzd, Pier Paolo Di Fioree,h,i, Sergio Masettoj, Sherri M. Jonesg, Marlies Knipperd, Matthew C. Holleyb, Guy P. Richardsonf, Bechara Kacharc, and Walter Marcottib,3 aInstitute for Science and Technology in Medicine, Keele University, Keele ST5 5BG, United Kingdom; bDepartment of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom; cLaboratory of Cell Structure and Dynamics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892; dDepartment of Otolaryngology, Tübingen Hearing Research Centre (THRC), University of Tübingen, 72076 Tübingen, Germany; eFondazione Istituto FIRC di Oncologia Molecolare (IFOM), 20139 Milan, Italy; fSchool of Life Sciences, University of Sussex, Brighton BN1 9QG, United Kingdom; gDepartment of Special Education and Communication Disorders, University of Nebraska, Lincoln, NE 68583; hIstituto Europeo di Oncologia, 20141 Milan, Italy; iDipartimento di Scienze della Salute, Universita’ degli Studi di Milano, 20122 Milan, Italy; and jDepartment of Physiological and Pharmacological Science, University of Pavia, Pavia 27100, Italy Edited by Michael Deans, Johns Hopkins University, Baltimore, MD, and accepted by the Editorial Board July 8, 2013 (received for review March 13, 2013) Mechanotransduction in the mammalian auditory system depends we do not have a complete molecular understanding of hair on mechanosensitive channels in the hair bundles that project bundle structure or how its growth and maintenance are con- from the apical surface of the sensory hair cells. Individual stereo- trolled. Recently, we showed that epidermal growth factor re- cilia within each bundle contain a core of tightly packed actin ceptor pathway substrate 8 (Eps8) (14) is located at the tips of filaments, whose length is dynamically regulated during develop- stereocilia where it regulates normal growth of the hair bundle ment and in the adult. We show that the actin-binding protein (15, 16). In mammals, the Eps8 family includes three Eps8-re- – GENETICS epidermal growth factor receptor pathway substrate 8 (Eps8)L2, lated proteins named Eps8L1 L3. Eps8L1 and L2 also show fi actin binding activity, displaying similar domain organization and a member of the Eps8-like protein family, is a newly identi ed hair – bundle protein that is localized at the tips of stereocilia of both 27 42% identity to Eps8 (17). Eps8L2 shares a common modular protein structure to Eps8, indicating a possible overlap in func- cochlear and vestibular hair cells. It has a spatiotemporal expres- tion (17). However, despite the identification of Eps8L2 several sion pattern that complements that of Eps8. In the cochlea, years ago, its function remains unknown. Given that Eps8L2 has whereas Eps8 is essential for the initial elongation of stereocilia, a remarkably similar expression pattern to that of Eps8, we hy- Eps8L2 is required for their maintenance in adult hair cells. In the pothesized that it may be important for the growth and/or absence of both proteins, the ordered staircase structure of the maintenance of hair cell stereocilia. We generated Eps8L2 KO hair bundle in the cochlea decays. In contrast to the early profound mice and studied the structure and physiology of their mecha- hearing loss associated with an absence of Eps8, Eps8L2 null- nosensory systems. We found that the mice have progressive mutant mice exhibit a late-onset, progressive hearing loss that is hearing loss, which is associated with progressive disorganization directly linked to a gradual deterioration in hair bundle morphol- of cochlear hair bundles and abnormal variations in the length and ogy. We conclude that Eps8L2 is required for the long-term main- width of stereocilia. We conclude that Eps8L2 is a newly identified tenance of the staircase structure and mechanosensory function stereociliary protein critical for hearing and for the normal func- of auditory hair bundles. It complements the developmental tion of mechanosensory hair bundles in adult mammals. role of Eps8 and is a candidate gene for progressive age-related hearing loss. Results Generation of Eps8L2 KO Mice. Eps8L2 KO mice were generated by deafness | sensory system | ion channel replacing the exons encoding the SH3 domain (exons XVI and XVII) with a neomycin cassette (Fig. S1A). Targeted ES cells were identified by PCR (Fig. S1B), and correct insertion was earing and balance depend on the transduction of me- verified at the 5′ and 3′ ends of the targeting vector by PCR and Hchanical stimuli into electrical signals. Transduction involves Southern blotting, respectively (Fig. S1 C and D). Injection of activation of mechanically gated ion channels near the tips of the two correctly targeted ES cells into blastocysts gave rise to off- stereocilia, specialized