The Nebraska Center for Molecular Biology of Neurosensory Systems: a Collaborative COBRE Project

The Nebraska Center for Molecular Biology of Neurosensory Systems: A Collaborative COBRE Project

Shelley Smith Professor of Pediatrics Director, Human Molecular Genetics Munroe Meyer Institute, University of Nebraska Medical Center

he National Center for Research Resources (NCRR) at the National Institutes of Health has developed the Institutional Development Award (IDeA) Tprogram to enhance biomedical research where NIH funding has been in the lower tier. Twenty-three states and Puerto Rico qualify for IDeA programs, including Nebraska, Kansas, and Oklahoma in our region. The Centers for Biomedical Research Excellence (COBRE) program is one of these mechanisms. COBREs are each organized around a central scientific theme, and the growth of research in that area is facilitated in three ways: support of core facilities to serve as resources, support of individual research projects primarily for junior faculty, and development of a mentoring program to ensure that the research projects result in independent NIH funding. In addition to building a network of successful researchers within a COBRE, regional interaction of COBREs and other IDeA programs is encouraged. In building the research researchers in the participating infrastructure of a state, a COBRE institutions. Thus, the Center can aid program has a particularly unique junior faculty in establishing an aspect: the research projects associated independent research program, and can with the center are generally junior also help make advanced technologies investigators who have not had previous available to researchers beyond the renewable NIH funding, and their Center. projects are designed to be the basis of The Nebraska Center for the grant applications that are funded after 2 Molecular Biology of Neurosensory -3 years of Center support. When Systems is built around the external funding starts, the projects characterization of the molecular rotate off Center funding and new mechanisms controlling the projects take their place. The mentoring development and maintenance of program provides advice to the neurosensory functions, particularly investigators to help ensure that he vision and hearing. Neurosensory cells projects are successful, and the core of the inner ear and retina are not facilities provide technical support for replaced after damage or degeneration, the investigators as well as for other so understanding of the regulation of

their development and the mechanisms in Omaha. The University of of their loss are important steps to the Nebraska Medical Center is the lead identification of potential avenues for institution and is the primary site for intervention. the administrative and scientific cores Currently, COBRE projects are and has research programs in retinal funded for 5 years for the first phase, and development and disorders. are eligible to apply for a second 5-year Creighton University has a long- phase. While the focus of the first phase is standing program in the regulation of the support of new investigators, the development of the auditory system, purpose of the second phase is and Boys Town National Research consolidation and strengthening of the Hospital has strengths in the Center, and more senior researchers can identification and characterizations of be supported in addition to junior genes causing hearing and vision investigators. Our Center is currently loss, particularly Usher Syndrome. starting its second phase. The administration of the center is Administration of a multi- handled by co-PIs from each institutional center institution and an Administrator who Our Center for Neurosensory together comprise an Executive Systems is a collaboration of 3 institutions Committee (Table 1).

Table 1: Executive Committee for the Center for Neurosensory Systems, Phase I PI: Shelley D. Smith, Ph.D., University of Nebraska Medical Center Co-PI: Bernd Fritsch, Ph.D., Creighton University Co-PI: Edward Walsh, Ph.D., Boys Town National Research Hospital Administrator: Melanie Schrack, University of Nebraska Medical Center Advisor: Kirk Beisel, Ph.D., Creighton University

Although a co-PI structure is not investigators and cores and for the typical structure for a COBRE recruitment of additional faculty program, we found this to be very members with interests within the advantageous in coordinating the scope of the Center. The potential for support of the participating pilot funding and mentoring as well as institutions. The Executive Committee the presence of state-of-the-art core manages the overall direction of the facilities can be effective recruitment Center, overseeing the progress of the tools, and the co-PIs are responsible for investigators on the research projects, keeping administrators and reviewing the core facilities, and department chairs aware of the selecting new projects as the initial strengths of the Center in building projects rotate off COBRE funding. The research. co-PIs also act as liaisons with the COBRE projects also have an administrations of their institutions, to External Advisory Committee made up keep them informed of progress in the of individuals who have national and Center and ensure their support for the international standing in research 60

