Different whirlin splicing isoforms play unique roles in the inner ear and retina

Usher Syndrome (USH) is the leading cause of deaf-blindness worldwide. Patients with USH have hearing loss, balance problems, and retinal degeneration. To date, eleven genes have been associated with USH. Interestingly, mutations in several USH genes lead to discrete diseases as well. Despite extensive studies on USH in the past, over one-hundred years since USH was originally identified, the molecular function of USH genes remains understudied. This incomplete understanding greatly limits therapeutic development for USH. To understand the molecular mechanisms underlying variable disease manifestations, I studied an USH2 gene, DFNB31, which causes USH2D when mutated in its N-terminal region, and autosomal recessive nonsyndromic deafness type 31 (DFNB31) when mutated towards its C-terminal region. DFNB31 encodes a protein called whirlin that was previously shown to have multiple mRNA variants in human and mouse tissues. I hypothesized that whirlin isoforms have unique functions and disruption of different whirlin isoforms is the cause of various disease manifestations. To test this hypothesis, I utilized region-specific whirlin antibodies and Dfnb31 mouse models that mimic human DFNB31 mutations and disease outcomes. I found that alternative splicing and alternative use of promoters produce several Dfnb31 mRNA variants that are translated to full-length (FL), N-terminal and C-terminal whirlin isoforms, which localize at different subcellular positions in the inner ear hair cells and retinal photoreceptors. Studies in Dfnb31 mutants show that FL-whirlin isoform is required at the hair cell stereociliary bases and retinal photoreceptor periciliary membrane complex to form a stable USH2 protein complex, whereas C-whirlin isoform is required at the stereocilia tips for stereociliary elongation. I found that mutations in N-terminal region of Dfnb31 lead to loss of FL- and N-whirlin isoforms and cause USH2D-like symptoms. On the other hand, mutations in the C-terminal region of Dfnb31 lead to loss of FL- and C-whirlin isoforms and cause DFNB31-like symptoms. Considering the presence of multiple splicing isoforms for several other USH genes and the variable phenotypes caused by mutations in these genes, differential disruption of the splicing isoforms is likely the mechanism underlying different disease manifestations upon mutation in these USH genes. In addition, I found vestibular deficits in Dfnb31 mutants, which was surprising because USH2 patients were thought to have normal vestibular function. My findings present a rationale for vestibular analysis of all USH2 patients at the clinics to comprehend the pathogenesis and mechanism of USH. In summary, my findings will help improve differential diagnosis between USH and its related diseases and is expected to contribute to the development of USH therapies.

DIFFERENT WHIRLIN SPLICING ISOFORMS PLAY UNIQUE ROLES IN THE INNER EAR AND RETINA by Pranav Dinesh Mathur A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Neurobiology and Anatomy The University of Utah August 2016 Copyright © Pranav Dinesh Mathur 2016 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of ____________________Pranav Dinesh Mathur___________ has been approved by the following supervisory committee members Jun Yang , Chair Gary C. Schoenwolf , Member ___ 5/10/2016_____ Date Approved 5/09/2016_____ Date Approved Wolfgang Baehr , Member 5/09/2016______ Suzanne L. Mansour , Member ___ 5/09/2016______ Date Approved Date Approved Michael Robert Deans , Member ___ 5/12/2016______ Yukio Saijoh , Member ___ 5/10/2016______ and by Monica L. Vetter Date Approved Date Approved , Chair of the Department/College/School of ______Neurobiology and Anatomy___________ and by David B. Kieda, Dean of The Graduate School. ABSTRACT Usher Syndrome (USH) is the leading cause of deaf-blindness worldwide. Patients with USH have hearing loss, balance problems, and retinal degeneration. To date, eleven genes have been associated with USH. Interestingly, mutations in several USH genes lead to discrete diseases as well. Despite extensive studies on USH in the past, over one-hundred years since USH was originally identified, the molecular function of USH genes remains understudied. This incomplete understanding greatly limits therapeutic development for USH. To understand the molecular mechanisms underlying variable disease manifestations, I studied an USH2 gene, DFNB31, which causes USH2D when mutated in its N-terminal region, and autosomal recessive nonsyndromic deafness type 31 (DFNB31) when mutated towards its C-terminal region. DFNB31 encodes a protein called whirlin that was previously shown to have multiple mRNA variants in human and mouse tissues. I hypothesized that whirlin isoforms have unique functions and disruption of different whirlin isoforms is the cause of various disease manifestations. To test this hypothesis, I utilized region-specific whirlin antibodies and Dfnb31 mouse models that mimic human DFNB31 mutations and disease outcomes. I found that alternative splicing and alternative use of promoters produce several Dfnb31 mRNA variants that are translated to full-length (FL), N-terminal and C-terminal whirlin isoforms, which localize at different subcellular positions in the inner ear hair cells and retinal photoreceptors. Studies in Dfnb31 mutants show that FL-whirlin isoform is required at the hair cell stereociliary bases and retinal iii photoreceptor periciliary membrane complex to form a stable USH2 protein complex, whereas C-whirlin isoform is required at the stereocilia tips for stereociliary elongation. I found that mutations in N-terminal region of Dfnb31 lead to loss of FL- and N-whirlin isoforms and cause USH2D-like symptoms. On the other hand, mutations in the C-terminal region of Dfnb31 lead to loss of FL- and C-whirlin isoforms and cause DFNB31-like symptoms. Considering the presence of multiple splicing isoforms for several other USH genes and the variable phenotypes caused by mutations in these genes, differential disruption of the splicing isoforms is likely the mechanism underlying different disease manifestations upon mutation in these USH genes. In addition, I found vestibular deficits in Dfnb31 mutants, which was surprising because USH2 patients were thought to have normal vestibular function. My findings present a rationale for vestibular analysis of all USH2 patients at the clinics to comprehend the pathogenesis and mechanism of USH. In summary, my findings will help improve differential diagnosis between USH and its related diseases and is expected to contribute to the development of USH therapies. iv "Be more dedicated to making solid achievements than in running after swift but synthetic happiness." Late Dr. APJ Abdul Kalam v TABLE OF CONTENTS ABSTRACT……………………………………………………………………………...iii ACKNOWLEDGEMENTS…………………………………………………..………....viii Chapters 1. PROLOGUE………………………………………………………………………1 References…………………………………………………………………6 2. USHER SYNDROME: HEARING LOSS, RETINAL DEGENERATION AND ASSOCIATED ABNORMALITIES…………………..…………………….…...8 Abstract……………………………………………………………………9 Introduction………………………………………………………………..9 Genes and loci identified in USH patients…….………………………….11 Expression of USH genes………………………………………………...12 USH proteins exist in multiprotein complexes…………………………...14 Functional studies of USH gene products………………………………...15 Insights from current literatures about USH genes in various tissues…….17 Current progress in therapeutic studies on USH………………………….17 Current gaps in understanding and treating USH…………………………18 Summary…………………………………………………………………19 Acknowledgements………………………………………………………19 References………………………………………………………………..19 3. DISTINCT EXPRESSION AND FUNCTION OF WHIRLIN ISOFORMS IN THE INNER EAR AND RETINA: AN INSIGHT INTO PATHOGENESIS OF USH2D AND DFNB31………………………..………………………………………….24 Abstract…………………………………………………………………..25 Introduction ...……………………………………………………………25 Results ...…………………………………………………………………26 Discussion………………………………………………..………………34 Materials and methods…………………………………………………...37 Acknowledgements ………………………...…………………………....38 References …………………………………………………….…………38 vi 4. A STUDY OF WHIRLIN ISOFORMS IN THE MOUSE VESTIBULAR SYSTEM SUGGESTS POTENTIAL VESTIBULAR DYSFUNCTION IN DFNB31-DEFICIENT PATIENTS……………………………………………...41 Abstract…………………………………………………………..………42 Introduction ………………………………………...………….….……..42 Results ……………………………………………………...….….……..43 Discussion…………………………………………………….