7^th Annual

2014 Midwest Eye Research Symposium

Sponsored by The Stephen A. Wynn Institute for Vision Research

MERS 2014 took place on August 8^th at
The University of Iowa Medical Education and Research Facility

Keynote Speaker/Session Chairs

Michael E. Boulton, Ph.D. - Keynote Speaker
Dr. Boulton is the Merrill Grayson Professor of Ophthalmology and the Director of Basic and Translational Research at the Eugene and Marilyn Glick Eye Institute at the Indiana University School of Medicine. His research interests focus on oxidative damage and vascular changes in age-related macular degeneration (AMD) and other retinal degenerations. He is the author of over 200 peer-reviewed publications, a fellow of ARVO, serves on the editorial boards of numerous journals, and participates in NEI study sections.

Daniel Goldman, Ph.D. - Chair, Session I
Dr. Goldmann is the Bernard W. Agranoff Collegiate Professor of Neuroscience at the University of Michigan, a Professor in the Department of Biological Chemistry, and a Research Professor at the University of Michigan Molecular and Behavioral Neuroscience Institute. One of his main research interests is to identify stratgies for retinal and optic nerve regeneration. His recent work demonstrated that Mueller cells can dedifferentiate into multipotent retinal stem cells following retinal injury.

Elena V. Semina, Ph.D. - Chair, Session II
Dr. Semina is a Professor and the chair of the Division of Developmental Biology in the Department of Pediatrics at the Medical College of Wisconsin in Milwaukee. She leads the Human Developmental Genetics Laboratory and studies genes involved in ocular development and the disorders caused by mutations in these genes.

Seongjin Seo, Ph.D. - Chair, Session III
Dr. Seo is an Assistant Professor and the Department of Ophthalmology and the Stephen A. Wynn Institute for Vision Research at the University of Iowa. His research is focused on the the proteins that form the primary cilium and the pathophysiology that results from defects in these protein. In particular he investigates ciliopathies that affect trafficking into the photoreceptor outer segment, a highly specialized primary cilium.

A trafficking signal in the N-terminus of Na+/K+-ATPase regulates the compartment in which the sodium pump is localized

Joseph G. Laird, Yuan Pan and Sheila A. Baker
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa

Purpose: Na+/K+-ATPase (NKA) is responsible for maintaining the electrochemical gradient that fuels ion transport and neuronal signaling. NKA has a restricted subcellular localization in polarized cells. For instance, in many epithelial cells it is found in basolateral membranes. Photoreceptors are unique in that the alpha-3 beta-2 isozyme of NKA is restricted to an apical compartment called the inner segment. It is not known how NKA is selectively retained in the inner segment. The purpose of this study was to determine if the alpha-3 subunit of NKA contains a sequence specific targeting motif necessary for the localization of this protein in photoreceptors.

Methods: GFP tagged alpha subunits were subcloned behind the Xenopus opsin promoter to direct expression to rod photoreceptors in transgenic Xenopus laevis tadpoles. The subcellular location of the transgenically expressed proteins was determined by confocal analysis of retinas labeled with anti-GFP antibodies.

Results: GFP-alpha-3 was found in the plasma membrane of the inner segment, similar to the endogenous protein. However, GFP-alpha-4 was found in the outer segment compartment. Exchanging the cytoplasmic N-terminus of alpha-3 and alpha-4 reversed the localization pattern. A series of chimeras and deletion mutants was used to pinpoint a 14 amino acid sequence critical for this differential localization pattern.

Conclusions: We have identified a novel targeting motif required for the trafficking of NKA in photoreceptors.

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The R345W mutation in the extracellular matrix Efemp1 causes amplified circadian and photophobic responses to light.

Bui, Gabrielle; Stasheff, Steven; Mullins, Robert; Singh, Pratibha; Koehn, Demelza; Anderson, Michael; Stone, Edwin; Thompson, Stewart.
Stephen A. Wynn Institute for Vision Research, University of Iowa, Iowa City, Iowa.

Purpose: The R345W mutation in EFEMP1 causes Malattia Leventinese, an autosomal dominant eye disease with features similar to age-related macular degeneration. In mice, Efemp1R345W has no discernable pathology by standard measures of structure and function, with normal retinal thickness and a normal electroretinogram. However, one study showed an unexplained abnormality in light adaptation in Malattia Leventinese patients. We therefore hypothesized that Efemp1R345W will cause abnormal responses to light in the irradiance-measurement circuits of the eye.

Methods: Irradiance measurement function was measured using circadian phase shifting, negative masking behavior, and the pupillary light reflex. Mechanism was investigated by Efemp1 expression and multielectrode recording of retinal output.

Results: Circadian phase shifts and negative masking had increased amplitude of responses to light, but the pupillary light reflex was not different from wild-type. Efemp1 was expressed in the ganglion cell layer of the retina, identifying a plausible mechanism. Further, melanopsin driven responses to light were shown to be amplified in the Efemp1R345W retinal ganglion cells.

Conclusions: Our results show that Efemp1R345W amplifies of responses to light in at least one type of intrinsically photosensitive retinal ganglion cells (ipRGCs). Circadian phase shifts and negative masking are mediated by M1 ipRGCs but the pupillary light reflex is primarily mediated by M2 ipRGCs. Therefore this effect appears to be ipRGC type specific. Finally, our data show that Efemp1R345W disrupts retinal irradiance measurement by a mechanism quite distinct from any outer retina degeneration.

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CEP290 gene addition rescues ciliogenesis in LCA patient cells

Burnight E.R.¹, Wiley, L.A.¹, Drack, A.V.¹, Braun, T.A.¹, Anfinson, K.R.¹, Kaalberg, E.E.¹, Halder, J.A.¹, Mullins, R.F.¹, Stone, E.M.^1,2, and Tucker, B.A.^1
1Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, and ²Howard Hughes Medical Institute, University of Iowa, Department of Ophthalmology, Iowa City, IA

Purpose: The purpose of this study was to generate induced plurtipotent stem cell (iPSC)-derived photoreceptor precursor cells from patients with CEP290-associated Leber Congenital Amaurosis (LCA). Additionally, we aimed to produce a lentiviral vector expressing full-length CEP290 to investigate gene replacement strategies in patient-derived cells.

Methods: Fibroblast-derived iPSCs were generated from the retinal degenerative mouse model CEP290rd16 and patients with molecularly confirmed CEP290-associated LCA. Mouse and human iPSCs were differentiated into photoreceptor precursor cells using our previously developed step-wise differentiation protocol. An HIV-1 lentiviral vector containing human CEP290 under the control of the CMV promoter was packaged and used to transduce LCA patient fibroblast cultures. Following serum-starvation and immunolocalization, cilia were counted using confocal microscopy.

Results: A lentiviral vector containing CMV-driven human full length CEP290 was constructed. Following transduction of patient-specific, iPSC-derived, photoreceptor precursor cells, RT-PCR analysis revealed vector-derived expression. In cultures derived from LCA patients, fewer cells formed cilia compared to unaffected controls. Cilia that were formed were shorter in patient derived cells than in cells from unaffected individuals. Importantly, lentiviral delivery of CEP290 rescued the ciliogenesis defect.

Conclusions: The successful construction and viral transfer of full length CEP290 brings us closer to the goal of providing gene- and cell- based therapies for patients affected with this common form of LCA.

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Using zebrafish to examine vertebrate retina development

Rebecca Chowdhury, Lauren Laboissonniere, Annie Wester, Jeffrey Trimarchi

Iowa State University

Purpose: Math5 is essential for ganglion cell development and its absence also leads to changes in other early-generated cell fates in the retina. To study the gene networks operating during retinal cell fate decisions, single cell profiling on microarrays was performed on Math5+ cells and genes with a developmental expression pattern similar to Math5 were identified. From the single cell transcriptomes, we chose candidate genes that we predict are involved in the cell fate acquisition and differentiation of early-generated retinal neurons. These candidate genes include Trim9, Rassf4, Ebf3 and Uchl1.

Methods: To study these genes, we are using both a loss-of-function and a gain-of function approach. Mutating these genes in zebrafish using TALENs or CRISPRs will enable us to study the phenotypic changes associated with these mutations. To study gain of function, we will overexpress these genes in the developing mouse retina using electroporation or viral transduction.

Results: We have generated TALENs/CRIPSRs to these genes and are currently injecting them into zebrafish to create mutations. With these mutant fish, we will study the role of these genes in the development of the zebrafish retina. Additionally we have produced overexpression constructs and tested them in cell culture. We are currently electroporating these plasmids into E14.5 retinas and examining the development of early-produced retinal neurons.

Conclusions: We have identified a cohort of genes with a similar expression pattern as Math5 during mouse retinal development. Through a complementary gain and loss of function approach we are investigating their role(s) in retinal cell fate acquisition.

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ONH shape features derived from phenotypic and genotypic data yield improved glaucoma progression biomarkers

Mark A. Christopher, Li Tang, John H. Fingert, Todd E. Scheetz, Michael D. Abramoff

Departments of Biomedical Engineering, Electrical Engineering, and Ophthalmology and Visual Sciences Stephen A. Wynn Institute for Vision Research, University of Iowa

Purpose: To discover unknown relationships between optic nerve head (ONH) structure, genetics, and glaucoma

Methods: Computational methods were applied to a large-scale glaucoma dataset to identify features that can be used to model ONH structure. The dataset consisted of phenotypic and genotypic data large from a cohort (n = 1056) of participants at risk for developing primary open angle glaucoma (POAG). The primary phenotypic data consisted of stereo fundus images from which three-dimensional ONH structure was inferred. Genetic data consisted of genotypes for a set of known POAG associated single nucleotide polymorphisms. Principal component and linear discriminant analysis techniques were applied to identify ONH structural features that explained both the observed variance in ONH structure and contributions from genetic factors. Features were evaluated based on their associations with POAG related variables and their power to predict the development of POAG.