related to the Center. Their guidance person at an annual 2 ½ day meeting in ensures that the research of the Center Omaha, and again in 6 months via e-mail. is at the forefront of the field, At the annual meeting, each project incorporating research questions and investigator presents a 15-minute summary technologies that are current and of their project and progress, followed by a competitive. The EAC for the 5-minute question period. New projects are Neurosensory Center is shown in Table 2. also presented for approval for funding. They review Center projects twice a year, in

Table 2: External Advisory Committee, Center for Neurosensory Systems, Phase I Robb Krumlauf, Ph.D., Stowers Institute for Medical Research, University of Kansas School of Medicine Cynthia C. Morton, Ph.D., Harvard University and Brigham & Women’s Hospital Suzanne L. Mansour, Ph.D., University of Utah Kathryn Albers, Ph.D., University of Pittsburg Guy Richardson, Ph.D., University of Sussex, UK

The next day, the committee meets each of the investigators, and both of the with the co-PIs and the core directors to EAC reports are submitted to the NCRR discuss the progress of the Center, and then with the annual progress report. meets with each investigator individually The structure of the EAC and its for at least 30 minutes to discuss their evaluations will be unchanged in Phase II of projects and give advice. On the third day, the Center for Neurosensory Systems, with the EAC meets again with the co-PIs to the exception that Dr. Robb Krumlauf will present their evaluation of the Center and to rotate off the committee and will be start to draft their report. The meeting replaced by Dr. Jane Johnson, University of culminates in a seminar presented by one of Texas Southwestern Medical Center, Dallas, the EAC members which is open to the TX. One member each year will rotate off entire research community of the 3 and be replaced, thus helping to maintain institutions and helps to publicize the the continuity of guidance of the center, but interests of the Center. The EAC finalizes its also adding new input. report over the next month and sends it to Core Facilities the PI, who then distributes it to the Similar to other NIH Centers, the investigators. The EAC designed a format COBRE programs have an Administrative for progress report from each of the project Core and several scientific cores. The cores investigators for submission to them 6 for the Center for Neurosensory Systems are months later. The committee member’s listed in Table 3. evaluations of these reports are shared with

Table 3: Core Facilities Administration Director: Shelley D. Smith Administrator: Melanie Schrack Mouse Genome Director: Michael Salbaum (years 1-3), Kay-Uwe Wagner Engineering (years 4-5) Supervisor: Judith Stribley Molecular Phenotyping Director: Claudia Kappan, (years 1-3); Bernd Fritzsch (year 4-5) Supervisor: Anita Jennings Gene Expression Director: James Eudy

The Administrative core is based at investigators on publications, presentations UNMC and is responsible for the at national meetings and the annual EAC financial aspects of the Center and its meeting, and grant applications. Mock subcontracts to Creighton University study sections also help the investigators and Boys Town National Research understand the grant review process. The Hospital. It also oversees the activities of mentor and the EAC members can also the Center, including a monthly Journal identify researchers and potential Club, a seminar series, the annual collaborators outside of Omaha, helping the meetings with the EAC, and a mentoring junior faculty members network in their program. field of research. Funds from the Center also The mentoring program is a vital part support travel of each investigator to one of a COBRE. Each of the junior faculty national meeting a year to present their investigators has a primary mentor to give research. advice on their research and on their career The 3 scientific cores provide development. The primary mentor should specialized services that would generally be be a senior researcher who is familiar with beyond the expertise and financial resources the area of research but is not a primary of an individual lab and cover the basic collaborator, thus ensuring the progression of research of the projects in our independence of the junior faculty member. center. The Mouse Genome Engineering Primary mentors are expected to talk with core provides a variety of mouse models, the investigator at least once a month and to including transgenic, knock-out, and review manuscripts and grant proposals. To conditional knock-outs of specific genes, compensate for the amount of time that is allowing the examination of in vivo function anticipated, a mentor receives salary of a genetic system in development. The support from the Center, usually 10% FTE. Molecular Phenotyping core supplies an Several secondary mentors are also analysis of normal development and the designated for each research project, and results of genetic changes at the histological these individuals have more direct expertise level, with techniques such as antibody the research project of the investigator. optimization and immunohistochemistry, in Secondary mentors may change as the situ hybridization, apoptosis assays, requirements of the project progresses. competition experiments, double-label The co-PIs of the Center also act as studies, and whole mount preparations. The mentors, and together with the mentors and Gene Expression core provides microarray scientific core directors, they advise the expression analysis using standard or 62

