2015 Midwest Eye Research Symposium
Sponsored by the Iowa Glaucoma Center and the University of Iowa Department of Ophthalmology and Visual Sciences
MERS 2015 took place on August 7th, 2015 at
|Aparna Lakkaraju:||The garbage catastrophe in the retinal pigment epithelium.|
|Kathleen Chirco:||Monomeric C-reactive protein and altered choroidal vasculature in AMD|
|Kimberly Toops:||Complex anomalies in late endosome and lysosome trafficking in stressed RPE cells|
|Chaid Schwarz:||Adhesion Strength of Descemet's Membrane in Healthy and Diabetic Donor Corneas|
Session II - Session Chair Jason Meyer, Ph.D.
|Jason Meyer:||Modeling Retinal Development and Disease with Human Pluripotent Stem Cells|
|Allison Songstad:||Generation of induced pluripotent stem cell-derived choroidal endothelial cells for the treatment of AMD|
|Ankur Jain:||CRISPR-Cas9 Mediated Genome Editing of Myocilin in Hereditary Glaucoma|
|Kazuya Oikawa:||Early optic nerve changes in a spontaneous feline glaucoma model|
Session III - Session Chair Sheila Baker, Ph.D.
|Sheila Baker:||Escaping the ER - Early steps in the trafficking of HCN1 channels|
|Emily Patterson:||The Use of the Color Assessment and Diagnosis (CAD) Test in Congenital Achromatopsia|
|Lauren Laboissonniere:||Single cell transcriptomics of defined retinal ganglion cell types|
|Rebecca Chowdhury:||The role of Trim9 in vertebrate retina development|
|Sara Adelman||Post-natal development of Intraocular Pressure in Feline Congenital Glaucoma (FCG) and in Normal Cats|
|Bailey Banach||Microfabrication of Retinal Biomimetic Structures Using Two Photon Polymerization|
|Erin Burnight||Using RNA-mediated Genome Editing to Create an Animal Model of Retinal Dystrophy for Analysis of in vivo CRISPR/CAS9 Treatment Efficacy.|
|Mark Christopher||Improved prediction of incident primary open angle glaucoma using automated analysis of optic nerve head structure|
|Poppy Datta||Accumulation of non-outer segment proteins in the outer segment underlies photoreceptor degeneration in Bardet-Biedl syndrome|
|Laura Dutca||Factors that influence retinal outcomes in a mouse model of blast-induced traumatic brain injury|
|Sarah Gardner||Restoration of Vision in a Mouse Model of Congenital Stationary Night Blindness|
|Allison Garmager||Neurodegeneration and capillary alterations in human donor eyes with minimal diabetic retinopathy|
|Joe Giacalone||RPGR ORF15: From Clinical Sequencing to Treatment|
|Wesley Goar||Detailed Inspection of Bardet-Biedl Syndrome Exomes|
|Oliver Gramlich||The cellular immune system of glaucomatous mice is capable of promoting RGC death|
|Ying Hsu||The role of the BBSome in retinal function during postnatal eye development and adulthood|
|Chunhua Jiao||Neuroretinal Degeneration in mice with streptozotocin-induced Diabetic Mellitus|
|Brodie Johnson||Morphological differences of microglia in human retina|
|Devi K. Karumanchi||Early diagnosis of diabetes through the eye|
|Jamil Khan||Inter-Subject Variability and Repeatability of In Vivo Foveal Avascular Zone Measurements by OCT Angiography|
|Joseph Laird||Identification of a VxP targeting signal in the flagellar Na+/K+-ATPase|
|Carly Lewis||Retinal cellularity near superficial vasculature in a mouse model of glaucoma|
|Rachel Linderman||Examining Interocular Symmetry in Vascular Density|
|Timur Mavlyutov||Subcellular localization of the sigma-1 receptor in retinal neurons - an electron microscopy study|
|Kacie Meyer||Identification of imprinted retinal genes using RNA sequencing|
|Sarah Ohlemacher||Elucidating Retinal Ganglion Cell Development and Disease Using Human Pluripotent Stem Cells|
|Brandon Rea||A new method for quantifying pain expression during CGRP-induced migraine in a mouse model|
|Madia Russillo||Comparison of Fluorescein Angiography and Optical Coherence Tomography (OCT) Angiography in Retinal Vascular Disease|
|Todd Scheetz||Resolving genotype-phenotype mismatches in whole-exome based genetic testing|
|Anthony Scott||VIZN: Rapid, high throughput, in-vivo analysis of vision candidate genes using the zebrafish|