…….……48 Materials and methods……………………………………….…….…….52 Acknowledgements ………………………………………………....…...53 References……………………………………………………….…….....53 5. DISCUSSION………………………………………………………….….……..56 Introduction……………………………………………………….……..57 Mutations in DFNB31 cause variable disease manifestations .…….…...61 Dfnb31 mouse models recapitulate USH2D and DFNB31 diseases…………………………..…………………………………..…..63 Whirlin has different spatiotemporal expression patterns in the inner ear and retina ..………………………………………………………...………....69 Whirlin forms different multiprotein complexes in the inner ear and retina..……………………………………………………………….…...74 Correlation of genotypes with phenotypes conveys the function of different whirlin isoforms …………...……………………………………….....…79 Whirlin isoforms have functions outside the inner ear and retina………82 Previous attempts to rescue Dfnb31 mutant phenotypes were only partially successful………………………………………………………………...83 Conclusion and future directions………………………………………...84 References………………………………………………………..………88 vii ACKNOWLEDGEMENTS First and foremost I would like to thank, with all my heart, my mentor Dr. Jun Yang for accepting me in her laboratory and giving me this incredible project to work on. I always wanted to do a research with high translational significance and Jun gave me this beautiful project that I was able to develop and contribute to science. Her guidance, persistence, and cooperation helped me throughout my research. She always welcomed my ideas and provided me the freedom to experiment out and test my hypothesis. In addition, she helped me improve my train of thought, which will help me become an independent scientist one day. I would like to specially acknowledge Dr. Yukio Saijoh, my previous employer and current thesis committee member. I worked in Yukio's lab as a laboratory technician for about a year. This experience not only helped me get into the graduate school, but also trained me to understand how science is done. Over the past several years, he has been more than a mentor to me. He is someone to whom I can run anytime with my problems. He always found time to listen to me and provided me a valid and most appropriate solution for my scientific/nonscientific worries. I would also like to thank other members of my supervisory committee, Drs. Suzanne Mansour, Gary Schoenwolf, Michael Deans and Wolfgang Baehr, who have given me valuable inputs and have been generous with their time. viii I would also like to take this opportunity to thank my undergraduate mentor, Dr. Arun Sirothia from the Nagpur Veterinary College, MAFSU, and my master's mentors, Drs. GVPPS Ravi Kumar and Gurvinder Singh Brah from the College of Veterinary Sciences, GADVASU in India. They were the ones who instilled in me a desire to do research in Cell Biology and Genetics. They also provided me with basic concepts of Genetics and my first hands-on experience with several molecular biology techniques. A great deal of thanks goes to my current and previous lab-mates, Junhuang, Qian, Ali Sharif, Cris, Jesse and Christin, who helped me with experiments and were always enthusiastic to talk about science. I would also like to thank Ali Almishaal, my scientific collaborator-cum-friend who helped me in my project by doing ABRs and DPOAE tests. Eventually, I also helped him in several experiments on his project. This gave me an idea about how collaboration works. I hope we both benefited by this collaboration and get more publications. I am sincerely thankful to my parents and parents-in-law, who always stood by me through thick and thin. My parents were my first source of inspiration. My father provided me my first ever understanding about Biology and Mathematics, and always encouraged me to pursue a scientific career. Ever since I met my parents-in-law, they have always motivated me and kept praying for my success. Last, but certainly not the least, I am thankful for my best friend and wife Deepti for her love, patience and endless support. Deepti moved to Salt Lake with me when I started my PhD program in 2010. She cooperated with me and always helped me, especially those days when I was terribly busy and stressed. She always believed in me and stood by me during this entire process. I will always be grateful to her kindness. ix CHAPTER 1 PROLOGUE 2 Background and significance of the study in this dissertation The overall goal of the research in this dissertation is to address the molecular functions of Usher syndrome (USH) proteins, and Chapters 2 to 4 represent my specific accomplishments in the process. Diseases that affect the retina and inner ear, two main sensory organs of the human body, significantly affect the patient's social life and psychological health. USH accounts for more than 50% of inherited deaf-blindness cases, with an estimated frequency of 1:6000 (Kimberling et al., 2010). USH patients exhibit a variety of symptoms. Based on the severity of hearing loss, USH is categorized into three clinical types, namely, USH1, USH2 and USH3. USH1 patients have congenital profound sensorineural hearing loss; USH2 patients display moderate to severe levels of congenital sensorineural hearing loss; and USH3 patients have progressive deafness. All USH patients undergo retinal degeneration (RD). However, the onset of RD is variable, with adolescence being the earliest age reported. There are eleven known genes that can cause USH. Most proteins encoded by these USH genes interact with one another to form complexes that carry out various functions in the inner ear and retina. To comprehend the complexity of USH genes, their known functions, available animal models, and current research progress, I co-authored a review paper that will serve as Chapter 2 of this dissertation (Mathur & Yang, 2015). Chapter 2 will thoroughly introduce the readers to various USH proteins, their functions, interaction complexes, disease outcomes, unsolved problems in USH research, and the current state of therapeutic advancement. A major limitation in the path of therapeutic advancement is the lack of complete knowledge of USH protein functions at the molecular level. Specifically, the function of USH proteins, in complex with other proteins at different subcellular 3 locations of various sensory organs, remains obscure. Furthermore, it remains unknown how mutations in most USH genes cause multiple discrete diseases. Addressing these questions is important since, despite several efforts to study USH genes and to develop therapies, there is no cure for USH to date. An understanding of the molecular function of USH proteins will aid disease treatment, early differential diagnosis, and patient rehabilitation. Moreover, it will advance our understanding of the cell-specific function of USH proteins. Study design and results To reveal the molecular mechanisms underlying USH, I studied an USH2 gene, DFNB31, which encodes a protein called whirlin. Mutations in the 5' region of DFNB31 are associated with USH2D, a subtype of USH, while mutations in the 3' region cause DFNB31, a subtype of nonsyndromic autosomal recessive deafness (DFNB) (Audo et al., 2011; Besnard et al., 2012; Ebermann et al., 2007; Mburu et al., 2003; Nishiguchi et al., 2013; Tlili et al., 2005). The variation in the disease outcome upon mutations within the same gene impacts differential diagnosis and development of therapies. Therefore, the objective of my research in Chapter 3 was to understand the genotype-phenotype correlation upon mutations in Dfnb31. To address this question, I utilized Dfnb31 mouse mutants that mimic the region and outcome of human DFNB31 mutations. A mouse Dfnb31neo/neo mutant, generated by replacing the 5' region of Dfnb31 exon1 including the translation start codon with a Neor cassette, mimics the mutation region and disease outcome of USH2D (Yang et al., 2010), whereas a Dfnb31wi/wi mutant mouse that harbors a 592-bp deletion in the coding region of Dfnb31 exons 6-9 mimics DFNB31 patients 4 (Mburu et al., 2003). Furthermore, similar to human DFNB31, mouse Dfnb31 is also known to yield multiple mRNA transcripts, likely as a result of alternative splicing and/or presence of multiple promoters in the gene (Belyantseva et al., 2005; Mburu et al., 2003; Wright, Hong, & Perkins, 2012). In my study, I found different spatiotemporal expressions and subcellular localizations of Dfnb31 mRNA variants and whirlin protein isoforms in the organ of Corti hair cells and retinal photoreceptors. My studies show that a mutation in Dfnb31 