custom chips. By comparing changes in full project funding. This could be used to gene expression at different times in construct a mouse model, for example, to development or as a result of genetic determine if the model is viable or has a changes, researchers can identify candidate phenotype that can be analyzed. We also genes and assess their effects on the provided bridge funding to lead into or expression of other genes, thus highlighting supplement the externally funded project genetic regulatory networks. that is expected to result from the Center Research Projects project. This funding was designed to COBRE programs are designed to complement the funded project and to help support several research projects lasting for ensure renewal of the grant. The research 2-3 years. Our Center supported 4-5 projects projects supported by our Center in Phase I at about $150,000 per year. We also are given in Table 4, and the funding developed a funding mechanism to allow outcomes are shown in Table 5. The new investigators to conduct seed projects, research projects in Phase I started out generally $15,000-$25,000 per year, to with a slightly broader determine the feasibility of a proposal for Table 4: Research Projects Investigator Position and Institution Title of Research Project Laura Hansen, Ph.D. Associate Professor, Biomedical Role of EGFR and erbB2 in the Sciences, Creighton University regulation of skin innervation School of Medicine Garrett Soukup, Associate Professor, Biomedical MicroRNA regulation of Ph.D. Sciences, Creighton University neurosensory development School of Medicine Kristen Drescher, Associate Professor, Medical Role of Neuregulins in Myelin Ph.D. Microbiology and Immunology , Repair in the CNS and PNS Creighton University Janee Gelineau-van Assistant Professor, Genetics, Cell Role of Microphthalmia- Waes, D.V.M, Ph.D. Biology & Anatomy, UNMC associated Transcription Factor (Mitf) in Development of the Retinal Pigment Epithelium (RPE) and Inner Ear (stria vascularis) Yunxia Lundberg, Staff Scientist, Boys Town National Formation and Regulation of Ph.D. Research Hospital Otoconia Neena Haider, Ph.D. Assistant Professor, Department of Functional characterization Genetics, Cell Biology & Anatomy, ofNr2e3 in the developing and UNMC adult photoreceptor cells Dr. Sonia Rocha- Assistant Professor, Oral Biology, The role of the E2F2 modulation Sanchez, Ph.D. Creighton University School of of Rb1 in cochlear hair cells and Dentistry supporting cells to mediate hair cell regeneration You-Wei Peng, M.D., Staff Scientist, Boys Town National Mechanisms of Retinal Ph.D. Research Hospital Degeneration in Usher Syndrome Type IIa Sumitra Assistant Professor, Characterization of retinal side- Bhattacharya, Ph.D. Ophthalmology and Visual population cells Sciences, UNMC