|Kevin Snyder||Functional Measures of Glaucoma Progression in a Spontaneous Feline Congenital Glaucoma (FCG) Model|
|Levi Sowers||Thalamic involvement in headache related photophobia|
|Akshayalakshmi Sridhar||Robust derivation of patient-specific retinal neurons via mRNA reprogramming|
|Aaron Street||Controlled release liposomal drug delivery for treating ocular angiogenesis|
|Li Xuan Tan||Mechanisms of membrane repair in polarized epithelia|
|Nicole Tatro||Extracting phenotypes from historic Goldmann visual fields|
|Alana Trotter||High Resolution Imaging in Juvenile Pigmentary Retinopathy|
|S. Scott Whitmore||Differential transcript expression and alternative splicing in three regions of the human retina|
|Luke Wiley||Genome editing and gene replacement: towards the treatment of Batten disease.|
|Brittany Williams||Characterization of human splice variants of the voltage-gated Cav1.4 Ca2+ channel|
|Huan Yang||Sigma-1 receptor ablation impairs mitophagy in neuronal and non-neuronal cells|
|Kasra Zarei||A Mobile Phone Application for Quantifying Flicker Fusion Threshold|
|Wei Zhu||Transplantation of iPSC-derived TM cells maintains outflow facility in a mouse model of glaucoma|
Adelman,S.A.1,2 D. Shinsako,1 J.A. Kiland,1 E.A. Hennes-Beean,1 J.A. Jens,3 N.M. Ellinwood,3 A. Ludwig,3 V. Yaccarino,1 G. Ben Shlomo,4 and G.J. McLellan.1,2,5
1Dept. of Ophthalmology and Visual Sciences, School of Medicine and Public Health and 2Dept. of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, WI ; 3Dept. of Animal Sciences and 4 Department of Veterinary Clinical Sciences, Iowa State University, Ames, IA; 5 McPherson Eye Research Institute, Madison, WI.
Purpose: Glaucoma is leading cause of blindness in humans and domestic animals, with intraocular pressure (IOP) identified as the most consistent risk factor for progressive vision loss. FCG is a spontaneous large animal model of recessively inherited glaucoma due to LTBP2 mutation. The goal of this study was to determine how IOP changes with age, early in life, in normal cats and cats with FCG.
Methods: IOP measurements were taken 2-3 times per week, from eyelid opening until three to six months of age using rebound tonometry (TonoVet, ICare Oy, Finland). IOP was measured bilaterally in 29 normal cats and 58 cats with FCG. IOP values for the right and left eyes were averaged for subsequent analyses. One-way ANOVA and Tukey-Kramer post-tests were used to compare mean IOP measurements between different age groups and between control and FCG groups (Instat v3.0, Graphpad Software, San Diego, CA). A value of p <0.05 was considered significant.
Results: Mean IOP at 8 weeks of age was significantly higher in glaucomatous (19.5 mmHg; 95% CI= 15.7, 23.3mmHg) than in normal kittens (10.0 mmHg; 95% CI= 7.4, 12.6mmHg). At 3 months of age, IOP in normal cats plateaued upon reaching adult values while IOP in glaucomatous cats continued to increase over the first 6 months of life.
Conclusions: These results provide a range of IOP values for normal kittens from eyelid opening to six months of age and confirm an early onset of IOP elevation in kittens with inherited FCG.
Baker, SA, Laird, JG, Yamaguchi, DM, Bhattarai, S, Drack, AV, Pan, Y
Departments of Biochemistry and Ophthalmology & Visual Sciences, Wynn Institute for Vision Research, University of Iowa, IA USA 52242
Purpose: Photoreceptor signaling depends on the coordinated action of multiple ion transporters and channels. Photoreceptor outer segments, inner segments, and synapses all contain a unique inventory of ion channels. How various ion channels are sorted into the correct compartment is completely unknown. In this study we have investigated the mechanisms controlling the trafficking of a representative inner segment localized channel HCN1. HCN1 opens in response to voltage changes caused by light and conducts an influx of cations that filters the light response and helps to reset the photoreceptor back to the dark-adapted state.