Results: Several statistically significant associations between the identified ONH structural features and variables of interest were found. These include associations with participant demographics (age, ethnicity), POAG risk factors (cup-to-disc ratio, central corneal thickness, presence of a peripapillary crescent) and the development of POAG. The inclusion genetic data into the identification of ONH structural features also led to a significant increase the accuracy of predicting POAG development.

Conclusions: The methodologies developed here identified novel ONH features with significant associations with POAG and related variables. These features show promise as biomarkers that can be used in future work to predict POAG early and reveal unknown genetic causes..

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Large Scale Exome Sequencing For Inherited Eye Disease

Adam P. DeLuca, Todd E. Scheetz, Edwin M. Stone
University of Iowa, Stephen A. Wynn Institute for Vision Research

Purpose: Whole exome sequencing is used to perform clinical genetic testing for a variety of inherited eye diseases and discover novel disease genes.

Methods: Whole exome sequencing has been obtained for 577 patients, and continues on an ongoing basis at a rate of one exome every 18 hours. Exome capture was performed using the Agilent v5 kit modified with custom targeting baits, sequenced on an Illumina Hiseq, and analyzed using BWA, GATK, and Conifer.

Results: With varying degrees of experimental validation per patient, causative genotypes have been discovered and validated in 128/577 (22%) patients. The 5 most common disease-causing genes in this cohort are USH2A (11), PRPF31 (9), BBS1 (6), PDE6B (6), and RHO (6). Each patient on average harbors 10.2 rare, non-synonymous or splice site variants in known retinal disease genes.

Conclusions: Exome sequencing is a powerful tool for clinical genetic testing, but of equal importance, patients with negative exome results are enriched for novel disease causing genes. Large cohorts of negative samples are needed to statistically distinguish these novel disease genes from private mutations and noise in the data.

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Adoptive cell transfer from a glaucoma mouse model induces retinal ganglion cell loss in healthy recipient mice

Qiong Ding, Oliver W. Gramlich, Markus H Kuehn
The University of Iowa, Department of Ophthalmology and Visual Sciences

Purpose: There is mounting evidence that autoimmune processes can contribute to the pathophysiology of glaucoma in some patients. Data has also been presented to indicate that experimental induction of a retinal ganglion cell (RGC) directed autoimmune response in rodent models can lead to a RGC loss. Here we demonstrate that the recently described B6-Sh3pxd2 nee (nee) mouse model of glaucoma spontaneously develops an autoimmune response that can be transferred to normal recipient mice.

Methods:Spleen cells (5 x 10⁶) from 2 months old nee or wild type (WT) mice were harvested and transferred to 2 months old WT mice (N=20) by tail vein injection. Mice were sacrificed after 4 month and serum samples and eyes were obtained. The number of surviving gamma synuclein immunoreactive RGC was determined in whole mount preparations of the retina. Differences between groups were evaluated using a one way ANOVA test. Additionally, eyes of 2 months old nee and WT mice were fixed in paraformaldehyde and used for immunohistochemical detection of CD4 and CD8.

Results:Histological evaluation revealed an intact retina despite significant RGC loss in 2 month old nee mice. Immunohistochemistry invariably detected CD4 and CD8 positive cells along the basal surface of the retina and in the optic nerve head in nee mice, while these cells were absent in WT controls. Transfer of WT splenocytes to WT recipients did not cause a decline in RGC numbers (2447.22 vs 2527.41 RGC/mm2, p=0.69). However, spleen cells harvested from glaucomatous nee mice caused a significant decline in RGC numbers in normal recipient mice (1898.00 RGC/mm2, p=6.7x 10^-5).

Conclusions:Our data indicate that in this mouse model CD4 and CD8 positive T-cells invade the retina and the optic nerve head, leading to the development of an autoimmune response. We further demonstrate this immune response can be transferred to healthy animals and cause RGC loss in healthy eyes in the absence of elevated IOP or other stressors.

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Blast-mediated traumatic brain injury results in abnormal retinal ganglion cell physiology

L.M. Dutca^1,2, F.R. Blodi³, M. Shankar³, M. Anderson^1,4, A. Hedberg-Buenz⁴, R.H. Kardon^1,2, S.F. Stasheff³, M.M. Harper^1,2
1Center of Excellence for Prevention and Treatment of Visual Loss Iowa City Veterans Administration Medical Center; Departments of ²Ophthalmology and Visual Science, ³Pediatrics (Neurology), ⁴Molecular Physiology and Biophysics The University of Iowa, Iowa City, IA

Purpose: To analyze the in vivo and in vitro function of retinal ganglion cells (RGCs) after blast-mediated traumatic brain injury.

Methods: Mice were exposed to an overpressure wave (20 PSI) directed to the head using a custom-built blast chamber. Analysis of in vivo RGC structure and function was performed using optical coherence tomography (OCT) and pattern electroretinography (PERG) at 7 days, 5 weeks and 4 months post-blast. Spontaneous activity and light-evoked responses of individual RGCs from freshly dissected retinas were monitored using a multielectrode array (MEA). RGCs were quantified on hematoxylin and eosin stained whole mounted retinas. Dendritic arborization of GFP labelled RGCs was also analyzed.

Results: In vivo, PERG amplitudes were decreased at 7 days compared to baseline amplitudes, recovered to normal at 5 weeks and significantly decreased 4 months post-blast. Decreased thickness of RGC complex layer was observed by OCT at all time points. In vitro, spontaneous activity of RGCs as determined by MEA analysis, was significantly increased at all the time points analyzed compared to controls. The median amplitude of responses to light ON-set and OFF-set increased significantly at 5 weeks post-blast exposure, while no significant differences were observed at other time points. RGC counts and dendritic analysis revealed a significantly decreased RGC density and abnormal dendritic fields at 4 months post-blast.

Conclusions: Exposure to blast induces significant alterations in RGC physiology. A better understanding of retinal changes after blast exposure will help in the development of improved clinical testing and treatment.

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Different Treatment Paradigms of 830 nm Photobiomodulation are Retinoprotective in a Rodent Model of Retinitis Pigmentosa

Janis Eells¹, Heather Schmitt², Phyllis Summerfelt³, Adam Dubis⁴, Joseph Carroll⁴, Sandeep Gopalakrishnan^5
1BIOMEDICAL SCIENCES, COLLEGE OF HEALTH SCIENCES, UW-MILWAUKEE, MILWAUKEE; ²PATHOLOGY, UW-MADISON, MADISON;³OPHTHALMOLOGY, MEDICAL COLLEGE OF WISCONSIN, MILWAUKEE; ⁴OPHTHALMOLOGY, UNIVERSITY COLLEGE LONDON, LONDON; ⁵COLLEGE OF NURSING, UW-MILWAUKEE, MILWAUKEE

Purpose: Our laboratory has shown that that 830 nm photobiomodulaton (PBM) protects against retinal dysfunction and photoreceptor cell death in the P23H rat when administered early in the course of the disease. The purpose of the present study was to determine if 830 nm PBM would continue to protect against retinal dysfunction and photoreceptor cell death as the animals matured.

Methods: P23H rats were treated once per day with 830 nm light (180 s; 25 mW/cm2; 4.5 J/cm2) using a light-emitting diode array (QDI, Barneveld WI) from [1] postnatal day p10 to p25, [2] p10 to p40 or [3] p20 to p40. Sham-treated rats were restrained for 180 seconds, but not exposed to 830 nm light. Retinal function and morphology were assessed at p30 or p45 by electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT).

Results: 830 PBM preserved retinal function and retinal morphology in 830 nm light-treated animals in comparison to the sham-treated group in each treatment protocol. ERG responses were greater in 830 nm light-treated P23H rats compared to sham-treated P23H rats. SD-OCT imaging showed that 830 nm PBM also preserved the structural integrity of the retina in each treatment protocol.

Conclusions: Our findings confirm that the retinoprotective effects of 830 PBM observed early in the course of retinal degeneration in the P23H rat persist as the animals mature. Based on our findings and on other studies documenting the neuroprotective actions of PBM in experimental and clinical studies, we propose that photobiomodulation is an innovative, non-invasive therapeutic approach for the treatment of retinal degenerative disease.

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Genetic Mutation in NHE1 Mice Causing Anophthalmia or Other Abnormalities of the Eye

Samantha Ellingson¹, Amy Cook¹, Markus H. Kuehn^1,2
1The Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, The University of Iowa ²Center of Excellence for Prevention and Treatment of Visual Loss Iowa City Veterans Administration Medical Center

Purpose: To identify the gene or group of genes responsible ocular malformations in a new mouse model (NHE1). These abnormalities include missing two eyes or one eye since birth, and deformities on the retinal surface.

Methods: Whole exome sequencing was carried out on DNA from a NHE1 mouse without eyes and non-synonymous mutations were identified. The association of these sequence changes with the observed phenotype was then tested in additional affected and control mice through Sanger sequencing and melt-map analyses. Additionally, retinas were examined using in vivo imaging (Micron III) and histochemistry.

Results: Mice from this strain frequently lack one or both eyes. Alternatively, some mice develop a less severe phenotype, characterized by retinal abnormalities that can be detected by fundus photography. The trait is inherited in an autosomal dominant pattern. Exome sequence analysis identified approximately 58 heterozygous non- synonymous sequence variations. We are currently in the process of evaluating and verifying these in additional animals.