interest on nervous system maintenance established that miRNAs also influence and development, including central and the structural development of the inner peripheral innervations. Subsequently, ear. By determining the roles of miRNAs as the Center grew in numbers, the in normal ear development and funded projects became more focused on function, the capacity of these genetic development of the auditory and visual regulatory elements to guide specific cell systems. The projects and a brief fates and functions might eventually summary of their results are as follows: contribute to therapeutic strategies Laura Hansen, Ph.D., Creighton designed to stimulate hair cell University School of Medicine regeneration and hearing restoration. Role of EGFR and erbB2 in the regulation of Kristen Drescher, Ph.D., Creighton skin innervation University School of Medicine Epidermal growth factor receptor Role of Neuregulins in Myelin Repair in (EGFR) family members Erbb2/HER2, the CNS and PNS Erbb3/HER3, and Erbb4/HER4 are The purpose of this study was to necessary for proper peripheral nervous investigate the genetic response to system development. The role of EGFR infection to determine which pathways in peripheral nervous system mediate protective responses. To development, however, has never been accomplish these studies, TMEV- investigated in vivo. Dr. Hansen’s data induced models of demylination were demonstrated that EGFR is required for modified to precisely identify where development of proper innervation to demylination first occurs. Using this the dorsal skin in a cell autonomous model, the effects of conditional knock- manner through changes in the cell outs of the genes erbB2 and EGRF were behavior of the DRG neurons. tested individually and together. Dr. Hansen also had a project in the Janee Gelineau-van Waes, D.V.M, COBRE-funded Nebraska Center for Ph.D., University of Nebraska Medical Cell Signaling, which was funded at the Center same time our Center was funded. At Role of Microphthalmia-associated the first meeting with our EAC, it was Transcription Factor (Mitf) in Development of recommended that in the second year the Retinal Pigment Epithelium (RPE) and her project should incorporate more cell Inner Ear (stria vascularis) signaling experiments, so funding was Although mutations in the transferred entirely to the other COBRE. Microphthalmia-Associated Garrett Soukup, Ph.D., Creighton Transcription Factor (MITF) are known University School of Medicine to cause retinal disorders and MicroRNA regulation of neurosensory sensorineural hearing loss in humans development and mice, the signaling pathways MicroRNAs have been involved have not been characterized. demonstrated to play fundamental roles This research demonstrated that Mift in developmental processes including may regulate iron homeostasis leading cell proliferation, fate specification and to the hypothesis that retinal damage is organ morphogenesis. This work due in part to oxidative stress. This led

to two tests of therapies; restriction of mutant mouse, rd7, lacking the retina dietary iron to pregnant dams carrying transcription factor Nr2e3, which is a normal and Miftvit embryos, and the model for the human Enhanced S Cone ability of transplanted hematopoetic Syndrome characterized by a retinal stem cells to replace the damaged RPE. degeneration and an increase in the Since excess iron accumulation has been function of blue cone cells. This project implicated in several neurodegenerative demonstrated that Nr2e3 is important in diseases as well as retinal degeneration, the development of rod and cone cells this research could have clinical and in maintaining their function in the implications beyond the effects of MITF adult retina. Dr. Haider also identified 4 mutation. potential loci for modifier genes, with Yunxia Lundberg, Ph.D., Boys Town strong evidence that one of these is the National Research Hospital Nr1d1 gene. Identification of modifiers Formation and Regulation of Otoconia will not only pinpoint proteins that Over 6 million adults in the United interact with Nr2e3 pathways, but will States suffer from disorders of balance highlight potential therapies. and dizziness, disorders which are Dr. Sonia Rocha-Sanchez, Ph.D., particularly prevalent in the elderly and Creighton University School of Dentistry a significant cause of morbidity through The role of the E2F2 modulation of Rb1 in falling and fractures. A significant cause cochlear hair cells and supporting cells to of balance disorders is the degeneration mediate hair cell regeneration and dislocation of otoconia, bio-crystals Rb1 is required for normal hair cell in the vestibular system. We generated a (HC) cell cycle control, including mutant mouse model in which the differentiation and mitotic predominant mammalian otoconial quiescence. Through control of Rb1 via protein, otoconin-90 (Oc90), is absent. modulation of the transcription factor The mutant mice had giant otoconia E2F2, we propose to regulate the which were not attached to the sensory proliferation of supporting cells, creating epithelium, resulting in poor balance the potential for their trans- and head-tilting. This suggests that differentiation into hair cells. During perturbed otoconial proteins can lead to pilot funding, we produced a loose and dislocated otoconia, which is conditional knockout of E2F2 and mice the cause of benign paroxysomal carrying a conditional E2F2 transgene to positional vertigo (BPPV), the most allow us to study under- and over- common form of dizziness. expression of E2F2. Analysis of the Neena Haider, Ph.D., University of knockout phenotype showed abnormal Nebraska Medical Center number and patterning of cochlear outer Functional characterization of Nr2e3 in hair cells and abnormal innervation. We the developing and adult photoreceptor cells also performed a microarray analysis to The goal of this project is to identify potential downstream genes understand the mechanisms through which could be additional targets for which photoreceptors are generated and regulation. maintained. The studies utilize the