Methods: HCN1 trafficking was measured with a biotinylation assay. Visual function was measured by ERG. The subcellular localization of a series of HCN1 mutants was determined by confocal imaging of transgenic tadpoles.
Results: TRIP8b is an accessory subunit of HCN1. In the retina of knockout mice, HCN1 levels are reduced but trafficking to the inner segment plasma membrane, and visual function, was unaltered. A comprehensive screen for trafficking signals in HCN1 expressed in transgenic tadpoles revealed two trafficking signals ? a di-arginine endoplasmic reticulum (ER) retention signal and a novel ER export signal.
Conclusions:TRIP8b is not essential for the trafficking of HCN1 in the retina. Two new trafficking signals were identified in HCN1 and both function in the ER. This work leads to a model where these two signals work in counterbalance to maintain the proper equilibrium of HCN1 levels between the ER and the plasma membrane. This work also challenges the dogma that newly synthesized membrane proteins only move through the ER passively.
Banach, Bailey B.1,2, Worthington, Kristan S.1, Wiley, Luke A.1, Stone, Edwin M.1, Mullins, Robert F.1, Tucker, Budd A.1
1Wynn Institute for Vision Research, University of Iowa, Iowa City, IA 52240 2Department of Biomedical Engineering, University of Iowa, Iowa City, IA 52240
Photoreceptor cell death in retinal degenerative diseases is usually preceded by loss of the underlying retinal pigment epithelium (RPE) and choriocapillaris. Thus, it is evident that multiple cell types of the outer retina are critically interdependent. Recent data suggests that the use of stem cells to achieve regeneration of any of these outer retinal layers is feasible. However, current cell delivery methods typically result in significant cell loss (more than 99%) and exhibit limited cellular integration following transplantation. We address this challenge by combining stem cell therapy methods with tissue engineering technologies such as two-photon polymerization. In this work, two-photon lithography parameters such as laser power and scanning speed were modulated to optimize resolution and printing speed for three commercially available photoresists. The effect of design parameters such as line spacing, hatching distance, and contour style on cell growth and alignment were also investigated. For these cell interaction studies, structures were fabricated using the commercially available photoresist IP-Dip, seeded with mouse induced pluripotent stem cells, stained with phalloidin, and imaged using confocal microscopy. Before adding cells, the structures were imaged using scanning electron microscopy. After identifying appropriate laser powers at maximum scanning speeds, we were able to also identify a maximum hatching distance (0.35 μm, regardless of line spacing and contour style), above which structural integrity was lost. Furthermore, the structures were able to support attachment of cells, indicating that the selected material is biocompatible. Interestingly, at the selected seeding density, only slight differences in attachment and alignment were observed between samples with different fabrication parameters. These studies lay the foundation for future work involving two-photon polymerization of support structures for photoreceptor, RPE, and choroidal endothelial cells.
Burnight E.R.1, Wiley L.A.1, Hsu P.D.2, Ochoa D.1, Wiley J.S.1, Halder J.A.1, Zhang F. 2, Mullins, R.F.1, Stone, E.M.1, and Tucker, B.A.1
1Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, and 2Broad Institute and McGovern Institute for Brain Research, Massachusetts Institute for Technology, Cambridge, MA.
Purpose: Mutations in CEP290 are the most common cause of LCA, a severe form of inherited retinal dystrophy for which no treatment exists. In vivo CRISPR-based genome editing has emerged as a new potential treatment strategy. The purpose of this study is to employ the CRISPR/Cas genome editing technology to generate a murine model carrying a deep intronic splice mutation in Cep290, which can be used to test in vivo CRISPR-based treatment strategies.
Methods: The human CEP290 intron 26 was aligned to murine Cep290 sequence (mmIVS26) using ClustalW. Small guide RNAs were designed and cloned into the bicistronic CRISPR/Cas9 expression plasmid. Efficiency of CRISPR/Cas9 modification was assessed via Surveyor nuclease assay and TA cloning/Sanger sequencing. Donor template containing ~500 bases of mmIVS26 homology flanking the 5' splice donor site of murine IVS26 was cloned and co-delivered with the bicistronic CRISPR/Cas9 expression plasmid into murine iPSCs. Homology-directed repair was detected via PCR and Cep290 transcript analysis was performed by rt-PCR.