Conclusions: The NHE1 mouse is a novel model of congenital eye disease. We anticipate that continued sequence analysis and morphologic analysis will lead to the identification of a novel gene causing anophthalmia and other abnormalities in the eye.

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Brain-Derived Neurotrophic Factor Protects Retinal Ganglion Cells in Mice with Sustained Ocular Hypertension

Feng, Liang^1,2, Chen, Hui¹, Suyeoka, Genn¹, Liu, Xiaorong^1,2
Departments of ¹Ophthalmology and ²Neurobiology, Northwestern University Evanston, IL 60208

Purpose: Glaucoma is a group of neurodegenerative diseases characterized by retinal ganglion cell (RGC) death. Growing evidence suggest that brain-derived neurotrophic factor (BDNF), one of the survival factors for neuronal development and function, acts as a neuroprotective agent in the diseased condition. Yet little is known about whether BDNF signaling protects retinal circuitry and visual function against the insult of glaucoma. In this study, we thus investigated whether and how BDNF preserves RGCs and vision in a mouse model of experimental glaucoma.

Methods: Using an inducible Cre-mediated recombination system, we generated a transgenic mouse line to over-express BDNF in a temporally- and spatially-controlled manner. To activate BDNF downstream signaling, we also applied a small molecule named 7,8-dihydroxyflavone (7,8-DHF), which is a selective agonist to TrkB, the high-affinity receptor of BDNF. With a previously established mouse model of experimental glaucoma (Feng et al., 2013), we examined the changes of RGC and vision loss when BDNF signaling was up-regulated.

Results: Over-expression of BDNF reduced the RGC and axon loss in ocular hypertensive mice. The gradual decrease of visual acuity in ocular hypertensive mice was also alleviated. Moreover, administration of 7,8-DHF through drinking water activated the BDNF/TrkB signaling in the retina, resulting in a similar protective effect on RGCs and vision in ocular hypertensive mice.

Conclusions: Our data demonstrated that up-regulation of BDNF/TrkB signaling reduced RGC loss and vision loss in mice with sustained IOP elevation. Our study provides a unique opportunity for therapeutic intervention in high-tension glaucoma.

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Retinal defects resulting from loss of Polo-like kinase

Jillian Goetz, Jeffrey Trimarchi
Department of Genetics, Development, and Cell Biology, Iowa State University

Purpose: During retinogenesis seven different cell types are generated in distinct yet overlapping timepoints from a population of retinal progenitor cells (RPCs). At any given time, RPCs are at various stages of development and differentiation. Therefore, any whole -tissue analyses run the risk of averaging out the intrinsic signals that drive a progenitor towards a specific fate. Previously, we have performed single cell transcriptome analyses of RPCs to identify candidate genes that may play roles in the generation of early-born retinal neurons. Polo-like kinase 3 (Plk3) is one candidate gene that is highly expressed in subsets of early retinal cells. Although previous studies outside of the retina have determined Plk3 and its family members play roles in cell cycle maintenance, our preliminary studies indicate the kinase plays a different role in the retina.

Methods: We have obtained a Plk3-KO mouse and investigated changes in the retinal morphology and transcriptome through immunohistochemistry, in situ hybridization and gene expression profiling. These experiments have been performed initially on adult mice and subsequently continued throughout retinal development.

Results: Plk3-KO mice exhibit exuberant processes leading to extensive destratification and disorganization of the retinal plexiform layers. Microarrays have revealed many potential candidate genes changed in Plk3-KO mice. Ongoing studies are being performed to link these gene changes to the phenotypes in the mice.

Conclusions: Single-cell transcriptomics revealed the Plk3 is expressed in a subset of RPCs during early retinal development. Studies of Plk3-KO mice have shown plexiform layer defects that we are further characterizing.

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Reprogramming Zebrafish Müller Glia for Retinal Repair

Daniel Goldman, Rajesh Ramachandran, Jin Wan and Xiaofeng Zhao
MOLECULAR AND BEHAVIORAL NEUROSCIENCE INSTITUTE AND DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF MICHIGAN, ANN ARBOR, MI, USA

Purpose: Identify secreted factors and signal transduction cascades regulating Müller glia reprogramming and retina regeneration in zebrafish

Methods: Gene expression and knockdown studies were used to identify secreted factors and signal transduction cascades that stimulate Müller glia reprogramming in the injured and uninjured retina. PCR and BrdU labeling were used to investigate the effect these secreted factors and signal transduction cascades had on Müller glia reprogramming and proliferation.

Results: We identified growth factors, cytokines and Wnts that are induced in the injured retina and sufficient to stimulate Müller glia reprogramming and retina regeneration. We found that these factors are produced by Müller glia and act in a synergistic fashion. Their activity requires MAPK, PI3K, beta-catenin and Stat3 signaling pathways and these pathways exhibit extensive crosstalk.

Conclusions: A variety of factors secreted by the injured retina have the potential to drive Müller glia reprogramming and retina regeneration. The local release of secreted factors by Müller glia residing at the injury site suggest Müller glia drive their own reprogramming. The synergistic action of these factors allows them to stimulate Müller glia proliferation at low concentrations. The extensive cross-talk exhibited by these factors suggests a feed-forward type of mechanism underlies Müller glia reprogramming and retina regeneration. These studies provide a fish-eye’s view of the reprogramming and regeneration process and suggest novel strategies for stimulating Müller glia reprogramming in mammals.

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Classical neuroinflammatory processes occur in the pathology of glaucoma

Oliver W. Gramlich¹, Markus H. Kuehn^1,2
1 Department of Ophthalmology and Visual Sciences, The University of Iowa; ² Center for the Prevention and Treatment of Visual Loss, , VA Medical Center, Iowa City, IA

Purpose: Retinal ganglion cell degeneration in glaucoma is linked to neuroinflammatory processes, including complement activation, and autoimmune involvement. This study aims to determine how autoantibodies, complement proteins and microglial activation interact in the complex pathology of glaucoma.

Methods: Microglia, IgG, and depositions of complement proteins were examined immunohistologically in donor retinae of 15 healthy subjects (CTRL) and 15 primary open angle glaucoma patients (POAG). Immunoproteomic studies analyzing neuroinflammatory processes were conducted in a second cohort.

Results: IgG depositions in the retinal ganglion cell layer are observed both in CTRL and POAG eyes, but the amount in glaucomatous samples is significantly increased (CTRL: 0.9+/-0.7 IgG/100 cells, POAG: 1.5+/-0.8 IgG/100 cells; p=0.036). C3 and C5b-9 are exclusively seen in glaucoma subjects and very few IgG accumulations co-localize with C5b-9 depositions or microglia. Furthermore, manifestations of a focal pro-inflammatory environment in degenerating retinae can be observed.

Conclusions: The pathology of glaucoma features many common neuroinflammatory mechanisms analogous to those in other neurodegenerative diseases. These are characterized by local complement accumulation and microglial activation under pro-inflammatory conditions. Whether retinal IgG autoantibodies, especially in combination with complement proteins or microglia, have profound pathogenic impact in glaucoma will require further investigation.

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Triplication of upstream regulatory sequences leads to gene dysregulation in patients with cavitary optic disc anomaly

Ralph J. Hazlewood^1,2, Benjamin R. Roos^1,2, Robert A. Honkanen³, Lee M. Jampol⁴, Stephen C. Gieser,⁵ Kacie J. Meyer ^1,2, Robert F. Mullins^1,2, Markus H. Kuehn^1,2, Young H. Kwon^1,2, Wallace L.M. Alward^1,2, Edwin M. Stone^1,2,6, John H. Fingert^1,2
1Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, ²Stephen A. Wynn Institute for Vision Research, University of Iowa, Iowa City, IA ³Department of Ophthalmology, State University of New York at Stony Brook, Stony Brook, New York, ⁴Department of Ophthalmology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois ⁵Wheaton Eye Clinic, Wheaton, IL ⁶Howard Hughes Medical Institute, Iowa City, IA

Purpose: To identify and functionally characterize the gene that causes autosomal dominant, congenital malformations of the optic nerve known as cavitary optic disc anomaly (CODA) in a multiplex family with 17 affected members.

Methods: The gene that causes CODA was previously mapped to a 13.5 Mb locus on chromosome 12q14. Members of the CODA pedigree was tested for copy number variations (CNVs) within this region with custom comparative genomic hybridization arrays. Confirmed CNVs were analyzed for their effect on downstream genes using a pGL3 luciferase reporter gene construct in HEK293T cells. Changes in protein and RNA expression were assessed by immunohistochemistry, western blot, and quantitative PCR.

Results: We identified a triplication of a 6Kb DNA segment upstream of matrix metalloproteinase 19 (MMP19) in all affected members of the CODA pedigree. No control subjects carried this mutation. Moreover, we detected an overlapping triplication in another small CODA pedigree. The luciferase reporter gene assay showed that the 6Kb sequence spanned by the CNV in CODA subjects functioned as a transcription enhancer, in particular, a 773 bp segment had a strong positive influence (8-fold higher) on downstream gene expression. Lastly, we detected robust and specific expression of MMP19 in optic nerve head.

Conclusions: We have identified a CNV mutation in the promoter sequence of the MMP19 gene that co-segregates with CODA in our large 17 member pedigree. Moreover we have shown that the CNV spans DNA sequences that powerfully enhance downstream genes (i.e. MMP19) and that MMP19 is expressed at the site of injury in the optic nerve. Together these data strongly suggest that overexpression of MMP19 in the optic nerve due to triplication of an upstream enhancer element may cause CODA.

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Investigating the influence of blast on cellularity in the retinal ganglion cell layer in a mouse model of blast-induced traumatic brain injury using a novel semi-automated technique.