leads to loss of different whirlin protein isoforms, depending upon the site of the mutation (Mathur, Zou et al., 2015). As a result, inner ear hair cells and/or retinal photoreceptors are differentially affected, resulting in either DFNB31 or USH2D. My study, combined with previous findings showing multiple splicing isoforms of several other USH genes and variable phenotypes caused by mutations of these USH genes, suggests that a parallel mechanism plays a role in those cases as well. Dfnb31 was previously reported to have multiple mRNA variants in the mouse vestibular hair cells (Belyantseva et al., 2005). In addition, a Dfnb31 mutant (Dfnb31wi/wi) shows circling and head-bobbing behaviors, suggestive of vestibular deficits (Holme et al., 2002). Since patients with mutations in DFNB31 do not report any balance issues, the role of whirlin in the vestibular system remains understudied. To understand the role of whirlin in the vestibular system, I examined the Dfnb31neo/neo and Dfnb31wi/wi vestibular hair cells. My studies revealed for the first time that a mutation in Dfnb31 leads to vestibular impairment, suggesting a possibility that USH2D and DFNB31 patients may have vestibular abnormalities (Mathur, Vijayakumar, et al., 2015). These results were published in Human Molecular Genetics and form Chapter 4 of this dissertation. The study described in Chapter 4 is significant, since, to date, vestibular abnormalities are thought to be 5 restricted only to USH1 and USH3 patients. My study suggests that USH2 patients need to be tested for vestibular problems in the clinics in the future. My findings have been further supported by a recent clinical study that reported the presence of vestibular pathologies in a majority of USH2 patients studied (Magliulo et al., 2015). Finally, my study in Chapter 4 also elucidates the molecular functions of Dfnb31 in the murine vestibular system. Conclusion Overall, I found that disruption of different whirlin isoforms by different mutations is the molecular basis underlying the genotype-phenotype correlation in mice, suggesting that a similar mechanism underlies the different disease manifestations in DFNB31deficient humans. Differential isoform disruption could also be the cause for a wide spectrum of disease manifestation in patients carrying mutations in other USH genes. For example, a mutation in CDH23, known to express three different protein isoforms, can cause either USH1D or DFNB12 (Lagziel et al., 2005; Schultz et al., 2005, 2011). Furthermore, I found that DFNB31-deficient patients may also have vestibular deficits in addition to hearing and/or vision defects. These findings are discussed in a broader context in Chapter 5 of this dissertation, written in a stand-alone review format. In addition, Chapter 5 explains how my research, described in Chapters 3 and 4, will inform accurate clinical diagnosis and therapeutic progress. Accurate differential diagnosis of patients with mutated USH genes is critical for parental counselling and initiation of rehabilitation and treatment. 6 References Audo, I., Bujakowska, K., Mohand-Saïd, S., Tronche, S., Lancelot, M.-E., Antonio, A., … Zeitz, C. (2011). A novel DFNB31 mutation associated with Usher type 2 syndrome showing variable degrees of auditory loss in a consanguineous Portuguese family. Molecular Vision, 17, 1598-606. Retrieved from http://www.pubmedcentral. nih.gov/articlerender.fcgi?artid=3123164&tool=pmcentrez&rendertype=abstract Belyantseva, I. a, Boger, E. T., Naz, S., Frolenkov, G. I., Sellers, J. R., Ahmed, Z. M., … Friedman, T. B. (2005). Myosin-XVa is required for tip localization of whirlin and differential elongation of hair-cell stereocilia. Nature Cell Biology, 7(2), 148-156. http://doi.org/10.1038/ncb1219 Besnard, T., Vaché, C., Baux, D., Larrieu, L., Abadie, C., Blanchet, C., … Roux, A.-F. (2012). Non-USH2A mutations in USH2 patients. Human Mutation, 33(3), 504-10. http://doi.org/10.1002/humu.22004 Ebermann, I., Scholl, H. P. N., Charbel Issa, P., Becirovic, E., Lamprecht, J., Jurklies, B., … Bolz, H. (2007). A novel gene for Usher syndrome type 2: Mutations in the long isoform of whirlin are associated with retinitis pigmentosa and sensorineural hearing loss. Human Genetics, 121(2), 203-211. http://doi.org/10.1007/s00439-006-0304-0 Holme, R. H., Kiernan, B. W., Brown, S. D. M., & Steel, K. P. (2002). Elongation of hair cell stereocilia is defective in the mouse mutant whirler. The Journal of Comparative Neurology, 450(1), 94-102. http://doi.org/10.1002/cne.10301 Kimberling, W. J., Hildebrand, M. S., Shearer, A. E., Jensen, M. L., Halder, J. A., Trzupek, K., … Smith, R. J. H. (2010). Frequency of Usher syndrome in two pediatric populations: Implications for genetic screening of deaf and hard of hearing children. Genetics in Medicine: Official Journal of the American College of Medical Genetics, 12(8), 512-6. http://doi.org/10.1097/GIM.0b013e3181e5afb8 Lagziel, A., Ahmed, Z. M., Schultz, J. M., Morell, R. J., Belyantseva, I. A., & Friedman, T. B. (2005). Spatiotemporal pattern and isoforms of cadherin 23 in wild type and waltzer mice during inner ear hair cell development. Developmental Biology, 280(2), 295-306. http://doi.org/10.1016/j.ydbio.2005.01.015 Magliulo, G., Iannella, G., Gagliardi, S., Plateroti, R., Plateroti, P., Iozzo, N., & Vingolo, E. M. (2015). Usher's syndrome: Evaluation of the vestibular system with Cervical and Ocular Vestibular Evoked Myogenic Potentials and the Video Head Impulse Test. Otology & Neurotology, 36(2), 1421-1427. http://doi.org/10.1097/MAO.0000000000000613 Mathur, P. D., Vijayakumar, S., Vashist, D., Jones, S. M., Jones, T. A., & Yang, J. (2015). A study of whirlin isoforms in the mouse vestibular system suggests potential vestibular dysfunction in DFNB31 -deficient patients. Human Molecular Genetics, (September), ddv403. http://doi.org/10.1093/hmg/ddv403 7 Mathur, P. D., Zou, J., Zheng, T., Almishaal, A., Wang, Y., Chen, Q., … Yang, J. (2015). Distinct expression and function of whirlin isoforms in the inner ear and retina: an insight into pathogenesis of USH2D and DFNB31. Human Molecular Genetics, 24(21), 6213-28. http://doi.org/10.1093/hmg/ddv339 Mathur, P., & Yang, J. (2015). Usher syndrome: Hearing loss, retinal degeneration and associated abnormalities. Biochimica et Biophysica Acta, 1852(3), 406-420. http://doi.org/10.1016/j.bbadis.2014.11.020 Mburu, P., Mustapha, M., Varela, A., Weil, D., El-Amraoui, A., Holme, R. H., … Brown, S. D. M. (2003). Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, cause deafness in the whirler mouse and families with DFNB31. Nature Genetics, 34(4), 421-8. http://doi.org/10.1038/ng1208 Nishiguchi, K. M., Tearle, R. G., Liu, Y. P., & et al. (2013). Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene. Proceedings of the National Academy of Sciences of the United States of America, 110(40), 16139-44. http://doi.org/10.1073/pnas.1308243110 Schultz, J. M., Bhatti, R., Madeo, A. C., Turriff, A., Muskett, J. a, Zalewski, C. K., … Friedman, T. B. (2011). Allelic hierarchy of CDH23 mutations causing nonsyndromic deafness DFNB12 or Usher syndrome USH1D in compound heterozygotes. Journal of Medical Genetics, 48(11), 767-75. http://doi.org/10.1136/jmedgenet-2011-100262 Schultz, J. M., Yang, Y., Caride, A. J., Filoteo, A. G., Penheiter, A. R., Lagziel, A., … Griffith, A. J. (2005). Modification of human hearing loss by plasma-membrane calcium pump PMCA2. The New England Journal of Medicine, 352(15), 1557-64. http://doi.org/10.1056/NEJMoa043899 Tlili, A., Charfedine, I., Lahmar, I., Benzina, Z., Mohamed, B. A., Weil, D., … Ayadi, H. (2005). Identification of a novel frameshift mutation in the DFNB31/WHRN gene in a Tunisian consanguineous family with hereditary non-syndromic recessive hearing loss. Human Mutation, 25(5), 503. http://doi.org/10.1002/humu.9333 Wright, R. N., Hong, D.-H., & Perkins, B. (2012). RpgrORF15 connects to the usher protein network through direct interactions with multiple whirlin isoforms. Investigative Ophthalmology & Visual Science, 53(3), 1519-29. http://doi.org/10.1167/iovs.11-8845 Yang, J., Liu, X., Zhao, Y., Adamian, M., Pawlyk, B., Sun, X., … Li, T. (2010). Ablation of whirlin long isoform disrupts the USH2 protein complex and causes vision and hearing loss. PLoS Genetics, 6(5), e1000955. http://doi.org/10.1371/journal.pgen.1000955 CHAPTER 2 USHER SYNDROME: HEARING LOSS, RETINAL DEGENERATION AND ASSOCIATED ABNORMALITIES Jun Yang wrote this review. My role was to read more literature and find out additional information that need to be incorporated. In addition, I generated figures for this review and helped revise each version of the manuscript. This is a precopyedited, author-produced PDF of an article accepted for publication in BBA Molecular Basis of Disease following peer review. Reprinted from Pranav Mathur and Jun Yang (2014) Usher syndrome: hearing loss, retinal degeneration and associated abnormalities. BBA - Molecular Basis of Disease. 