Results: Three of six sgRNAs successfully and efficiently modified the mmIVS26 locus as determined by Surveyor and TA cloning/Sanger sequencing assays. Successful homology-directed repair was detected via PCR. Cep290 expression analysis indicated a reduction in the amount of transcript in cells treated with both the sgRNA/Cas9 and donor template plasmids as compared to cells treated with the donor template alone.
Conclusions: These results provide a foundation on which to build an animal model of CEP290 LCA that can be used to evaluate in vivo genome editing-based therapies.
Chirco, Kathleen, Whitmore, S. Scott; Stone, Edwin M.; Tucker, Budd A.; Mullins, Robert F.
The Stephen A. Wynn Institute for Vision Research Department of Ophthalmology and Visual Sciences, The University of Iowa
Purpose: Previous studies found an association between increased total C-reactive protein (CRP) levels in the choroid, and AMD (Johnson, 2006; Bhutto, 2011). However, CRP exists in two functionally opposing conformations, pentameric CRP (pCRP) and monomeric CRP (mCRP). We aimed to address (a) which form of CRP is present in human choroid and (b) the physiological responses of choroidal endothelial cells to CRP.
Methods: Immunohistochemistry was utilized to study the localization and relative abundance of mCRP versus pCRP in genotyped human donor maculas. To assess the effect of mCRP and pCRP on endothelial cells, scratch closure and transepithelial resistance (TER) assays were performed using monkey choroidal endothelial (RF/6A) cells. Gene expression was assessed using RNA-seq analysis after CRP incubation with human choroid organ cultures.
Results: mCRP, but not pCRP, was more abundant in the choriocapillaris and Bruch 's membrane of donors with the high-risk CFH (Y402H) genotype versus those with the low-risk genotype. RF/6A cell migration rate and monolayer permeability increased with mCRP treatment compared to pCRP or medium alone. mCRP treatment in human organ cultures also led to a significant change in ENPEP, ANGPTL4, and ADAM12 gene expression.
Conclusions: Our data suggest that pro-inflammatory mCRP is the main form of CRP in the choroid of high-risk AMD eyes. Interestingly, while pCRP has a mild effect on choroidal endothelial cell function in vitro, mCRP significantly increases cell migration and monolayer permeability, in addition to altering gene expression. These data indicate an important role for mCRP in vascular dysfunction in AMD choroids.
Chowdhury, Rebecca, Goetz, Jillian, Laboissonniere, Lauren, Trimarchi, Jeff
Iowa State University
Purpose: Math5 is a highly conserved transcription factor that is essential for retinal ganglion cell (RGC) development in the developing mouse and zebrafish retina. Using single cell transcriptome profiling, we previously characterized individual mouse Math5+ cells and found several genes with similar expression patterns as Math5. Of these candidate genes, Trim9, an E3 ubiquitin ligase has been shown to be involved in axon branching in the mouse brain. Based on our array data, we hypothesize that Trim9 will play important roles in the cell fate determination and maturation of early retinal neurons.
Methods: We obtained a Trim9 knockout mouse and are now examining the phenotypic changes associated with the mutant retina. Currently, we are using in situ hybridization (ISH), immunohistochemistry (IHC) and transcriptome analysis to examine the early generated cell types of the retina, specifically ganglion cells and cone photoreceptors. To generate mutations in zebrafish Trim9, we used a specifically targeted CRISPR. IHC and ISH are being used to detect changes to different populations of retinal neurons that result from the loss of these genes in the mouse or zebrafish retina.
Results: The loss of Trim9 in the mouse retina possibly leads to reductions in small subsets of ganglion cells and cones.
Conclusions: Our experiments in mice and zebrafish indicate that Trim9 is a gene that plays an important role in subsets of early generated neurons. We are currently using our loss-of-function mutants to further characterize the precise role of Trim9 in these cells.
Christopher, Mark; Tang, Li: Fingert, John H.; Scheetz, Todd E.; Abramoff, Michael D.
1Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa
Purpose: To identify unknown relationships between optic nerve head (ONH) structure, commonly-used clinical measurements, and development of glaucoma.
Methods: Structural building blocks of ONH structure were identified and evaluated based on associations with clinical variables and accuracy in early prediction of primary open angle glaucoma (POAG). The primary data consisted of stereo fundus images, a set of clinical measurements, and follow-up evaluations to determine conversion to POAG for a large cohort of non-glaucomatous subjects (n = 1635). Three-dimensional ONH structure was determined for each subject using a stereo correspondence algorithm and building blocks of ONH structure were identified by applying principal component analysis to these structural measurments. The resulting ONH structural features were evaluated based on associations with clinical variables and with later development of POAG. These features were also used to build predictive models for early POAG prediction.