You-Wei Peng, M.D., Ph.D., Boys Town blindness in the world. We have National Research Hospital evidence that the short isoform of Mechanisms of Retinal Degeneration in usherin is a basement membrane protein Usher Syndrome Type IIa that interacts with α1β1 integrin on Usher syndrome type IIa is the most retinal pigment epithelial cells, and common of the Usher syndromes, some human USH2A mutations this making it the single most important interaction. We hypothesize that this genetic cause of combined deafness and interaction is essential for the retinal Table 5: External Funding of Research Projects Investigator Years of Grant awards Grant awards funding NIH/DOD/NSF Private foundations/other Hansen 1 R01 Health Futures Foundation, Nebraska LB595 Soukup 3 R01, EPSCOR (NSF) Drescher 3 DOD Multiple Sclerosis Society Gelineau-van 3 R21 Retina Research Foundation Waes Lundberg 3 R01, P50 (Deng, PI) Haider 3 R01 Hope for Vision Rocha- 1 R03 Deafness Research Foundation Sanchez Peng 1 Bhattacharya 1 Research to Prevent Blindness

pigment epithelium to function photoreceptor degeneration in the properly, with the resulting mutant retinas. dysfunctional cell signaling directly Sumitra Bhattacharya, Ph.D., University affecting basement membrane of Nebraska Medical Center metabolism and photoreceptor cell Characterization of retinal side- health, culminating in synaptic population cells malformations and photoreceptor Identification and characterization apoptosis. We have produced mouse of retinal progenitor cells offers the models of integrin and usherin possibility to treat degenerative eye deficiency, and both have progressive diseases using stem cell therapy. retinal degeneration along with delayed However, the success of this approach translocation of transducin and arrestin will largely depend upon the efficiency in the rod photoreceptors in response to of enrichment of retinal stem cells and light/dark adaptation. Furthermore, it their maintenance ex vivo. Enrichment was demonstrated that increased light of neural stem cells is challenging for the exposure produces more rapid lack of specific cell surface markers. We