1852(3):406-420, with permission from Elsevier. License obtained through RightsLink. The version of this record mentioned above is available online at: http://www.sciencedirect.com/science/article/pii/S0925443914003627 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 CHAPTER 3 DISTINCT EXPRESSION AND FUNCTION OF WHIRLIN ISOFORMS IN THE INNER EAR AND RETINA: AN INSIGHT INTO PATHOGENESIS OF USH2D AND DFNB31 Jun Yang designed the original study. My role was to take over this project when I joined the lab. I did a majority of the experiments in the manuscript. I analyzed the data, generated figures and wrote the methods section for my experiments. Jun Yang wrote the manuscript and I helped revise each version of the manuscript. This is a PDF of an article accepted for publication in Human Molecular Genetics following peer review. Reprinted from Pranav Dinesh Mathur, Junhuang Zou, Tihua Zheng, Ali Almishaal, Yong Wang, Qian Chen, Le Wang, Deepti Vashist, Steve Brown, Albert Park, Jun Yang. (2015) Distinct Expressions and Functions of Whirlin Isoforms in the Inner Ear and Retina: an Insight into Pathogenesis of USH2D and DFNB31. Human Mol Gen. 24(21):6213-28., with permission from Oxford University Press and Elsevier. License obtained through RightsLink. The version of this record mentioned above is available online at: http://hmg.oxfordjournals.org/content/24/21/6213.long 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 CHAPTER 4 A STUDY OF WHIRLIN ISOFORMS IN THE MOUSE VESTIBULAR SYSTEM SUGGESTS POTENTIAL VESTIBULAR DYSFUNCTION IN DFNB31-DEFICIENT PATIENTS Jun Yang and I designed the study. I did all the experiments and analyzed the data, except vestibular recordings part of the manuscript. I generated figures and wrote the methods section for my experiments. Jun Yang wrote the manuscript. Timothy Jones wrote the vestibular recordings part of the manuscript. I helped revise each version of the manuscript. This is a precopyedited, author-produced PDF of an article accepted for publication in Human Molecular Genetics following peer review. Reprinted from Pranav Dinesh Mathur, Sarath Vijayakumar, Deepti Vashist, Sherri M. Jones, Timothy A. Jones, Jun Yang. (2015) A Study of Whirlin Isoforms in the Mouse Vestibular System Suggests Potential Vestibular Dysfunction in DFNB31-Defecient Patients. Human Mol Gen 24 (24): 7017-30, with permission from Oxford University Press and Elsevier. License obtained through RightsLink. The version of this record mentioned above is available online at: http://hmg.oxfordjournals.org/content/24/24/7017.long 42 43 44 45 46 47 48 49 50 51 52 53 54 55 CHAPTER 5 DISCUSSION 57 Introduction Usher Syndrome (USH) is one of the major causes of deaf-blindness worldwide, with an estimated frequency of 1:6000 (Kimberling et al., 2010). USH is an autosomal recessive genetic disease and there is no cure to date. More than 16 loci have been associated with USH. In addition, it is predicted that there are several unidentified loci/genes associated with USH yet to be discovered (Vozzi et al., 2011). Based on the severity of the disease, USH has been subdivided into three categories. USH1 is the most severe, wherein patients have congenital hearing loss, early onset retinal degeneration, and vestibular disorder. USH2 is the most prevalent, wherein patients have moderate congenital hearing loss and retinal degeneration starting as early as adolescence. USH3 patients have progressive hearing loss, retinal degeneration, and variable vestibular deficits. Sensorineural hearing loss and vestibular deficit in USH patients occur primarily due to defective hair cells in the inner ear. In the cochlea, the coil-shaped organ of Corti is positioned on top of the basilar membrane, which resonates in response to vibrations induced by sound waves. The organ of Corti has three rows of outer hair cells (OHCs) and one row of inner hair cells (IHCs). Each sensory hair cell contains a stereociliary bundle composed of actin-based stereocilia, derived from microvilli, and a transient microtubule-based kinocilium. OHC stereocilia tips are inserted into the overlying tectorial membrane. Therefore, vibration of the basilar membrane develops a shear that deflects and stimulates the OHC stereocilia. The resulting mechanotransduction response induces longitudinal oscillation of the OHC cell body and thus amplifies the vibration of the basilar membrane. The amplified signals subsequently deflect the stereociliary bundle 58 of inner hair cells (IHCs) that synapse with the afferent spiral ganglion auditory nerve fibers and send the signal to the brain. Stereocilia in the vestibular hair cells (VHCs) sense movement of the head and they function in a similar manner to those of cochlear hair cells. The specialized stereocilia in the hair cells receive sound/head-movement inputs, which makes the organization, arrangement and development of these stereocilia crucial for normal hearing and balance. A developing hair cell has several stereociliary links, including kinociliary links, shaft links, tip links, and ankle links, that connect one stereocilium to its neighboring stereocilia (Fig. 5.1A). Tip links connect the tips of shorter stereocilia to the lateral wall of its neighboring taller stereocilia and are essential for ion channel opening during mechanotransduction. Kinociliary links connect the kinocilium with its neighboring stereocilia, while shaft links connect adjacent stereocilia to each other. These links are thought to be required for bundle development and maintenance (Goodyear, Marcotti, Kros, & Richardson, 2005). Ankle links are present at the stereociliary base transiently (postnatal day (P) 2 - 12) in the organ of Corti hair cells, and permanently in the VHCs. Ankle links are required for the organization of a developing organ of Corti hair cell bundle and are also thought to have a signaling function (Michalski et al., 2007). USH proteins are localized at different regions in the hair cell stereocilia, and are required directly or indirectly for receiving and processing sound/balance inputs. Retinal degeneration in USH patients occurs in the form of retinitis pigmentosa (RP), with night-blindness and tunnel vision as typical symptoms. These symptoms arise as a result of degeneration of rod photoreceptors in the retina. Cone photoreceptors degenerate in advanced RP stages and could lead to complete blindness. Photoreceptors 59 Figure 5.1: Structure of a rod photoreceptor and a developing inner ear hair cell. (A) A developing inner ear hair cell (P2-12) has ankle links present at the base of stereocilia. Other shaft links are also present. Tip links connect neighboring short stereocilia in the adjacent row and are required for mechanotransduction. (B) A rod photoreceptor has an outer segment with discs that contain rhodopsin. Rhodopsin receives light in the form of photons. The photoreceptor inner segment contains mainly organelles required for metabolism. The periciliary membrane complex (in blue) is located above the basal body in the apical region of inner segment, close to the outer segment. The cell body contains the photoreceptor nucleus, while the synaptic region contains ribbon synapses. 