Results: Several statistically significant associations between the identified ONH structural features and clinical measurements were found. These include associations with age, ethnicity, cup-to-disc ratio, central corneal thickness, refraction, and later development of POAG. Inclusion of the features into POAG prediction models also yielded a significant increase of accuracy over baseline (AUC = 0.722 vs. AUC = 0.599).
Conclusions: The methodologies developed here identified novel ONH structural features with significant associations with POAG and related variables. These features show promise for use in early screening for disease or as biomarkers for further study of POAG-related ONH changes.
Datta, Poppy1, Allamargot, Chantal2, Hudson, Joseph S.1, Andersen, Emily K.1, Bhattarai, Sajag1; Drack, Arlene, V1, Sheffield, Val C3,4, Seo, Seongjin1
Departments of 1Ophthalmology and Visual Sciences, 2Central Microscopy Research Facility, 3Pediatrics, The University of Iowa, 4Howard Hughes Medical Institute, Iowa City, Iowa.
Purpose: Photoreceptor degeneration is one of the major phenotypic components of Bardet-Biedl syndrome (BBS). BBS proteins have been thought to mediate protein trafficking to primary cilia and to the photoreceptor outer segment (OS). However, the precise role of BBS proteins in vivo is not well understood. In this project, we generated a new BBS mouse model (Lztfl1; aka Bbs17) and compared the histology, OS proteome and immunolocalization of vesicular trafficking related proteins to better understand the molecular functions of BBS proteins in photoreceptors and mechanisms of photoreceptor degeneration in BBS.
Methods: OSs from normal and Lztfl1 KO retinas were collected by sucrose gradient centrifugation and submitted for label-free quantitative proteomics. Data were collected in 4 independent experiments and t-test was used to determine proteins with significant changes. Quantitative changes, localization, and OS ultrastructure were examined by Western blotting, Immunofluorescence microscopy and Transmission electron microscopy respectively.
Results: As in human BBS patients and other BBS mouse models, photoreceptors showed early degeneration in Lztfl1 KO retina along with significant reduction in b-wave in combined ERG as well as in 5 Htz data. Using isolated OSs and quantitative proteomics analyses, most of the abundant OS proteins were found normal or minimally affected in KO retina. However, 138 proteins showed more than 3-fold enrichment in Lztfl1 mutant OS, while only 8 proteins showed more than 3-fold decrease. Among the enriched proteins, we found Stx3 and Stxbp1/Munc18-1 highly accumulated in the BBS mutant OS. Ultrastructure analysis also revealed large vesicles in the mutant OS, disrupting its lamellar structure.
Conclusions: Our findings suggest that accumulation (and consequent sequestration) of non-OS proteins in the OS is likely the primary cause of photoreceptor degeneration in BBS. Our data also suggest that a major function of BBS proteins in photoreceptors is to transport proteins from the OS to the cell body or to prevent entry of non-OS proteins into the OS.
Rudd, Danielle1 , Dutca, Laura1,2, Herlein, Judy1, Hedberg-Buenz, Adam 1,3 , Kardon, Randy H.1,2, Harper, Matthew1,2, Anderson, Michael1,3
1Center of Excellence for Prevention and Treatment of Visual Loss, Iowa City Veterans Administration Medical Center Departments of 2 Ophthalmology and Visual Science, 3 Molecular Physiology and Biophysics The University of Iowa, Iowa City, IA
Purpose: The effects of traumatic brain injury (TBI) are complex, heterogeneous and hard to predict. Patients with TBI suffer from visual problems, both acutely and chronically. This study will determine the influence that genetic heterogeneity and environmental factors have on the structure and function of the retina in blast-mediated TBI.
Methods: To establish the role that genetic factors play in the response of the visual system to TBI, a genetically complex mouse strain, the Diversity Outbred (DO), was used. The DO mice were exposed to blast injury in a custom-built overpressure chamber. Retinal structure and function were analyzed using optical coherence tomography (OCT) and pattern electroretinography (PERG) 4 weeks post-blast. The number of retinal ganglion cells was determined in whole mounted retinas with an automated software system.