Adam Hedberg-Buenz¹,⁴, Mark Christopher², Mona Garvin^1,2, Todd Scheetz^2,3, Laura Dutca¹, Randy Kardon^1,3, Matt Harper^1,3, Michael Anderson^1,3,4
1Center for the Prevention and Treatment of Visual Loss, VA Medical Center, Iowa City, IA. Departments of ²Engineering, ³Ophthalmology and Visual Sciences and, ⁴Molecular Physiology and Biophysics, The University of Iowa

Purpose: Blast-mediated injuries are the leading cause of combat-related injury in modern warfare. Visual dysfunction has been reported in Veterans with blast-mediated traumatic brain injury (TBI). We have previously shown retinal ganglion cells (RGC) are sensitive to blast exposure. However, the timeframe and magnitude of RGC loss is not yet understood. The purpose of these experiments is to develop a method to quantify cellularity and investigate the influence of blast on the retinal ganglion cell layer (GCL) after blast-induced TBI.

Methods: C57BL/6J mice were exposed to an overpressure wave directed to the head using a custom-built blast chamber (blast-injured). Mice placed in the chamber without blast were used as controls. At 4 months post-blast, retinas from blast-injured (n=16) and control (n =12) eyes were mounted whole, stained, and uniformly imaged. Retinal images were quantitatively assessed for cellularity in the GCL using a custom plugin for Image J. A semi-automated method using ImageJ was developed that quantifies GCL density. In the peripheral retina, blast-injured mice exhibit a significant decrease in density compared to controls (p = 0.03, 2-tailed Students t-test). In the central retina, blast-injured mice exhibit a trend of reduced density compared to controls.

Results: These findings demonstrate that this mouse model of blast-induced TBI involves a loss of GCL cellularity by 4 months post-exposure. Using this model and quantification method, our ongoing studies will test mechanisms contributing to this RGC susceptibility, with a long term goal of contributing to improved clinical testing and treatment of visual deficits to those suffering from TBI.

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CRISPR-Cas9 Mediated Genome Editing of Myocilin in Trabecular Meshwork Cells

Ankur Jain¹, Gulab Zode², Charles Searby¹, Abe Clark², Val C Sheffield^1
1Pediatrics, Carver College of Medicine, University of Iowa, Iowa ²North Texas Eye Research Institute, UNT Health Science Center at Fort Worth, Texas

Purpose: Glaucoma is a leading cause of irreversible blindness with elevated intraocular pressure (IOP) being the most important risk factor. Mutations in myocilin (MYOC) leading to MYOC misfolding and increased expression of MYOC by dexamethasone (DEX) have been associated with endoplasmic reticulum (ER) stress in the trabecular meshwork (TM), the tissue that maintains the aqueous humor outflow and regulates IOP. ER stress and/or death of the TM lead to ocular hypertension and glaucoma. We propose to relieve ER stress in human and mouse TM cells by targeting the MYOC gene using state of art CRISPR-Cas9 technology.

Methods: We generated TM cell lines overexpressing wild type and mutant human MYOC and looked for ER stress markers using quantitative PCR and western immunoblotting. MYOC was targeted by transient transfection of px330-CRISPR plasmids with guide RNAs targeting the MYOC gene (Exon1). Mouse TM cells were pretreated with Ad5px330-CRISPR virus and checked for DEX-mediated MYOC accumulation and ER stress.

Results: TM cell lines overexpressing mutant MYOC have increased accumulation of MYOC in the ER and increased ER stress as compared to wild type. Transient transfection reduces levels of MYOC and ER stress in these cells. Mouse TM cells have reduced levels of DEX-mediated ER stress when pretreated with Ad5-CRISPR as compared to null virus controls.

Conclusions: This is a proof of principle study indicating that targeting the MYOC gene can relieve ER stress in TM cells, rescuing their function. We plan to translate this approach to in vivo transgenic hMYOCY437H and DEX-induced ocular hypertension mouse models.

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Multifunctional antioxidants for oxidative changes and neuroprotection

Peter F. Kador^1-3, Hiroshi Kawada¹, and James Randazzo^1
1 Department of Pharmaceutical Sciences; ² Department of Ophthalmology, University of Nebraska Medical Center, Omaha, NE ³ Therapeutic Vision, Inc., Omaha, NE

Purpose: Synthesize orally active multifunctional antioxidants (MFAOs) possessing distinct free radical scavenging activity and independent metal attenuating activity and evaluate these compounds in ocular cell lines and animal models of cataract and retinal degeneration.

Methods: In vitro cell culture studies and in vivo animal models have been employed.

Results: In cell lines such as human retinal pigmented epithelial (RPE) cells, these compounds protect the cells against not only ROS generated by the Fenton Reaction but also mitochondrial damage induced by manganese chloride and neurotoxicity induced by Aβ:Zn complex formation. Interestingly, these compounds can remove Zn from the tightly bound Aβ:Zn complex but allow free Zn levels to be maintained in the cellular cytoplasm. In rodents MFAOs protect the lens against ROS generated by ER stress, gamma, and UV irradiation and in the retina, protect the photoreceptor cells against light damage, the accumulation of Abeta plaque, and possibly traumatic injury.

Conclusions: These studies suggest that MFAOs represent a new class of drugs for the treatment of age related ocular diseases such as cataract, macular degeneration, and other neurodegenerations.

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Prevalence of Structural Abnormalities of the Retinal Nerve Fiber Layer and Ganglion Cell Layer Complex by OCT in Veterans with Traumatic Brain Injury

Randy Kardon¹, Kelvin Lim¹, Mona Garvin¹, Ray Wang¹, Glenn Cockerham^4
1 UNIVERSITY OF IOWA AND VETERANS AFFAIRS MEDICAL CENTER, IOWA CITY, IA; DEPARTMENT OF OPHTHALMOLOGY AND VISUAL SCIENCES; ² VETERANS AFFAIRS MEDICAL CENTER AND UNIVERSITY OF MINNESOTA, MINNEAPOLIS, MN, DEPARTMENT OF PSYCHIATRY; ³ UNIVERSITY OF IOWA AND VETERANS AFFAIRS MEDICAL CENTER, IOWA CITY, IA; DEPARTMENT OF ENGINEERING, ELECTRICAL AND COMPUTER SCIENCE DIVISION; ⁴ DEPARTMENT OF VETERANS AFFAIRS AND PALO ALTO VA MEDICAL CENTER, PALO ALTO, CA

Purpose: To determine the prevalence of structural loss of neurons in the retina of veterans with a history of Traumatic Brain Injury (TBI) compared to an age matched control group of veterans.

Methods: Cirrus volume OCT scans of the optic nerve and macula were obtained in 74 veterans with a history of TBI and 45 control veterans from the Palo Alto and Minneapolis VA Polytrauma Centers. The volume scans were segmented in 3 dimensions to derive the thickness of the retinal nerve fiber layer (RNFL) and the retinal ganglion cell -inner plexiform layer complex (GCLC).

Results: Thinning of the average RNFL below the 5th percentile of control patients occurred in 14.8% in right eyes and 7.6% in left eyes from the Palo Alto VA cohort. Thinning of the average GCLC occurred in 24.5% in right eyes and 14.8% in left eyes. In the Minneapolis VA cohort, regression of GCLC thickness vs time since TBI showed a negative slope that was greater than predicted by age of the subject.

Conclusions: OCT analysis showed almost twice the prevalence of abnormalities in the retinal ganglion cell layer complex compared to the RNFL in veterans with TBI. There appears to be thinning of the retina dependent on time since TBI that may indicate progression of neuron loss after TBI. Ongoing investigations will determine correlation between structural loss and visual field loss and neurological deficits over time in individual patients who are retested.

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A non-canonical role for Cav1.4 channels in photoreceptor synapse maturation

Vasily Kerov^1,2, Joe Laird¹, Brittany Williams², Sheila Baker¹, Amy Lee²
Departments of ¹Biochemistry and ²Molecular Physiology and Biophysics, Carver College of Medicine, The University of Iowa

Purpose: Cav1.4 voltage-gated calcium channel is expressed in the synaptic terminals of photoreceptors (PR) cells and is responsible for the calcium flux into the cell which triggers synaptic vesicle release. Mutations in Cav1.4 or its regulatory subunit, calcium-binding protein 4 (CaBP4) lead to visual disorders including congenital stationary night blindness type 2 (CSNB2) suggesting the effect of altered calcium signaling. We investigated the nature of the disease using in vivo transfection of Cav1.4 and CaBP4 constructs into Cav1.4 KO mice in which synapse maturation is disrupted. Unexpectedly we find that Cav1.4 has a crucial role in the PR synapse that is independent of the channel conductance. Both, wild-type (Cav1.4 WT) and non-conducting channel (Cav1.4 3EQ) upon transfection into PRs of Cav1.4 KO mice rescued the mature synapse structure. Expression of a Cav1.4 mutant incapable of binding CaBP4 (Cav1.4.45A) did not rescue the synapse morphology, whereas Cav1.4.45A-CaBP4 fusion successfully rescued the terminals, indicating critical role of Cav1.4-CaBP4 interaction in the organization of PR synaptic scaffold. These results reveal new structural role for voltage-gated calcium channels in neurons and change our understanding of the effect of a number of CSNB2 mutations previously thought to be due to disrupted conducting properties of the channel.