demonstrated that retinal stem cells can Characterization of a MicroRNA be enriched as a “side population” (SP) misexpression model of age related deafness by Hoechst dye efflux assay, but this MicroRNAs (miRNAs) are ~22nt sorted population is still heterogeneous. noncoding RNAs that inhibit expression This project will characterize retinal SP of target mRNAs through cells in terms of their molecular complementary base pairing. Their phenotype and regulation towards functions are similar to transcription providing an approach to reproducibly factors, but their interactions with know enrich specific subpopulations of cells gene regulatory networks is unknown. and predict the functional outcomes To study the effects of miRNAs on following their transplantation in animal auditory development, we have models. generated transgenic miR-183fam Dr. Rocha-Sanchez, Peng and misexpression mouse lines, and one of Bhattacharya will continue their research these was found to show progressive projects for one more year into Phase II, loss of auditory hair cells. We and we have added two new projects: hypothesize that this is due to David Nichols, Ph.D., Assoc. Professor, perturbation of specific regulatory, Biomedical Sciences, Creighton University structural and/or metabolic pathways, School of Medicine including the Notch signaling pathway An analysis of the Lmx1a (Dreher) ligand Jagged1 (Jag1) and a downstream mutant inner ear effector transcription factor Sox2. Our preliminary results show that In the second year of Phase II, we members of the Dickkopf (Dkk) family of Wnt anticipate adding two new projects signaling modulators are altered in mice which will be presented to the EAC for mutant for the LIM homeobox transcription their approval. Taken together, the factor, Lmx1a, suggesting that Lmx1a Center projects have common themes in expression plays a role in the control of Wnt the identification of the regulatory signaling. To verify this, we will compare the networks in development and spatiotemporal distributions of Wnt’s 2b, 4, 5a, maintenance of function of neurosensory b and 7a, plus those of Dkk’s 1 and 2 in systems, and the potential for therapy wildtype and Lmx1a mutant mouse inner through regenerative methods, and we ears. These would then be compared with have reached a “critical mass” for alterations in the pattern of Wnt signaling in exchange of ideas and techniques. Most Lmx1a//TOPGAL mutant mice, accompanied importantly, all of the junior faculty by a molecular analysis of the genesis of the investigators who participated in the mutant stria vascularis and endolymphatic COBRE program for more than 2 years duct in the Lmx1a mutant. These studies will have received federal funding. determine the role of Lmx1a on the Regional Integration mechanism for endolymphatic homeostatis. The National Center for Research Michael Weston, Ph.D. Asst. Professor, Resources also funds other programs to Oral Biology and Biomedical Sciences, enhance research in IDeA states. Two Creighton University School of Dentistry major programs serve as a pipeline to research institutions: the Science

Education Partnership Awards (SEPA), about the programs and offer guidance. which is designed to promote and enhance Through these meetings, our Center for science education at elementary and Neurosensory Systems has found research secondary school levels, and the IDeA goals in common with the Center for Networks of Biomedical Research Epithelial Function at Kansas State Excellence (INBRE), which provides University and the Center for Visual support for students, faculty, and research Neuroscience in South Dakota. infrastructure for undergraduate institutions On alternate years, a national meeting in partnership with research institutions. In is held in Bethesda for all COBRE and Nebraska, a SEPA program headed by Dr. INBRE programs. This offers the Maurice Godfrey at UNMC provides opportunity to create collaborations on a training for teachers in rural and tribal national level, and also provides an schools. It also interacts with a SEPA opportunity for NCRR and NIH officials to program at the University of Nebraska- update the PIs on new initiatives and Lincoln that is based in the Natural History priorities. museum there. These programs are Conclusion designed to encourage students to go on to The Center for Neurosensory Systems college programs in the sciences, and has helped build an interactive group of COBRE faculty have assisted in SEPA researchers from 3 independent institutions, projects. The INBRE program headed by Dr. providing critical core facilities and bringing James Turpen at UNMC works with small them together to produce the critical mass colleges and tribal colleges and connects the that supports discussion and growth of faculty with research faculty at UNMC, UN- knowledge. The funding of research projects L, and Creighton University, and supports and provision of a mentoring program has summer research opportunities for resulted in independent funding for junior undergraduate students, encouraging them faculty who previously had not had that to consider graduate education. The INBRE level of funding, helping our institutions program has interacted with the COBRE “grow our own”. Through additional program by contributing to the funding of support from other funding sources such as the Gene Expression Core, and many of the the INBRE or the University of Nebraska, faculty of the COBRE and SEPA programs the core facilities enhance the research also participate in INBRE projects. infrastructure benefiting researchers at all 3 Regional collaboration between institutions. By networking with regional COBRE and INBRE programs is facilitated and national COBRE, INBRE, and SEPA by meetings of PIs, administrators, and researchers, the investment of the NCRR in researchers in Nebraska, Kansas, Oklahoma, IDeA states is leveraged further, so that the South Dakota, and North Dakota. The level of research quality is increased across meeting is hosted by an institution from one the region. of the states, giving them the opportunity to Acknowledgement present their facility to other researchers in This work is funded by NIH-NCRR the region. Poster sessions allow all of the P20 RR018788. researchers to exchange information, and NCRR officials also attend to learn more