To determine if environmental factors such as elevated temperature exacerbate the effects of blast-induced TBI, hyperthermia was induced in inbred mice prior to injury. Normothermic mice exposed to blast and sham served as controls. Retinal structure (OCT) and function (PERG) were tested at 1, 4, 8 and 16 weeks post-blast.
Results: The DO mice exhibited a wider range of responses to TBI indicating that genetic context plays a large role in phenotypic variability. Hyperthermia at the time of injury exacerbated the functional and structural deficits present after blast-induced TBI compared to controls.
Conclusions: Genetic and environmental factors, such as hyperthermia at the time of injury, influence the responsiveness of the visual system to blast-mediated TBI.
Gardner, Sarah, Laird, Joseph, Kerov,Vasily, Joiner, Mei-Ling, Lee, Amy, Baker, Sheila A.
Departments of Biochemistry and Molecular Physiology & Biophysics, University of Iowa
Purpose: Cav1.4 is a voltage-gated ion channel found in photoreceptor synapses. It is responsible for allowing Ca2+ into the synapse and neurotransmitter release across the synapse. However, Cav1.4 is also crucial to the development and organization of the synapse. Without Cav1.4, people are typically diagnosed with Congenital Stationary Night Blindness (CSNB) or Cone-Rod Dystrophy. The purpose of this project was to evaluate the use of a behavioral assay to measure visual function in a mouse model.
Methods: The rods of Cav1.4 KO mice were transfected using sub-retinal injection of plasmid DNA followed by an in vivo electroporation. Their vision was assessed using a modified Morris water maze, in which mice are trained to escape to a visible platform. Each mouse completed 30 trials (5/day/animal), where we measured the amount of time that it took them to escape to the platform.
Results: Every mouse was trained for four days before the trials began. WT mice learned to escape the maze in 2.1 ± 0.1 sec, Cav1.4 KO in 44.0 ± 4.1 sec (n=10) and electroporated Cav1.4 KO in 24.4 ± 4.7 sec (n=7). Retinas were collected and flat-mounted to measure the size and location of the transfected patch of retina in each animal. There was a positive correlation between transfection efficiency and improved visual behavior.
Conclusions: We have validated an assay able to measure vision dependent behavior. Future studies could use this assay to test vision of Cav1.4 KO mice treated with Cav1.4 KO variants to determine how this channel promotes synaptogenesis.
Garmager, Allison1, 1 Abramoff, Michael D1,2,3, Jiao, Chunhua1, Kuehn, Markus, H1,3, Sohn, Elliott H1
1Stephen A. Wynn Institute for Vision Research, Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa; 2Iowa Institute for Biomedical Imaging, University of Iowa; 3Veterans Administration, Iowa City, Iowa
Purpose: Recent studies suggest that neurodegeneration is progressive in diabetic eyes with little to no detectable diabetic retinopathy. We sought to determine whether neurodegeneration or vascular changes occur first in diabetic eyes with no or minimal retinopathy in human.
Methods: Donor eyes from the Iowa Lions Eye Bank were used. Whole mount retinal punches (4mm from inferior macula) were immunostained with UEA1 and γ-synuclein antibody to label vessel walls and ganglion cells respectively, and the entire tissue was assessed for capillary density and NFL thickness using a custom macro in Image J. Ganglion cell counts were averaged in 6-7 regions of each piece of retina using confocal imaging.
Results: 6 diabetic eyes (mean age 77.8 years) with no to mild NPDR and no history of ocular treatment for retinopathy were compared to 6 control eyes with no history of glaucoma or disease affecting the macula (mean age 76.3 years). NFL was significantly thinner (p=0.029) in the DM eyes compared to controls. There was no significant difference in retinal capillary density between DM and controls. The mean density of ganglion cells was reduced in the diabetic eyes compared to the controls. There was a negative correlation between ganglion cell density and capillaries in all eyes.
Conclusions: Retinal neurodegeneration precedes microvascular damage in human donors with diabetes mellitus and no to minimal microvascular retinopathy. DM causes progressive neuroretinal loss that can occur in the absence of microvascular manifestations of retinopathy. Retinal diabetic neuropathy is neurotoxic, rather than ischemic, in origin.