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Light aversive behavior in mice lacking Rgs9-/-

Adisa Kuburas¹, Stewart Thompson², Nikolai O. Artemyev¹, Randy H. Kardon^2,4, Andrew F. Russo^1,3,4
Departments of ¹Molecular Physiology and Biophysics, ²Ophthalmology and Visual Sciences, ³Neurology, University of Iowa, Iowa City, IA ; ⁴Veterans Affairs Health Care System, Iowa City, IA

Purpose: Mutations in the RGS9 gene lead to the visual disorder Bradyopsia, characterized by difficulty adapting to changes in luminance and photophobia. Rgs9 knockout (Rgs9-/-) mice exhibit slow photoreceptor deactivation, consistent with the symptoms observed in patients. In this study we wanted to test if Rgs9-/- mice show photophobia-like behavior.

Methods: As a surrogate for photophobia, we measured light aversive behavior in an operant assay. We also measured negative masking (suppression of wheel-running activity by light) and pupillary light reflex in Rgs9-/- mice.

Results: Our results show that Rgs9-/- mice spent less time in the light than wild-type C57BL/6J mice at all illumination levels. These mice also showed increased sensitivity to light in negative masking behavior. Both genotypes showed similar behavior in open field and predator odor-freezing assays, which suggests that light aversive behavior of the Rgs9-/- mice is not due to a general increase in anxiety. Constriction of the pupil showed that Rgs9-/- mice had an abnormally sustained response to light.

Conclusions: From these data we conclude that a single gene mutation that prolongs rod photoreceptor activation generates photophobia-like behavior in mice. The Rgs9-/- mouse model will be useful in studies of photophobia and for development of therapeutic strategies.

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Engineered TALEN induced mutations in Regulator of G-protein signaling 16

Lauren Laboissonniere, Jeffrey Trimarchi
Department of Genetics, Development, and Cell Biology Iowa State University

Purpose: Math5 is a bHLH transcription factor implicated in the generation of several retinal neurons, especially retinal ganglion cells. Single cell transcriptome profiling of Math5+ single cells showed a strong correlation between Math5 expression and that of the gene for regulator of G-protein signaling 16 (RGS16). Previous studies have determined the role of RGS16 in the pancreas and inflammatory system; however, no study has yet examined the role of RGS16 in cell fate determination of the retina.

Methods: Transcription activator-like effector nucleases (TALEN) are a useful technology to observe the role of a gene in the development of the retina. This technology can be used to engineer a mutation at a desired location within a particular gene. We have generated a TALEN specifically targeted against RGS16.

Results: We have injected our TALEN mRNA into zebrafish at the one cell stage and genotyped a selection of injected fish at 48 hours post fertilization (hpf). From these initial injected fish we identified those that transmit the mutation to their offspring. These fish will be crossed in the future and their retina phenotypes examined.

Conclusions: Single cell transcriptomics identified several genes that strongly correlate with Math5. We predict these genes will play an important role in cell fate determination. To assess that role, we have used TALEN technology to generate mutations against RGS16 in zebrafish.

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Interphotoreceptor Matrix Changes in Best Vitelliform Macular Dystrophy

Daniel Locker, Arlene V. Drack, Robert F. Mullins, Markus H. Kuehn
The Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, The University of Iowa

Purpose: Best disease is an autosomal dominant, juvenile onset macular dystrophy that is characterized by an unusual "egg yolk" lesion in the macula. This disease is caused by mutations in the gene BEST1 which is believed to act as an ion channel or ion channel regulator. Disturbances in ion homeostasis can cause dramatic changes in interphotoreceptor matrix (IPM) structure and function. To investigate wheather lack of BEST1 alters the IPM we examined the differences between light and dark adapted IPM states in a knock-in mouse model of Best disease.

Methods: Mice with a Tryp93Cys mutation in the Best1 gene control mice were sacrificed in light or dark adapted states. The eyes were immediately fixed in 4% paraformaldehyde, processed for cryosectioning and lectin-stained with biotinylated wheat germ agglutinin (WGA) visualized with Texas red avidin. Fluorescence was analyzed by intensity and area using ImageJ.

Results: WGA labeling intensity differs between dark and light adapted in both cohorts, but mean intensity and light-dark differences are increased in Best.

Conclusions: Mutations in the bestrophin gene appear to alter IPM physiology which may be related to the pathogenesis of Best's Disease. Further studies should be carried out to investigate how the dynamics changes observed may lead to photoreceptor loss in affected patients.

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Tumor necrosis factor alpha has an early protective effect on retinal ganglion cells after optic nerve crush

Mac Nair, Caitlin^1,2; Fernandes, Kimberly³; Schlamp, Cassandra¹; Libby, Richard^3,4,5; Nickells, Robert^1
1 Ophthalmology and Visual Sciences, University of Wisconsin - Madison, Madison, Wisconsin ²Cellular and Molecular Pathology Graduate Program, University of Wisconsin - Madison, Madison, WI ³Flaum Eye Institute, University of Rochester Medical Center, Rochester, NY ⁴Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, NY ⁵The Center for Visual Sciences, University of Rochester Medical Center, Rochester, NY

Purpose: The degeneration of retinal ganglion cells (RGCs) during glaucoma has been thought to occur in two waves. The first is axogenic, caused by injury to the optic nerve, and the second is somatic, thought to be caused by inflammatory cytokine production from retinal innate immune cells. One of the suspected cytokines is tumor necrosis factor alpha (TNFα). Despite strong evidence implicating TNFα in neurodegeneration, a direct injection does not trigger rapid RGC loss like acute optic nerve trauma. This suggests that our understanding of TNFα signaling is incomplete.

Methods: RGC death was induced by optic nerve crush in mice. Tnfα gene expression was determined by QPCR, and the role of TNFα following crush was examined through quantification of cell loss in Tnfα ^-/-� mice, or in wild type animals receiving an intraocular injection of exongenous TNFα either before or after crush. Signaling pathways downstream of TNFα were examined, including JUN protein accumulation and EGFP expression in NFκβ reporter mice.

Results: Optic nerve crush caused a modest increase in Tnfα gene expression, with kinetics similar to the activation of both macroglia and microglia. A pre-�injection of TNFα attenuated ganglion cell loss after crush, while ganglion cell loss was more severe in Tnfα^-�/- mice. Conversely, long-term exposure to TNFα induces extrinsic apoptosis in RGCs. Muller cells responded to exogenous TNFα by accumulating JUN and activating NFκβ.

Conclusions: Early after optic nerve crush, TNFα appears to have a protective role for RGCs, which may be mediated through Muller cells and NFκβ.

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BAX Oligomerization Kinetics and its Role in Retinal Ganglion Cell Death

Maes, ME, Schlamp, CL, and Nickells, RW.
University of Wisconsin-Madison Department of Ophthalmology

Purpose: Glaucoma is characterized by retinal ganglion cell (RGC) apoptosis. RGC death occurs through a BAX-dependent apoptotic pathway in which BAX oligomerization marks the committed step of this process. By understanding the mechanism of BAX function within the apoptotic pathway, we can identify the opportune time for therapeutic intervention during RGC death.

Methods: In vitro, BAX oligomerization kinetics were followed in living cells expressing fluorescently labeled BAX protein. Changes in BAX fluorescence at individual mitochondria were quantified using IMARIS 7.7. The curve fitting function in the SciPy library was used to fit these data. ONC was performed on mice after intraocular injection of AAV2-GFP-BAX. Three days post-ONC, retinas were whole mounted and monitored by confocal microscopy.

Results: BAX oligomerization followed a sigmoidal growth curve from which the time of initiation and rate of oligomerization could be calculated. Mitochondria in individual cells exhibited nearly identical nucleation times but varying rates of oligomerization. Cytochrome c release confirmed functional BAX oligomer formation. Bax oligomerization was visible in mouse retina three days post-ONC.

Conclusions: The kinetics of BAX oligomerization was similar between cell lines. Further studies of BAX oligomerization kinetics in vivo will help identify the opportune time for therapeutic intervention after RGC injury.

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Identification of quantitative trait loci that modify Cep290-mediated retinal degeneration

Kacie J. Meyer, Michael G. Anderson
Departments of ¹Molecular Physiology and Biophysics, and ²Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, IA; ³ Center for the Prevention and Treatment of Vision Loss, VA Medical Center, Iowa City, IA

Purpose: CEP290-mediated Leber Congenital Amaurosis (LCA) is an inherited retinal degeneration characterized by nystagmus and blindness that typically manifest in the first year of life. Currently, CEP290-mediated LCA has no treatment and unfortunately, CEP290 is not a particularly attractive therapeutic target due to its large size and lack of enzymatic activity. However, the variable expressivity of CEP290 LCA, even among individuals with similar CEP290 mutations, provides an important clue that treatment may be found through the manipulation of other genes capable of modifying the CEP290 LCA phenotype. The goal of this research is to use phenotype-driven studies in mice to identify these genetic modifiers of CEP290-mediated retinal degeneration, which might be manipulated in the future as therapeutic surrogates in lieu of CEP290 itself.

Methods: The retinas of Cep290-mutant mice resulting from a backcross (N2) and an intercross (F2) were phenotyped using Bioptigen optical coherence tomography. The mice were also genotyped on the Fluidigm platform with a genome-wide panel of 96 polymorphic genetic markers. Quantitative trait locus (QTL) analysis, which identifies genotype-phenotype correlations, was performed using R/qtl. Retinal fundi were photographed with a Micron III and retinal electrical response was measured by electroretinography.

Results: QTL analysis ofN2 and F2 mice has identified 5 significant QTL capable of modifying CEP290-mediated retinal degeneration. Cep290-mutant mice with modified retinal phenotypes have improved retinal lamination, retinal vasculature, and retinal electrical responses.

Conclusions: These experiments demonstrate that Cep290rd16retinal phenotypes are sensitive to genetic background and identify multiple tractable loci that modify CEP290-mediated retinal degeneration.