60 61 are special sensory neurons that sense light. Photons are received by rhodopsin present on the outer segment photoreceptors discs. The outer segment lacks protein production machinery and relies on the inner segment for its supply of proteins. The periciliary membrane complex (PMC) is located at the apex of the inner segment above the basal body and is supposedly involved in transporting protein cargos from the inner to the outer segment (Fig. 5.1B) (Yang et al., 2010a). USH proteins are localized at different locations within photoreceptors and are likely required for photoreceptor maintenance and function. Despite many years of research on USH genes, the molecular mechanisms by which their disruption causes hearing loss remains unexplained. Specifically, for most USH genes, mutations in different regions of the same genes can cause either USH or other discrete diseases (Table 5.1). For example, mutations in a recently identified USH1 gene, CIB2, may either lead to USH1J, a subtype of USH1, or DFNB48, a subtype of DFNB (nonsyndromic autosomal recessive deafness) (Riazuddin et al., 2012). Understanding molecular mechanisms underlying this variability will be essential for not only early and accurate diagnosis but also developing therapies. To address this issue, I studied an USH2 gene, DFNB31, that produces a protein called whirlin. Mutations in DFNB31 cause variable disease manifestations Mutations in DFNB31 lead to either USH2D, a subtype of USH2, or DFNB31, a subtype of DFNB. USH2D patients have a moderate level of hearing loss and retinitis pigmentosa (RP), while DFNB31 patients have profound sensorineural hearing loss and normal vision. The DFNB31 gene was identified as one of the causes of DFNB in 2003, 62 Table 5.1: USH genes other than DFNB31 showing different disease manifestations. Gene name Diseases associated USH subtype Other diseases MYO7A USH1B USH1C USH1C DFNB2, DFNA11, atypical USH DFNB18 CDH23 USH1D DFNB12 PCDH15 USH1F DFNB23 CIB2 USH2A USH1J USH2A DFNB48 Nonsyndromic RP ADGRV1 USH2C Febrile and afebrile seizures PDZD7 Digenic USH DFNB Total number References of protein isoforms found 1 (Liu et al. 1998; Liu et al. 1999; Liu et al. 1997; Riazuddin et al. 2008) 3 (Zubair M. Ahmed et al., 2002; Ouyang et al., 2002; Reiners et al., 2003; Verpy et al., 2000) 3 (Bork et al., 2001; Lagziel et al., 2005, 2009; Schultz et al., 2005, 2011) 4 (Z. M. Ahmed et al., 2006; Zubair M. Ahmed et al., 2003; Doucette et al., 2009) 3 (Riazuddin et al., 2012) 2 (Eudy, 1998; Rivolta, Sweklo, Berson, & Dryja, 2000; van Wijk et al., 2004) 1 (Nakayama et al., 2002; Randy McMillan, Kayes-Wandover, Richardson, & White, 2002; Skradski et al., 2001; Weston et al., 2004; Yagi et al., 2005) 3 (Ebermann et al., 2010; Schneider et al., 2009) 63 when the DFNB31 disease was studied in patients affected with prelingual sensorineural hearing impairment (Mburu et al., 2003; Mustapha et al., 2002). The affected individuals had a cysteine to threonine substitution in the 10th exon of DFNB31, changing the arginine codon to a stop codon (Arg778X) (Mburu et al., 2003). Another DFNB31 mutation in the 11th exon (Gly808AspfsX11) also leads to profound deafness (Tlili et al., 2005). On the other hand, patients with compound heterozygous nonsense (Gln103X) and splice site (Val280Met) mutations in the 1st and 2nd exons of DFNB31, respectively, have USH2D, with moderate congenital hearing loss and RP that begins by the age of 20 years (Ebermann et al., 2007). Patients with either a homozygous one base-pair deletion (Pro246HisfsX13) or compound hetereozygous mutations (Pro246HisfsX13; Tyr228fs) in DFNB31 also have USH2D symptoms (Audo et al., 2011; Besnard et al., 2012). Recently, a patient with a nonsense mutation (Gln54X) in DFNB31 exon1 showed a relatively late onset of RP, but was unavailable for hearing tests (Nishiguchi et al., 2013). None of these aforementioned patients self-reported any vestibular deficits. In summary, the clinical reports suggest that mutations in the N-terminal region of DFNB31 lead to USH2D, while mutations toward the C-terminal end cause DFNB31 (Fig. 5.2). Dfnb31 mouse models recapitulate USH2D and DFNB31 diseases To understand the aforementioned genotype-phenotype correlation in DFNB31deficient patients, I utilized mouse models with a series of mutations in the DFNB31 ortholog, Dfnb31, to mimic various human DFNB31mutations that lead to USH2D and DFNB31. For example, a Whrntm1Tili/Whrntm1Tili (MGI:4462398) mouse, hereafter referred to as Dfnb31neo/neo, was generated by replacing the 5'-region of Dfnb31 exon 1 64 Figure 5.2: Location and disease manifestation of human whirlin mutations. FL-whirlin mRNA is translated into a protein with three PDZ domains, a proline rich (PR) domain and a PDZ binding motif (PBM). Asterisks in red denote mutations in the N-terminal region that cause USH2D. Asterisks in blue denote C-terminal mutations that cause DFNB31. 65 66 with a Neor cassette to mimic mutations found in USH2D patients (Yang et al., 2010b) (Fig 5.3B). This mouse shows moderate hearing loss and retinal degeneration similar to those observed in USH2D patients. Another Dfnb31 mutant mouse Whrnwi/wi (MGI:1857090), hereafter referred to as Dfnb31wi/wi, has a spontaneous deletion between exons 6-9 (Fig 5.3C). This 592bp deletion in the transcript creates a frameshift leading to a premature translation termination upstream of the 3rd PDZ domain. The Dfnb31wi/wi mice are profoundly deaf and show no retinal degeneration, resembling the symptoms found in DFNB31 patients (Holme, Kiernan, Brown, & Steel, 2002; Mburu et al., 2003; Yang et al., 2010a). Therefore, the Dfnb31wi/wi mutation represents C-terminal region mutations found in DFNB31 patients. In addition, I also studied the inner ear of Dfnb31tm1a(EUCOMM)wtsi (referred to as Dfnb31tm1a/tm1a hereafter) mice (MGI:4432119) for the first time. This mouse carries a gene trap insertion composed of a targeting cassette, placed between exon 3 and exon 4. Since this mouse has a mutation toward the 3' region of Dfnb31, it mimics the mutation region in USH2D patients. I also analyzed Dfnb31wi/wi+BAC transgenic mice, generated by Mburu et al., (2003). To generate this mouse, a Bacterial Artificial Chromosome (BAC), containing the Dfnb31 gDNA from exons 4-12, was inserted into the genome of Dfnb31wi/wi mice (Fig 5.3E). It was previously thought that this BAC insertion completely recued the hearing loss in Dfnb31wi/wi mice (Mburu et al., 2003). I utilized these Dfnb31 mouse models to study whirlin expression in the inner ear and retina tissues, and to understand the pathogenesis of USH2D and DFNB31. 67 Figure 5.3: Dfnb31 mRNA variants in WT, Dfnb31neo/neo and Dfnb31wi/wi mice. (A) FL-, N-and C-terminal whirlin mRNA variants found in the WT mice. (B) Of these, only the C-terminal variant is found in the Dfnb31neo/neo mice, which has a Neor cassette (red box) in its exon1 3' region. (C) A deletion from exon 6 to 9 (red box) truncates both FL-whirlin mRNA and C-whirlin mRNA in Dfnb31wi/wi mice. Blue color denotes 3' and 5'-UTR; green color denotes CDS of whirlin mRNA; vertical yellow color line indicates a premature stop in truncated Dfnb31wi/wi mRNA fragments. (D) A targeting cassette (light blue box) in intron 3 of Dfnb31tm1a/tm1a gDNA disrupts the FL transcript. However, it is expected that C-terminal whirlin mRNA transcript will remain unaffected. L, loxP site. (E) Dfnb31wi/wi+BAC mice is also expected to express C-terminal whirlin mRNA transcripts in addition to transcripts expressed by Dfnb31wi/wi mice. 68 69 Whirlin has different spatiotemporal expression patterns in the inner ear and retina The presence of two different predicted promoter regions and alternative splicing in the DFNB31/Dfnb31 gene yields several mRNA transcripts in humans and mice (Belyantseva et al., 2005; Mburu et al., 2003; Wright, Hong, & Perkins, 2012), and these mRNA transcripts encode whirlin protein splice isoforms. My research identified and compared the Dfnb31 mRNA transcripts expressed in the organ of Corti, vestibular and retinal tissues of wildtype (WT), Dfnb31neo/neo and Dfnb31wi/wi mice. My results indicate that WT mice have transcripts encoding full-length (FL)-, N- and C-terminal whirlin proteins in the retina, but only transcripts encoding FL- and C-terminal proteins in the inner ear (Table 5.2). Dfnb31neo/neo mice lack all Dfnb31 transcripts except the transcript encoding C-terminal whirlin protein in the inner ear. Dfnb31wi/wi mice have truncated FLand C-whirlin mRNA transcripts in the inner ear and retina (Table 5.2; Fig. 5.3). FLtranscripts are expected to yield a FL-whirlin protein isoform having three PDZ domains, a PDZ binding motif (PBM) and a Proline Rich (PR) region. Transcripts encoding Cterminal whirlin have start sites in either exon 1 or exon 6, yet C-whirlin protein isoforms have only the PR, PDZ3, and PBM domains (Belyantseva et al., 2005; Mburu et al., 2003; Yang et al., 2010a). Finally, transcripts encoding N-terminal whirlin yield Nwhirlin isoforms that contain either PDZ1 or both PDZ1 and PDZ2 domains (Wright et al., 2012). Despite several studies on whirlin localization, a clear understanding of the localization of different whirlin isoforms was missing. For example, in one study, whirlin was shown to localize only at the stereociliary tips (Kikkawa et al., 2005). A different 70 Table 5.2: Different Dfnb31 mRNA variants present in the retina and inner ear of WT, Dfnb31neo/neo and Dfnb31wi/wi mice. Mouse Organs models WT Retina Cochlea Vestibular system Transcripts encoding Transcripts encoding Transcripts encoding FL- whirlin FL- whirlin FL- whirlin C- whirlin C- whirlin C- whirlin Transcript encoding Transcript encoding C- whirlin C- whirlin Transcripts encoding Transcripts encoding Transcripts encoding truncated N- whirlin truncated N- whirlin truncated N- whirlin truncated C- whirlin. truncated C- whirlin truncated C- whirlin N- whirlin Dfnb31neo/neo - Dfnb31wi/wi 71 study reported presence of whirlin at the stereociliary tips and bases (Delprat et al., 2005). A third study found whirlin localized at the OHC synaptic regions (van Wijk et al., 2006a). In my study, however, I could not see whirlin localization at the synapses. To confirm the presence of whirlin at the synapses, co-localization of whirlin with a known synaptic marker and use of the Dfnb31 mutant mice as a negative control are essential. Moreover, this variation could be due to the use of different antibodies and sample fixation procedures. To decipher the localization of different whirlin isoforms in the inner ear and retina, I generated antibodies specific to the different whirlin regions and utilized Dfnb31 mutants as controls in comparison to WT mice. I found that in the WT inner ear, both FL- and C-whirlin isoforms localize at the IHC and VHC stereociliary tips, whereas C-whirlin is the only isoform found in the OHC stereociliary tips. The only whirlin isoform found at the WT stereociliary base is FL-whirlin and it is present in all HC types (Fig. 5.4A,B) (Mathur, Vijayakumar, et al., 2015; Mathur, Zou, et al., 2015). In Dfnb31neo/neo mice, the FL-whirlin isoform at the stereociliary base is missing, but the Cwhirlin isoform remains localized at the stereociliary tips of OHCs, IHCs and VHCs. (Fig. 5.4D,E). Localization of whirlin isoforms in Dfnb31tm1a/tm1a and majority of Dfnb31wi/wi+BAC hair cells were similar to that of Dfnb31neo/neo hair cells (Fig 5.4D, E and J-L). Dfnb31wi/wi mice did not show any whirlin isoforms either at the stereociliary tips or at the stereociliary base of their inner ear hair cells (Fig 5.4G,H), suggesting that the truncated FL- and C-terminal whirlin Dfnb31 transcripts are probably degraded by nonsense-mediated mRNA decay (Mathur, Vijayakumar, et al., 2015; Mathur, Zou, et al., 2015). In summary, my findings show that a 3'-region mutation in Dfnb31neo/neo and Dfnb31tm1a/tm1a mice spares the C-terminal whirlin isoform, while a 5'-region mutation in 72 Figure 5.4: Localization of whirlin isoforms (green) in the inner ear and retina. (A) FL-whirlin is localized at the stereociliary base of developing (P2-P12) inner hair cells (IHCs) and outer hair cells (OHCs) in the organ of Corti. IHC stereociliary tips contain both FL- and C-whirlins, while OHC stereociliary tips contain only C-whirlin. (B) Localization of whirlin in vestibular hair cells (VHCs) is similar to that of IHCs as shown in A. (C) FL- and N-whirlins are localized at the periciliary membrane complex (PMC) of the WT photoreceptor cell. (D, E) Only C-whirlin is localized at the stereociliary tips of OHC, IHC and VHC in Dfnb31neo/neo mice. (F) No whirlin localizations were detected in Dfnb31neo/neo retinas. (G, H) Dfnb31wi/wi inner ear hair cells do not show any whirlin localizations. (I) Truncated N-whirlin fragment in Dfnb31wi/wi retinas localizes to the PMC. (J,K) 96% of Dfnb31wi/wi +BAC IHCs and only 33% of of Dfnb31wi/wi +BAC OHCs show localization of C-whirlin at their stereociliary tips. (L) Similar to Dfnb31neo/neo IHCs and OHCs, Dfnb31tm1a/tm1a show localization of only Cwhirlin at their IHC and OHC stereociliary tips. 73 74 Dfnb31wi/wi mice leads to loss of both FL- and C-whirlin isoforms in the inner ear hair cells (Fig 5.3 and Fig 5.4). Whirlin was previously found at the PMC in photoreceptors (Yang et al., 2010a); however, isoform-specific information on whirlin in the PMC was unknown. I found that the FL- and likely the N-whirlin isoforms are expressed and localize to the PMC in the WT (Fig. 5.4C). Dfnb31neo/neo mice did not show any whirlin isoforms in the retina (Fig 5.4F). In Dfnb31wi/wi mice, a truncated N-whirlin fragment (Fig. 5.3C) localized normally at the PMC (Fig 5.4I), although the expression level was low. This suggests that the truncated FL-whirlin mRNA transcript in Dfnb31wi/wi is translated into truncated Nwhirlin protein fragment. Together, these findings indicate that whirlin has multiple spatiotemporal expression patterns in the inner ear and retina. Whirlin forms different multiprotein complexes in the inner ear and retina Whirlin is a scaffold protein with three PDZ domains, which aid in assembling several large multiprotein complexes. Different whirlin protein isoforms likely form different complexes at unique subcellular locations in the inner ear hair cells and at the PMC of the photoreceptors. To understand the function of different whirlin isoforms and the mechanism underlying different phenotypes observed in Dfnb31 mutants, it is important to identify proteins that interact with whirlin and to determine how whirlin isoforms mediate these interactions. Proteins interacting with whirlin at the stereociliary tips in hair cells. Actin regulatory protein EPS8, actin motor protein myosin-XVa and a MAGUK scaffold 75 protein p55, bind to and colocalize with whirlin at the stereociliary tips to maintain the stereocilia length (Fig 5.5A) (Manor et al., 2011; Mburu et al., 2006). Loss of any of these proteins leads to short stereociliary length, as observed in Eps8 knockout (Eps8-/-), shaker2 (Myo15ash2/sh2), Dfnb31wi/wi and Dfnb31neo/neo mice (Holme et al., 2002; Mathur, Zou, et al., 2015; Mustapha et al., 2007; Zampini et al., 2011). The N-terminal region of EPS8 binds to the PDZ1, PDZ2 and PR domains of whirlin, while its C-terminal region binds to myosin-XVa (Manor et al., 2011). The C-terminal PDZ-binding motif of myosin-XVa interacts with the PDZ3 domain of whirlin and delivers whirlin to the tips of stereocilia (Belyantseva et al., 2005). By contrast, another study shows that whirlin PDZ1 and PDZ2 domains interact with the MyTH4-FERM domain of myosin-XVa and the whirlin PDZ3 domain binds to the SH3-MyTH4 domain of myosin-XVa (Delprat et al., 2005). My findings in Chapter 3 suggest that only C-terminal whirlin region is required for the interaction between myosin-XVa and whirlin, and that both whirlin N- and Cterminal regions are involved in the interaction between EPS8 and whirlin (Mathur, Zou, et al., 2015). The GUK domain of p55 interacts with whirlin PDZ3 (Mburu et al., 2006, 2010; Yang, Le, Song, & Sokolov, 2012). In addition, 4.1R, one of the four 4.1 protein isoforms, and CASK, other MAGUK proteins similar to P55, are shown to localize at the OHC stereociliary tips. Localization of P55 and 4.1R is sporadic at E17.5 (embryonic day 17.5) OHC stereociliary tips and their signals get completely ablated at P5 in Dfnb31wi/wi mice (Jing-Ping et al., 2005; Mburu et al., 2006). Because only C- whirlin is localized at the stereociliary tips of OHCs (Mathur et al. 2015), it is likely that 4.1R and CASK also interact with the PDZ3 of C-whirlin. Interaction of CASK with the whirlin C-terminal region is thought to be required for the actin cytoskeleton reorganization in neurons 76 Figure 5.5: Interacting partners of whirlin at various subcellular regions. (A) Whirlin interacts with EPS8, MYOXVA, P55, CASK and 4.1R proteins at the stereociliary tips of the inner ear hair cells. (B) Whirlin interacts with ADGRV1, usherin and PDZD7 at the ankle-link complex region of the stereociliary bases in the inner ear hair cells. (C) At the periciliary membrane complex of the retina, whirlin interacts with ADGRV1 and usherin. A fourth protein (indicated as ‘?'), required to form a stable USH2 protein complex is still unkown. 