microvilli that form the hair bundles that spring with high coat chimaerism in both cases. Chimaeras were project from the surface of sensory hair cells (1). Stereocilia have bred for germ-line transmission, and the resulting heterozygous a cytoskeletal core composed of tightly packed, cross-linked, and mice were intercrossed to obtain Eps8L2 KO mice, which were uniformly polarized actin filaments (2, 3). Stereociliary length is regulated to ensure the characteristic staircase-like structure of each bundle, whose overall size and shape depends on location along the sensory organ (4). In the mammalian cochlea, hair Author contributions: P.P.D.F., G.P.R., B.K., and W.M. designed research; D.N.F., S.L.J., U.M., L.R., A.T., N.O., J.O., R.J.G., S.V., Y.D., C.M.H., C.F., S.M.J., and W.M. performed research; bundles usually include three rows of stereocilia coupled by D.N.F., S.L.J., U.M., L.R., A.T., N.O., J.O., R.J.G., S.V., Y.D., C.F., S.M.J., M.K., G.P.R., and W.M. several types of extracellular links (2, 5). The embryonic and analyzed data; and S.M., M.C.H., G.P.R., B.K., and W.M. wrote the paper. postnatal development of the bundle involves elongation and The authors declare no conflict of interest. thickening of stereocilia, as well as elimination of redundant This article is a PNAS Direct Submission. M.D. is a guest editor invited by the Editorial stereocilia (4, 5). Board. In the adult cochlea, the height of stereocilia within each row is similar, not only within a single hair bundle but also between Freely available online through the PNAS open access option. the bundles of adjacent hair cells, indicating a sophisticated level 1D.N.F., S.L.J., U.M., and L.R. contributed equally to this work. of control over growth (5, 6). Stereociliary growth and mainte- 2Present address: KtedoGen srl, 20138 Milan, Italy. nance involves actin-binding proteins such as espin (7, 8), plastin 3To whom correspondence should be addressed. E-mail: w.marcotti@sheffield.ac.uk. (9), twinfilin 2 (10), gelsolin (11), and unconventional myosin This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. motors including myosin XVa (12) and myosin IIIa (13). Currently, 1073/pnas.1304644110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1304644110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 born and weaned at the expected Mendalian ratio (Fig. S1E). Eps8L2 remained at the tips of all cochlear hair cell stereocilia Absence of Ep8L2 protein in lung tissue from Eps8L2 KO mice in Eps8 KO mice (Fig. 1E), which are short and show a relatively was verified by Western blotting (Fig. S1F). similar height among rows (15, 16). Despite the similar expres- sion profile and high homology of the two genes (17), there is Eps8L2 Is Localized at the Tips of Hair Cell Stereocilia. Immunofluo- very little functional redundancy between the two proteins in rescence showed that Eps8L2 was expressed at the tips of cochlear cochlear hair cells. Although the localization of Eps8 requires hair cell stereocilia (Fig. 1; Fig. S2) and that it was absent in hair the motor protein myosin XVa (15, 16), we found that in shaker- cells from Eps8L2 KO mice (Fig. S2). The distribution of Eps8L2 2 mice, which are deficient in myosin XVa (18), Eps8L2 was still overlapped with that of Eps8 (15, 16) as previously suggested in present at the stereocilia tip (Fig. 1F). other systems (17). However, the spatiotemporal expression pat- terns in cochlear hair cells were different. Eps8 labeling in the Progressive Hearing Loss in Eps8L2 KO Mice. Eps8L2 KO mice shortest rows of stereocilia diminished quickly during postnatal (males and females) have progressive hearing loss, in contrast to maturation but remained high in the longest row, whereas Eps8L2 the early profound hearing loss that occurs in the absence of labeling was high in the middle and shortest rows of fully grown Eps8. Auditory brainstem responses (ABRs) reflect the activity stereocilia and was hardly detectable in the longest rows (Fig. 1A). of the afferent auditory neurons downstream of the inner hair The differential distribution of each protein to different rows of cells (IHCs). WT Eps8L2 littermates had normal thresholds for stereocilia was present as early as P2 (Fig. 1B), and it appeared to click, noise, and pure-tone evoked ABRs at about 2 mo of age intensify with age and location along the cochlea (Fig. 1A). In (Fig. 2 A–C) and at 5–7 mo of age (Fig. 2 D–F). The thresholds vestibular hair, Eps8 and Eps8L2 were more obviously colocalized for click stimuli were 18.3 ± 2.9 dB SPL at 2 mo, 16.5 ± 4.5 dB at the tips of most stereocilia in similar length-proportional SPL at 5 mo, and 16.5 ± 3.9 dB SPL at 7 mo.
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