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Poster #29

Trafficking of HCN1 channels in the early secretory pathway: Discovery of a di-arginine ER retention signal using Xenopus photoreceptors as a model system

Yuan Pan, Joseph G. Laird, David M. Yamaguchi and Sheila A. Baker
Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa

Purpose: Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels carry Ih which contributes to neuronal excitability and signal transmission in the nervous system. In photoreceptors, HCN1 is primarily localized in the membrane of inner segments and functions by keeping light responses transient. Controlling the trafficking of HCN1 is an important aspect of its regulation, yet the details of this process are poorly understood. Our goal is to identify regulatory elements that control HCN1 trafficking in photoreceptors.

Methods: Various regions of HCN1 were fused to a membrane reporter and expressed in transgenic Xenopus rods. Localization of the transgenically expressed proteins was visualized with confocal microscopy. Functions of wildtype and mutant HCN1 were studied in HEK293 cells using immunocytochemistry and biotinylation approaches.

Results: We found that HCN1 contains an ER localization signal and through a series of deletion constructs, identified the responsible di-arginine ER retention signal. This signal is located in the intrinsically disordered region of the C-terminus of HCN1 and negatively regulates surface expression of HCN1.

Conclusions: We report a new mode of regulating HCN1 trafficking: through the use of a di-arginine ER retention signal that monitors processing of the channel in the early secretory pathway.

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Perfusion model - model to predict "in-vivo" transport of injected drug surrogate

Anita Penkova^1,2, Komsan Rattanakijsuntorn¹, Rex Moats², Susan Lee^3,4, Michael Robinson^3,4, Satwindar S Sadhal^1,2
1Aerospace & Mechanical Engineering Department, University of Southern California, Los Angeles, CA; ² SAIRC, Saban Research Center, Children’s Hospital Los Angeles, Los Angeles, CA; ³ Biomedical Engineering Department, University of Southern California, Los Angeles, CA; ⁴ Global Pharmaceutical Sciences, Allergan, Inc., 2525 Dupont Dr, Irvine, CA

Eye diseases such as Age-related Macular Degeneration (AMD) and Diabetic Retinopathy (DR) affect a large segment of the US population, projected to rise substantially by the year of 2020. According NIH statistics and data in 2020 the number of people having AMD will reach up to 3 million. While many advances have been made for drug development, proper delivery to specific ocular regions such as the retina is still a significant medical, pharmaceutical and fluid dynamics problem. Among the challenges and key components of the research designs are:

1. Further development of drug delivery methods that target the retina and minimize the losses to unaffected regions.

2. The development of mathematical models considering various transport mechanisms to effectively predict drug concentration distribution of the injected (delivered) surrogate.

3. Close replication of living conditions for ex vivo eye samples: we have created perfusion model on “ex-vivo” bovine eyes for the purpose of mimicking a living eye. With the convection current established we can study the preferential (directional) movement of the injected surrogate in the human vitreous for example on which we have obtained preliminary data

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Histone deacetylase 3 (HDAC3) plays an important role in retinal ganglion cell death after acute optic nerve injury

Heather M. Schmitt¹, Heather R. Pelzel², Cassandra L. Schlamp¹, Robert W. Nickells¹*
¹Department of Ophthalmology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI ²Department of Biology, University of Wisconsin-Whitewater, Whitewater, WI

Purpose: Optic nerve damage initiates a series of early atrophic events in retinal ganglion cells (RGCs) that precede the BAX-dependent committed step of the intrinsic apoptotic program. Nuclear atrophy, including global histone deacetylation, heterochromatin formation, collapse of nuclear structure, and the silencing of normal gene expression, comprise an important obstacle to overcome in therapeutic approaches to preserve neuronal function. Several studies have implicated histone deacetylases (HDACs) in the early stages of neuronal cell death, including RGCs. Importantly, these neurons exhibit nuclear translocation of HDAC3 shortly after optic nerve damage.

Methods: RGC-specific conditional knockout of Hdac3 was achieved by transducing the RGCs of Hdac3fl/fl mice with an adeno-associated virus serotype 2 carrying CRE recombinase and GFP (AAV2-Cre/GFP). Controls included similar viral transduction of Rosa26fl/fl reporter mice. Optic nerve crush (ONC) was then performed on eyes.

Results: The ablation of Hdac3 in RGCs resulted in significant amelioration of characteristics of ONC-induced nuclear atrophy such as H4 deacetylation, heterochromatin formation, and the loss of nuclear structure. RGC death was also significantly reduced. Interestingly, loss of Hdac3 expression did not lead to protection against RGC-specific gene silencing after ONC, although this effect was achieved using the broad spectrum inhibitor, Trichostatin A.

Conclusions: Although other HDACs may be responsible for gene expression changes in RGCs, our results indicate a critical role for HDAC3 in nuclear atrophy in RGC apoptosis following axonal injury. This study provides a framework for studying the roles of other prevalent retinal HDACs in neuronal death as a result of axonal injury.

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Whole Exome Sequencing in Developmental Ocular Disorders

Elena V. Semina, Linda M. Reis, Brett Deml, Eric Weh, Rebecca C. Tyler
DEPARTMENT OF PEDIATRICS AND CHILDREN'S RESEARCH INSTITUTE, MEDICAL COLLEGE OF WISCONSIN, MILWAUKEE, WI

Purpose: A large number of genes are known to play an important role in eye development and disruption of these genes results in predictable ocular conditions. We aimed to identify the proportion of developmental ocular disorders which can be explained by mutations in known genes as well as to identify novel causative mutations in cases that lack changes in the previously reported factors.

Methods: We enrolled 214 probands with developmental ocular conditions including MAC (59), Axenfeld-Rieger anomaly/syndrome (22), congenital/juvenile cataracts (43), congenital/juvenile glaucoma (12), Peters anomaly/syndrome (45), or other ASD (33). We undertook genetic screening by a combination of single gene sequencing, quantitative PCR, array comparative genomic hybridization, and whole exome sequencing.

Results: Pathogenic mutations were identified in 85 probands (40%; 74 single gene mutation and 12 copy number variants); analysis of additional possible pathogenic variants is ongoing. The highest mutation detection rate was for Axenfeld-Rieger anomaly/syndrome with pathogenic mutations identified in 73%; the lowest rate was for non-familial cataracts with no pathogenic mutations identified; mutation detection rates for the other diagnoses ranged from 30-50%. Novel phenotype/genotype correlations were suggested for several genes. Analysis of known-gene-negative cases identified several strong candidates, including MAB21L2 in a family affected with coloboma, microcornea and cataracts.

Conclusions: The genetic and phenotypic heterogeneity of ocular disorders makes genetic diagnosis challenging; use of whole exome sequencing is effective in identifying causative mutations in known genes and facilitating novel gene discovery.

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Loss of Lztfl1, a negative regulator of the BBSome ciliary trafficking, causes BBS-like phenotypes in mice

Poppy Datta¹, Joseph S. Hudson¹, Emily Andersen¹, Sajag Bhattarai¹, Daniel R. Thedens², Kevin E. Bugge^3,4, Arlene V. Drack¹, Val C. Sheffield^3,4, Seongjin Seo¹
UNIVERSITY OF IOEWA DEPARTMENTS OF ¹OPHTHALMOLOGY AND VISUAL SCIENCES, ²RADIOLOGY, ³PEDIATRICS, ⁴HOWARD HUGHES MEDICAL INSTITUTE, IOWA CITY, IA

Purpose: Bardet–Biedl Syndrome (BBS) is a genetically heterogeneous ciliopathy characterized mainly by retinitis pigmentosa, polydactyly, and obesity. Recently, Leucine-zipper transcription factor-like 1 (Lztfl1) was identified as a negative regulator of the BBSome, a complex composed of 8 BBS proteins, and mutations in Lztfl1 have been found in several human BBS patients. This study is aimed to determine the roles of Lztfl1 in vivo and verify that loss of Lztfl1 function leads to BBS.

Methods: Lztfl1 mutant mouse line was generated using ES cells from Knockout Mouse Project (KOMP) repository. Retinal degeneration was studied by H&E staining of retinal sections and electroretinogram (ERG). Magnetic resonance imaging (MRI) was used to study obesity and hydrocephalus. Scanning and transmission electron microscopy (SEM and TEM) were used for ultrastructural analyses of cilia. H&E staining was used to examine testis and kidney abnormalities.

Results: Lztfl1mutant mice displayed severely reduced ERG and progressive loss of photoreceptor cells. These animals also displayed a significant increase in body weight and body fat mass. MRI and SEM studies revealed that Lztfl1 mutant mice have hydrocephalus and a reduced number of ependymal cilia in the brain ventricle. Finally, severe reduction in the number and motility of sperm flagella was observed in Lztfl1 mutants despite normal BBSome assembly.

Conclusions: Our findings indicate that Lztfl1is essential for maintaining photoreceptor cells, energy homeostasis, and normal cilia/flagella functions and verify that loss of LZTFL1 causes BBS. Further biochemical and proteomics studies are aimed to identify interacting partners and related cellular pathways.

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Generation of iPSC derived vascular endothelial cells for the treatment of AMD

Allison E. Songstad¹, Cathryn M. Cranston¹, Miles J. Flamme-Wiese¹, Edwin M. Stone¹,², Robert F. Mullins¹, Budd A. Tucker^1
1Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, and ²Howard Hughes Medical Institute, University of Iowa, Department of Ophthalmology, Iowa City, IA

Purpose: Age-related macular degeneration (AMD), the most common cause of incurable blindness in the western world, is characterized by the dysfunction and eventual death of choroidal endothelial, retinal pigment epithelial (RPE), and photoreceptor cells. Stem-cell based treatment strategies designed to replace both photoreceptor and RPE cells are currently a major scientific focus, and success of these approaches will undoubtedly also require replacement of choroidal vasculature. The Purpose of this study was to generate a Tie2 GFP iPSC reporter line to develop efficient vascular endothelial cell (EC) differentiation and transplantation protocols.