77 78 (Yap et al., 2003). However, the exact function of interactions of whirlin with P55, CASK and 4.1R in hair cells remains unknown. Proteins interacting with whirlin at the stereociliary base in hair cells. ADGRV1, usherin and PDZD7 bind to one another and to whirlin to form the ankle-link complex (ALC) toward the stereociliary base (Fig. 5.5B). This complex is required for stereociliary bundle organization. Several studies have confirmed the existence and function of this USH2 protein complex in vivo (Grati et al., 2012; Michalski et al., 2007; van Wijk et al., 2006b; Yang et al., 2010b; Zou et al., 2015). PDZ1 of whirlin binds to the PDZ2 of PDZD7 and the cytoplasmic C-termini of usherin and of ADGRV1. The whirlin PDZ2 can bind to usherin, but not ADGRV1, C-terminus (Chen, Zou, Shen, Zhang, & Yang, 2014). I found that loss of whirlin at the stereociliary base leads to partial mislocalization of ADGRV1 to the stereociliary tips, while usherin and PDZD7 remain relatively unaffected (Zou et al., 2015). ADGRV1 along with other proteins forms and stabilizes ankle links of the developing hair cell stereocilia. Ankle links are essential for stereociliary organization and the typical ‘V'-shape stereociliary bundle. (McGee et al., 2006; Michalski et al., 2007). Together, these findings suggest that loss of whirlin at the stereociliary base cause partial destabilization of ankle links. ADGRV1 is also implicated in G-protein-coupled receptor (GPCR) signaling pathway (Hu et al., 2014; Weston, Luijendijk, Humphrey, Möller, & Kimberling, 2004). Therefore, it is likely that formation of the ALC is required for efficient and proper GPCR signaling. Whether or not loss of whirlin from the ALC affects GPCR signaling remains to be studied. Proteins interacting with whirlin at the PMC in photoreceptors. ADGRV1 and usherin are also colocalized with whirlin at the PMC in photoreceptors (Fig. 5.5C) (Yang 79 et al., 2010a). Our in vitro studies demonstrated that formation of a stable USH2 complex requires a fourth protein apart from whirlin, usherin and ADGRV1 (Chen et al., 2014). Interestingly, PDZD7, the fourth USH2 protein required for stable USH2 complex formation in the inner ear ALC, was not found in the retina (Zou et al., 2014), which suggests that a fourth unknown protein exists in the retina for stabilization of the USH2 complex. The RPGR (Retinitis Pigmentosa GTPase regulator) isoform RPGRorf15, which is preferentially expressed in the retina, colocalizes and interacts with whirlin at the connecting cilium of photoreceptors. Specifically, PDZ1 and PDZ2 of whirlin were shown to interact with the C-terminal region of RPGRorf15 (Wright et al., 2012). Furthermore, RPGR is thought to be essential for the protein trafficking and mutations in RPGR lead to RP (Hong et al., 2000). However, my localization studies on whirlin suggest that whirlin is localized at the PMC and not at the connecting cilium. Therefore, the functional significance of the whirlin-RPGRorf15 interaction remains unexplored, and whether RPGRorf15 interacts with other USH2 proteins present at the PMC is yet to be tested. Further studies are required to identify all the components of the USH2 protein complex at the PMC in the retina. Correlation of genotypes with phenotypes conveys the function of different whirlin isoforms Phenotypes caused by the absence of whirlin isoforms at a specific subcellular location in the retina or the inner ear convey information about the function of the whirlin isoforms. For example, whirlin isoforms are completely missing in Dfnb31neo/neo mouse retinas. This causes mislocalization of ADGRV1 and usherin at the PMC, and is likely 80 the cause for retinal degeneration in Dfnb31neo/neo mice. In contrast, Dfnb31wi/wi retinas retain the truncated N-terminal whirlin fragment and this partially rescues the normal localizations of ADGRV1 and usherin to the PMC (Mathur, Zou, et al., 2015). The partial rescue of the USH2 complex at the PMC is likely sufficient to prevent or delay retinal degeneration in Dfnb31wi/wi mice. A 2014 study, however, reported that Dfnb31wi/wi mice had delayed transducin translocation from the outer segment to the inner segment upon light stimulation and that light exposure can induce photoreceptor degeneration (Tian et al., 2014). This study, however, did not include the Dfnb31neo/neo mice. It would, therefore, be interesting to see whether Dfnb31neo/neo mice have a more severe transducin translocation and photoreceptor degeneration phenotype compared to Dfnb31wi/wi mice upon light stimulation. Localization of both FL- and C-whirlins at the stereociliary tips is essential for IHC stereociliary elongation, whereas only C-whirlin is required for OHC stereociliary elongation. Loss of FL-whirlin but normal C-whirlin localization at the Dfnb31neo/neo IHC stereociliary tips leads to relatively shorter IHC stereocilia in Dfnb31neo/neo mice compared to WT. Dfnb31neo/neo OHCs, like WT OHCs, have only the C-whirlin isoform localized at their stereociliary tips and, as a result, Dfnb31neo/neo OHCs have normal stereocilia lengths (Mathur, Zou, et al., 2015). Dfnb31wi/wi mice lack both FL- and Cwhirlins at their IHC and OHC stereociliary tips and, therefore, have significantly short IHC and OHC stereocilia compared to Dfnb31neo/neo and WT mice (Fig 5.4G). As a result, Dfnb31wi/wi mice have a more severe hearing loss compared with Dfnb31neo/neo, Dfnb31tm1a/tm1a and Dfnb31wi/wi+BAC mice that harbor C-whirlin isoform at their hair cell stereociliary tips (Fig. 5.4D, J-L) (Mathur, Zou, et al., 2015). At the stereociliary base, all 81 Dfnb31 mutants lack the FL-whirlin isoform. OHC stereocilia disorganization is similar in Dfnb31neo/neo, Dfnb31tm1a/tm1a, Dfnb31wi/wi+BAC and Dfnb31wi/wi mice, and these mice have similar distortion products of otoacoustic emissions (DPOAE) responses, which specifically indicates the function of OHCs (Mathur, Zou, et al., 2015). This observation suggests that FL-whirlin is required at the OHC ALC for the stereociliary bundle organization during development. The function of FL-whirlin at the stereociliary base needs to be further investigated. Localization of whirlin isoforms in VHCs is the same as that in IHCs, and Dfnb31neo/neo VHC stereocilia are taller than Dfnb31wi/wi but shorter than WT VHC stereocilia (Fig. 5.4E,H) (Mathur, Vijayakumar, et al., 2015). This suggests that in terms of stereociliary elongation, the function of FL- and C-whirlins at stereociliary tips in VHCs is similar to that in IHCs. Consistently, Dfnb31wi/wi mice have overt vestibular behaviors (Holme et al., 2002), whereas Dfnb31neo/neo mice do not (Mathur, Vijayakumar, et al., 2015). However, both Dfnb31neo/neo and Dfnb31wi/wi mice show similar severe to profound loss of linear vestibular evoked potential (VsEP) responses. One reason for this discrepancy could be that the central nervous system (CNS) compensates in Dfnb31neo/neo mice, owing to some uncharacterized functional role of C-whirlin in the murine CNS. Another possibility could be the presence of tall VHC stereocilia in the peripheral region of Dfnb31neo/neo cristae. Since linear VsEPs test only the otolith organs, measurements of angular VsEP measurements are required to test this hypothesis. 82 Whirlin isoforms have functions outside the inner ear and retina My studies on Dfnb31 mutant VHCs suggest the possibility that whirlin functions in the CNS. Consistent with this, several recent studies emphasize the expression and role of whirlin in the CNS. Loss of whirlin disrupts the axonal domain organization and causes paranodal abnormalities during development in both central and peripheral nervous systems (Green, Yang, Grati, Kachar, & Bhat, 2013). Another study revealed that Dfnb31tm1a/tm1a mice have elevated nociceptive thresholds (White et al., 2013). Subsequently, it was found that whirlin is selectively expressed in proprioceptive sensory neurons, where it functions in afferent firing in response to touch (de Nooij et al., 2015). Recently, both C-terminal and FL-whirlin isoforms were found to associate with and increase the stability and clustering of TRPV1, a thermosensory channel, at the cell membrane of nociceptive neurons (Ciardo et al., 2016). Dysc (dyschronic), the closest homolog of whirlin, is required in Drosophila for locomotor behavior and circadian rhythm (Jepson et al., 2012). Dysc is expressed at the presynaptic region and is essential in synaptic development and output. Dysc mutants show increased evoked and spontaneous synaptic transmission (Jepson et al., 2014). Together, these findings suggest that whirlin isoforms have multiple functions in the inner ear, retina, and nervous system, and that different mutations in Dfnb31 may affect some isoforms while sparing other isoforms. Unaffected isoforms localize and function normally in their respective tissues. Disruption of different whirlin isoforms is, therefore, a likely explanation for different disease manifestations and genotypephenotype correlation. Furthermore, expression