Methods: Tie2 GFP mouse fibroblasts were isolated and reprogrammed into induced pluripotent stem cells (iPSCs) via viral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc. iPSC potency was characterized via RT-PCR, immunocytochemistry, and teratoma assays. Tie2 GFP iPSCs were differentiated into embryoid bodies using a co-culture method with monkey choroidal ECs.

Results: Tie2 GFP iPSCs expressed the pluripotency markers Nanog, Oct4, Sox2, Klf4, and c-Myc as determined by RT-PCR, and immunocytochemistry. Following both embryoid body formation and transplantation into immune compromised SCID mice, undifferentiated cells formed tissues specific to each of the three embryonic germ layers. Pluripotent iPSCs subjected to co-culture with the monkey choroidal EC line RF/6A differentiated into vascular ECs that expressed choroidal endothelial markers and were morphologically indistinguishable from native choroidal ECs.

Conclusions: We successfully generated Tie2 GFP iPSCs and used them to develop vascular EC differentiation protocols. We have set the stage for future studies focused on investigation of disease pathophysiology and EC replacement.

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Compositional Studies of Human Retinal Lipofuscin: Wet versus Dry Age Related Macular Degeneration

Tournear, Jennifer; Dillon, James; Gaillard, Elizabeth
Northern Illinois University

Purpose: This study aims to further elucidate the chemical composition of human retinal lipofuscin including the investigation of fluorophores and photooxidative byproducts in order to better understand AMD.

Methods: Human retinal lipofuscin is isolated from human donor eyes diagnosed with either wet or dry AMD according to the method previously described by Feeney-Burns. The organic soluble fraction of lipofuscin is collected, dried, and reconstituted using methanol for use in high performance liquid chromatography tandem mass spectrometry (LC/MS) coupled with a photo diode array and fluorescence detector (Surveyor LC with PDA, Thermo Finnigan LCQ Advantage MS, Surveyor FL). Tandem mass spectrometry data is analyzed for the investigation of chemical composition specific to wet and dry AMD.

Results: Total ion current chromatograms observed from LC/MS analysis suggests unique chemical composition for tissue diagnosed as wet versus dry AMD. The extensively studied fluorophore, A2E, was not observed in lipofuscin extracts diagnosed as dry AMD. However, A2E and its derivatives were observed in extracts diagnosed as wet AMD and verified by analysis of fragmentation patterns.

Conclusions: The lipofuscin extracted from tissue diagnosed as wet and dry AMD suggest different chemical composition. Analysis of an undefined AMD lipofuscin extract shows similarity to data obtained for dry AMD. These data support the hypothesis that wet and dry AMD are two distinct diseases. Understanding the chemical composition and fluorophores found in these samples can aid in furthering the treatment, diagnosis and prevention of wet and dry AMD.

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Active Phenotype Acquisition for the Genetic Characterization of Heritable Retinal Diseases Wagner

Wagner, Alex; Hector, Michael; Stone, Edwin; Braun, Terry
The University of Iowa

Purpose: This research is focused on the active acquisition of patient information for rapid and accurate diagnosis of heritable retinal dystrophies. The goal of this project is to use these signs and symptoms to inform the physicians of likely subclasses of disease, and by extension the most likely genetic causes of the patient�s disorder. These predictions are used to inform genetic test

Results: and actively inspire alternative hypotheses in the diagnosis of retinal dystrophies.

Methods: Expert knowledge is codified in a Bayesian Network expert system. This system is cost-aware and used to inform what questions are most beneficial for differential diagnosis given current knowledge. Phenotype and genotype information of patients that have a heritable retinal dystrophy and an identified genetic cause are collected as case studies in our TRIPOD system.

Results: We demonstrate the construction and utility of our TRIPOD system and the use of example cases with an expert system encoding known heritable retinal dystrophies.

Conclusions: We have developed a system for the storage of patient case data for learning new relationships between phenotype and disease.

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Functional characterization of Prickle2 and BBS7 identify overlapping phenotypes yet distinct mechanisms

Trudi A. Westfall, Xue Mei, Qihong Zhang, Val C. Sheffield, Alexander G. Bassuk and Diane C. Slusarski
University of Iowa, Department of Biology

Purpose: Ciliopathies are genetic disorders that are caused by dysfunctional cilia and affect multiple organs. One type of ciliopathy, Bardet-Biedl Syndrome, is a rare disorder characterized by obesity, retinitis pigmentosa, polydactyly, mental retardation and susceptibility to cardiovascular diseases. The Wnt/Planar Cell Polarity (PCP) has been associated with cilia function and ciliogenesis in directing the orientation of cilia and basal bodies. Yet the exact relationship between PCP and ciliopathy is not well understood.

Methods: We examined interactions between a core PCP component, Prickle2 (Pk2), and a central BBS gene, Bbs7, using gene knockdown in the zebrafish embryo. Several organs and processes were monitored for BBS7 and Pk2 specific defects. We evaluated formation of the axon tracks in the retina, polarization of the neural tube, and cilia formation in the Kupffer's Vesicle. In addition, we monitored intracellular transport rates using a melanosome trafficking assay.

Results: pk2 and bbs7 knockdown both disrupt the formation and ciliogenesis of Kupffer's Vesicle, but do not display a synergistic interaction. We find that bbs7 activity is not required for pk asymmetric localization in the neural tube, but does alter neural tube polarity. Knockdown of pk2 and similarly, ift22, an anterograde intraflagellar transport component, both suppress bbs7-related retrograde melanosome transport delay. Notably, pk2 knockdown larvae show a delay in anterograde transport.

Conclusions: These data suggest a novel role for Pk2 in directional intracellular transport and our analyses show that PCP and BBS function independently, yet result in overlapping phenotypes when knocked down in zebrafish.

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Spatial gene expression of human retina and RPE/choroid

Whitmore, S. Scott, Wagner, Alex H. Drack, Arlene V., Stone, Edwin M., Tucker, Budd A., DeLuca, Adam P., Zeng, Shemin, Braun, Terry A., Mullins, Robert F., Scheetz, Todd E.
Stephen A. Wynn Institute for Vision Research, The University of Iowa

Purpose: To evaluate the spatial transcriptomics of the posterior pole of human eyes, we performed differential gene expression analysis on temporal, macular, and nasal regions of neural retina and RPE/choroid using RNA sequencing (RNA-Seq).

Methods: Human donor eyes were obtained through the Iowa Lions Eye Bank after family consent in accord with the tenets of the Declaration of Helsinki. RNA was extracted from temporal, macular, and nasal punches of retina and RPE/choroid from four human donor eyes. Sequencing was performed at the Genomics Division of the Iowa Institute of Human Genetics. Read mapping and data analysis were performed with the Tuxedo software suite.

Results: Neural retina was clearly separable from RPE/choroid at the transcriptome level. Many of the most differentially expressed genes were transcription factors. Within the retina, photoreceptor cell-specific genes were enriched at the periphery, and ganglion cell and amacrine cell genes were modestly enriched in the macula. Enrichment for endothelium related genes was observed in macular RPE/choroid.

Conclusions: For both neural retina and RPE/choroid, more genes are differentially expressed between the macula and peripheral regions than between the two peripheral punches. Our dataset suggests potential transcriptional regulators of spatial identity within the human retina and RPE/choroid.

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Using stem cells to develop gene therapy for Batten Disease

Luke A. Wiley¹, Budd A. Tucker¹, Kristin R. Anfinson¹, Dalyz Ochoa¹, Luan M. Streb¹, Jeaneen L. Andorf¹, Louisa M. Affatigato¹, Arlene V. Drack¹, Edwin M. Stone^1,2
1Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa; ² Howard Hughes Medical Institute, Iowa City, IA

Purpose: Batten Disease, or juvenile neuronal ceroid lipofuscinosis (JNCL), is a devastating autosomal recessive lysosomal storage disorder characterized by early onset blindness, epilepsy, cognitive decline and death. This study will use patient-specific induced pluripotent stem cells (iPSCs) as a model system to investigate JNCL pathophysiology and to develop drug, gene and autologous cell-based therapies.

Methods: IPSCs were generated via transduction of human dermal fibroblasts from patients with JNCL using Oct4, Sox2, c-Myc and KLF4. IPSC potency was characterized via rt-PCR, immunoblotting, immunocytochemistry (ICC), and embryoid body formation. IPSCs were differentiated into retinal neurons using our previously published protocol. A combination of rt-PCR, Western blotting, ICC, and confocal microscopy were used to evaluate disease phenotype and efficacy of the developed gene therapeutic approach.

Results: Patient-specific iPSCs were generated from three patients with molecularly-confirmed Batten disease. After 60 days of differentiation, retinal neurons were identified via ICC analysis targeted against OTX2, recoverin and cone opsins. As observed in vivo, differentiated retinal neurons harbored the one-kilobase deletion in CLN3 and loss of normal full-length transcript and protein. Accumulation of autofluorescent lysosomal storage material was accompanied by intense LAMP-1 expression. Full-length CLN3 was cloned and packaged into lentiviral vectors and tested for the ability to drive expression of full-length CLN3.

Conclusions: Retinal neurons generated from JNCL patients recapitulate cardinal aspects of the disease in vitro. Genetic correction of the disease phenotype in patient-specific iPSCs will pave the way for combined gene and autologous cell replacement-based therapeutic trials for the treatment of Batten disease.