and function of whirlin in the murine nervous system suggest that patients with mutations in DFNB31 might have 83 abnormalities in the nervous system. Previous attempts to rescue Dfnb31 mutant phenotypes were only partially successful Gene therapy appears to be one of the most promising therapeutic approaches in human medicine, and several attempts have been made to rescue the phenotypes of whirlin mutant mice using gene replacement therapy. Adeno-Associated Virus (AAV) carrying FL-whirlin cDNA was able to successfully restore the USH2 complex at the PMC in Dfnb31neo/neo retinas (Zou et al., 2011). Dfnb31neo/neo mice completely lack whirlin in their retina and the N-terminal region of whirlin is sufficient to stabilize and restore the normal localization of usherin and ADGRV1 in the retina (Chen et al., 2014; Mathur, Zou, et al., 2015), suggesting that AAV-carrying N-whirlin may also be able to rescue normal localizations of usherin and ADGRV1, and potentially to prevent retinal degeneration in Dfnb31neo/neo mice. Dfnb31 restoration studies in the inner ear (Belyantseva et al., 2005; Chien et al., 2015; Mburu et al., 2003) have been unsuccessful in completely rescuing hearing or stereociliary bundle morphology. The Dfnb31wi/wi+BAC mouse does not circle and has longer stereocilia and better hearing compared with Dfnb31wi/wi mouse. However, Dfnb31wi/wi+BAC mice still have the abnormal ‘U'-shaped OHC stereociliary arrangement and impaired hearing function comparable to Dfnb31neo/neo mice (Mathur, Zou, et al., 2015; Mburu et al., 2003). This phenotype probably results from the absence of FL-whirlin, which is required for ALC stabilization and IHC stereociliary elongation. Gene gun transfection of VHCs with GFP-FL-whirlin cDNA was able to elongate 84 stereocilia and rescue the missing staircase pattern in Dfnb31wi/wi mice to some extent (Belyantseva et al., 2005). However, whether the rescued hair cells had stereociliary lengths similar to those of WT hair cells was not studied. My studies showed that both FL- and C-whirlins are required for stereocilia elongation, with FL-whirlin playing a major role in VHC stereocilia elongation. Furthermore, the presence of C-whirlin isoform alone is sufficient to partially bring back the missing staircase pattern in the VHC stereocilia of Dfnb31wi/wi mice (Mathur, Vijayakumar, et al., 2015). In a very recent study, Chien et al., packaged FL-whirlin cDNA into an AAV8 vector and delivered this AAV particle to the inner ear hair cells of Dfnb31wi/wi mice. This AAV FL-whirlin particle partially increased the stereocilia length in IHCs but not OHCs. Moreover, the hearing loss in Dfnb31wi/wi mice could not be restored (Chien et al., 2015). Based on my findings in Chapter Three, the partial rescue observed by Chien et al., is likely due to the absence of C-whirlin at the OHC stereociliary tips, which contributes to normal OHC stereociliary lengths and hearing function (Mathur, Zou, et al., 2015). Conclusion and future directions My studies on Dfnb31 in this dissertation suggest that disruption of different isoforms might be a mechanism underlying the different disease manifestations observed in mutations in other USH genes as well. Nine out of eleven currently known USH genes show variable disease manifestations when mutated and, similar to Dfnb31, most of these USH genes express multiple splicing isoforms (Table 5.1). Furthermore, a high percentage of similarity between human and mouse genes provides a rationale that findings from the study of USH genes in mice can possibly be extended to answer 85 questions about human USH. For example, analysis of human DFNB31 cDNA sequences (AB040959; AL11028; AK022854 and AL110228) predicts that human whirlin is also expressed as FL-, N- and C-terminal protein isoforms, similarly to mice. However, the information about the promoter regions that lead to expression of multiple DFNB31/Dfnb31 variants still remains unknown. Nonetheless, with an 88% amino acid sequence identity between mouse and human whirlins that rises to 94.4% in the PDZ domains (Mburu et al., 2003), it is likely that there is little difference in the function of whirlin isoforms in humans and mice. My findings in Chapters 3 and 4 are, therefore, valuable for early differential diagnosis of USH2D and DFNB31, genetic counselling of parents and educational planning of newborns with mutated DFNB31 gene. This is important because retinal degeneration in USH2D patients does not occur until about 20 years of age, and prior knowledge of USH2D will allow ample time for treatment and preventive measures. Abnormal vestibular responses in Dfnb31neo/neo mice (Mathur, Vijayakumar, et al., 2015), which represent USH2D patients, suggest for the first time that USH2 patients may have vestibular deficits. In support of this finding, a recent study reported abnormal vestibular responses in 8 out of 11 USH2 patients examined. These eight patients never self-reported any balance problems (like other USH2 patients), but later recalled occasional vertigo attacks (Magliulo et al., 2015). Therefore, vestibular tests of other USH2 mouse models and all USH2 patients in clinics are important. The role of other USH2 proteins in vestibular function and the role of the ALC in VHCs also need to be studied. This is important because to date USH2 patients are considered to have normal vestibular function. However, my findings, coupled with those of Magliulo et al., suggest 86 that vestibular deficits in USH2 patients may not be ruled out even if USH2 patients do not self-report vertigo attacks. Therefore, USH2 patients need testing and counselling about a potential vestibular dysfunction in them. Moreover, for a clear understanding of the USH pathogenesis and mechanism, all the affected systems need to be studied. Therapeutic strategy using my study. Among the several therapeutic measures studied to treat USH, only viral-mediated gene therapy has shown promise, due to its high efficacy and relative safety. However, all previous attempts to rescue the hearing and inner ear morphology in Dfnb31 mutants by putting the Dfnb31 cDNA or gene back into them have only been partially successful, mostly due to incomplete knowledge about the expression and function of different whirlin isoforms. My studies elucidated the role of each whirlin isoform in the retinal photoreceptors and inner ear hair cells. A rescue study utilizing the isoform-specific information discussed in this dissertation may be promising for future therapy. Specifically, Dfnb31neo/neo mice that show a moderate level of hearing loss and retinal degeneration should receive a therapy which provides the FLwhirlin isoform to the Dfnb31neo/neo ALC and PMC regions. On the other hand, Dfnb31wi/wi inner ear hair cells require both FL- and C-whirlin isoforms for complete rescue of morphology and hearing. Adeno-associated virus serotype 2 (AAV2) is in clinical trials to deliver the packaged DNA to retinal photoreceptors (Hauswirth et al., 2008). AAV2 has also been shown to transduce both OHC and IHC with an overall efficiency better than other AAV serotypes tested (Stone, Lurie, Kelley, & Poulsen, 2005). The ability of AAV2 to transduce photoreceptors, OHCs, and IHCs makes it a promising candidate for gene therapy for USH. With a packaging capacity of about 4.7kb, AAV2 can carry the DFNB31 cDNA variants, but packaging other USH genes 87 with large full-length cDNA may be challenging. However, a knowledge of the function of the various isoforms of other USH genes may help address this issue. For example, a FL-usherin cDNA is about 16kb, which is beyond the packing capacity of AAV2, but the shorter usherin isoform cDNA is about 4.5kb in size and can be packaged in AAV2. Therefore, to understand the molecular function of the USH genes that show variable disease manifestations and have multiple isoforms using a study design similar to my Dfnb31 study described in this dissertation may be promising in order to make therapeutic progress. 88 References Ahmed, Z. M., Goodyear, R., Riazuddin, S., Lagziel, A., Legan, P. K., Behra, M., … Friedman, T. B. (2006). The Tip-Link Antigen, a Protein Associated with the Transduction Complex of Sensory Hair Cells, Is Protocadherin-15. Journal of Neuroscience, 26(26), 7022-7034. http://doi.org/10.1523/JNEUROSCI.116306.2006 Ahmed, Z. M., Riazuddin, S., Ahmad, J., Bernstein, S. L., Guo, Y., Sabar, M. F.,Friedman, T.B., & Wilcox, E. R. (2003). 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