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Nanostructured Photopolymers as Stem Cell Scaffolds for Photoreceptor Regeneration

Worthington, Kristan S.^1;2; Bartlett, Alexandra M.²; Wiley, Luke A.¹; Stone, Edwin M.^1,34; Guymon, C. Allan²; Tucker, Budd A.^1
1Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Vision Sciences, Carver College of Medicine ²Department of Chemical and Biochemical Engineering, College of Engineering ³Howard Hughes Medical Institute ⁴. Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, (all at University of Iowa, Iowa City, IA)

Purpose: The degeneration of photoreceptors is difficult to treat due to the limited regeneration of relevant cell types. Although retinal progenitor cell injection to the sub-retinal space has been shown to restore function to retinas with early-stage degeneration, this success does not translate to advanced disease states due to an overall lack of the support necessary for restoring retinal function. Stem cell scaffolds offer a solution to this lack of support, but the effect of physical properties (i.e. nanostructure) on cell/material interactions are not well understood.

Methods: Nano-porous cell scaffolds were synthesized by lyotropic liquid crystalline (LLC) templating of photopolymerizable poly(ethylene glycol) pre-polymers. Morphology was characterized using scanning electron microscopy (SEM), small-angle x-ray scattering (SAXS), and polarized light microscopy (PLM). Surfactant removal and swelling were both examined gravimetrically. Further, the effect of nanostructure on cell/material interaction was investigated by seeding the materials with murine induced pluripotent stem (MiPS) cells, after which growth and differentiation were monitored using SEM and immunohistochemsitry.

Results: Polymers templated with surfactants manifested nano- and micro-structure. The presence of this structure improved the diffusion properties of the material and altered the mechanical properties, influencing cell/material interactions.

Conclusions: This work shows that the nanostructure of photopolymers plays an important role in cell/material interactions and can be successfully manipulated to meet the needs of photoreceptor regeneration applications. An optimized material of this kind could lead to the successful transplantation of replacement cells and ultimately, restoration of retinal function in patients who suffer from retinal degeneration.

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Corneal nerve structure damage associated with peripheral neuopathy in streptozotocin-treated C57Bl/6J diabetic mice: effect of glycemic control

Matthew S. Yorek^1,4, Alexander Obrosov², Hanna Shevalye², Sergey Lupachyk², Matthew M. Harper^1,3,4, Randy H. Kardon^1,3,4 Mark A. Yorek^1,2,4,5
1Department of Veterans Affairs Iowa City Health Care System, Iowa City, IA,²Department of Internal Medicine, University of Iowa, Iowa City, IA, ³Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA,⁴Veterans Affairs Center for the Prevention and Treatment of Visual Loss, Iowa City, ⁵Fraternal Order of Eagles Diabetes Research Center, University of Iowa, Iowa City, IA

Purpose: The CDC reported in 2011 that 4 million diabetics experienced visual impairment. Our study detailed neuropathic changes in the diabetic murine eye in an effort to improve diagnostic and treatment outcomes.

Methods: C57BL/6J mice were treated with streptozotocin (150 mg/kg) to induce diabetes. Treatment groups received insulin pellet implants to control glycemia. Groups consisted of poor glycemic control, good glycemic control, and poor to good control of glycemia. Animals were scrutinized via standard blood serum assays, thermal and mechanical sensitivity, and sensory and motor nerve conduction velocity. Imaging and microscopy tests included spectral domain optical coherence tomography for quantification of retinal nerve fiber layer thickness, corneal confocal microscopy (CCM) to assess nerve fiber length, footpad intraepidermal nerve fiber density, and corneal epithelial nerve volume.

Results: We observed decreased retinal nerve fiber layer thickness in diabetic animals. Diabetic corneal nerves have reduced epithelial volume, diminished surface area in the central whorl region, and showed shorter nerve lengths via CCM. The use of insulin to moderate blood glucose had measureable benefits. The data show animals receiving glycemic control faired better than untreated diabetics in all tests completed, however, insulin was unable to normalize outcome measures.

Conclusions: Sensitive and non-invasive endpoints are needed clinically to more effectively identify and treat diabetic neuropathy. Our study demonstrates that CCM, a non-invasive test, is sufficiently sensitive to detect changes in corneal innervation in a mouse model and may serve as an effective endpoint in grading diabetes mellitus.

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Validation of a Novel Software to Study Axonal Loss in Mice Models of Glaucoma

Kasra Zarei^1,2,3, John H Fingert^2,4, Kathy Miller², Michael G Anderson^4,5, Adam Hedberg-Buenz⁵, Todd E Scheetz^1,2,3,4, Michael D Abramoff^1,2,4, Mark Christopher^1,3
Departments of ¹²Biomedical Engineering, ²Ophthalmology and Visual Sciences,³Center for Bioinformatics and Computational Biology, ⁴Stephen A. Wynn Institute for Vision Research, ⁵Department of Molecular Physiology and Biophysics

Abstract: Glaucoma is the second leading cause of blindness in the world today. Full axon counting of whole optic nerve cross-sections is the most desirable and accurate method to quantify glaucomatous damage, but such analysis is unfeasible and very time-extensive. We have developed image-analysis software that determines automated whole optic nerve axon counts. Furthermore our software can construct optic-nerve heat maps that can be used to also determine regional patterns of axonal loss. Our method yields accurate axon counts compared to human truth counts at 100x magnification (r^2 = 0.95). Our method also yields accurate whole optic nerve axon counts compared to extrapolated whole nerve counts from the same human truth counts at 100x magnification (r^2 = 0.86). Using this novel software approach to quantify axon counts, we have discovered that the area of optic nerve cross sections is variable and not constant as assumed for prior analyses. As a result, we have determined that there is no significant difference in total axon counts between wild-type mice and transgenic TBK1 mice. However, after normalizing the counts for optic nerve cross-sectional area, we have determined that there is a 10% significant difference (P=0.003) in axon density (i.e. axons per unit area). Thus, we believe that future studies must employ a new metric such as axon density to accurately assess axonal loss in mice models of glaucoma.

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Characterization of BEST1 transcript isoforms and miRNA levels between the human macular and extramacular retinal pigment epithelium

Shemin Zeng, Arlene V. Drack, Budd A. Tucker, Edwin M. Stone and Robert F. Mullins
Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, IA

Purpose: Best disease is an early onset macular dystrophy caused by mutations in the BEST1 gene. The molecular basis of the regional pathology of Best disease is poorly understood. This study aimed to determine the regional distribution of RNA transcript isoforms of BEST1 and to assess whether DNA methylation of BEST1 CpG island (CGI) and four miRNAs with potential to regulate BEST1 are involved in the differential expression between the human macular and extramacular retina pigment epithelium (RPE).

Methods: DNA or RNA was extracted from RPE/choroids from fixed or frozen human eye samples. Transcript isoforms were quantified by real time quantitative PCR (qPCR) using isoform-specific primers. Potential BEST1 regulating miRNAs were bioinformatically identified and measured using TaqMan-miRNA Assay. DNA methylation was evaluated using bisulfite-specific sequencing (BSP). Results were compared between macular and extramacular punches.

Results: The qPCR analysis indicated that the relative abundance of three BEST1 isoforms (isoform-1, 3 and 4) was significantly lower in macular than extramacular RPE (P<0.05). In BSP analysis, BEST1 methylation was found at the 18th to 19th CpG site of its 3'-terminal, but the methylation pattern was not significantly different between macular and extramacular RPE. Three of four miRNAs evaluated as potential regulators of BEST1 (hsa-miR-107, hsa-miR-204 and hsa-miR-211) were expressed at higher levels in macular than extramacular RPE-choroid (P<0.05).

Conclusions: The lower levels of three BEST1 transcript isoforms and higher levels of three miRNAs were found in human macular RPE. This reciprocal relationship suggests that miRNAs may repress BEST1 expression in macula. Strategies to increase macular BEST1 expression may be beneficial in Best disease.

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Characterization and utility of iPSCs-derived TM cells for maintaining TM function in vivo

Wei Zhu, Qiong Ding, Budd A. Tucker, Markus H. Kuehn
The Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, The University of Iowa

Purpose: To rescue TM cell loss affecting pathological process of primary open-angle glaucoma (POAG) patients, induced pluripotent stem cells (iPSCs) become the optimum choice of stem cells-based therapy. The primary goal of this study was to characterize iPSCs-derived TM (iPSC-TM) cells and apply the differentiated cells to rebuild the injured TM tissue in vivo.

Methods: Mouse iPSCs were differentiated by culture for up to 14 days in media conditioned by primary TM cell. The appearance of iPSC derived TM cells (iPSC-TM) was confirmed using morphological and immunohistochemical approaches and cells with continued expression of the stem cell marker SSEA-1 were removed. Purified iPSC-TM were injected into the anterior chamber of Tg-MYOCY437H mice and the IOP was monitored by rebound tonometry.

Results: 14 days post differentiation mouse iPSC-TM cells resemble primary TM cells based on morphology, and the transcriptional and protein expression levels of LAMA4, TIMP3, Nanog4 and Sox2. Following injection into the anterior chamber Tg-MYOCY437H mice, iPSC-TM establish themselves in the trabecular meshwork. These studies are ongoing, but early demonstrate that within one week after transplantation of iPSC-TM the IOP in treated eyes decreased from 18.68 to 15.8 mmHg (N=12).

Conclusions: The effects of mouse iPSCs-derived TM cells on mediating the IOP in vivo provided a new approach of stem cell-base therapy to rescue TM cell loss in POAG patients.

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