Giacalone, J.C.1, Wiley, L.A.1, Streb, L.A.1, Daggett, H.T.1, Burnight, E.R.1, Mullins, R.F.1, Tucker, B.A.1, Stone, E.M.1,2
1Stephen A. Wynn Institute for Vision Research, Ophthalmology and Visual Sciences, 22Howard Hughes Medical Institute, University of Iowa, Department of Ophthalmology, Iowa City, IA
Purpose: Retinitis Pigmentosa is a genetically heterogeneous disorder characterized by progressive photoreceptor degeneration. In X-linked Retinitis Pigmentosa (XLRP), a large number of disease causing mutations reside within the gene, RPGR. Interestingly, RPGR undergoes alternative splicing, and one variant, containing ORF15, has a significant role in XLRP. Specifically, the 3 prime end of the ORF15 transcript is a highly repetitive, purine rich region that is susceptible to mutations. To date, we have screened 152 families using a cloning-based sequencing technique, which resulted in 27 positive families with 45 affected individuals, 13 of whom are female. In a subset of 15 patients with extensive clinical information, the age at diagnosis ranged from 4 to 48 years. Given the clinical impact of this mutational hotspot, treatment options are urgently needed.
Methods: Skin biopsies were obtained from molecularly confirmed XLRP patients with terminating variants residing within the ORF15 region of RPGR. Patient-specific iPSCs were generated from dermal fibroblasts. Adeno-associated viral (AAV) constructs carrying the ubiquitous form of RPGR and a non-ORF15 retinal-specific isoform under the control of the cytomegalovirus promoter were cloned, packaged and transduced into iPSC-derived retinal cells. CRISPR/Cas9 constructs targeting the ORF15 region and wild-type donor homology directed repair constructs were cloned.
Results: The ubiquitous form of RPGR and a retinal specific isoform transduce iPSC-derived retinal neurons and drive full-length and retinal specific RPGR expression as shown by rt-PCR, immunoblotting and immunohistochemistry. Specific and efficient small guide RNA targeting of ORF15 modified specific RPGR locus was achieved in HEK293t cells. A combined CRISPR/CAS and homology directed repair approach in patient-specific iPSCs and iPSC-derived retinal precursor cells was performed.
Conclusions: Terminating variants within the ORF15 have the potential to produce severe clinical phenotypes. The recent advances in the fields of gene therapy and genome editing present potential avenues for treating the diversity of cases observed. We have shown that genome editing via the CRISPR/Cas9 system can successfully target ORF15. Additionally, gene therapy may present another means of treatment.
Goar, W.A., A.P. DeLuca; S.S. Whitmore; D.Y. Nishimura; V.C. Sheffield; T.E. Scheetz
Stephen A. Wynn Institute for Vision Research
Purpose: Bardet-Biedl syndrome (BBS) is a rare autosomal recessive ciliopathy caused by mutations within at least 19 genes. Characteristics of BBS include retinitis pigmentosa, postaxial polydactyly, obesity, renal failure, and learning difficulties. By using whole exome sequencing (WES) in a cohort of 36 unrelated patients with BBS and related ciliopathies we hoped to discover both novel BBS genes and novel disease-causing mutations.
Methods: Samples for exome sequencing were obtained from BBS patients and were sequenced using the Illumina HiSeq 2500 platform. The raw reads were analyzed using our standard exome pipeline. Resulting variants were filtered based upon variant quality, predicted function, and population prevalence from several resources (1000 genomes, ExAC, and local resources).
Results: We found 26 functionally-relevant genes with variants that could be contributing to the BBS phenotype. We used our own ocular RNA sequencing data along with the Illumina Body Map data to determine if the candidate genes were expressed in the proper tissues. This resulted in 18 candidate genes of which 3 are known BBS genes. The best candidate (CEP97) was seen in two unrelated individuals with two different mutations in the same exon, and is known to interact with a BBS gene.
Conclusions: As expected, no common mutations were found in our first set of 36 exomes from individuals with BBS. We are currently following up on the putative mutations on additional patients and cohorts. During the detailed re-analysis, we identified multiple exons of BBS genes that are not captured, or are imperfectly captured, by existing exome reagents. We are, therefore, in the process of designing augmented capture baits to fully interrogate the set of BBS genes.
Gramlich, Oliver W.1,2, Ding, Qiong1, Kuehn, Markus H.1,2
1The University of Iowa Department of Ophthalmology and Visual Sciences and 2Center for the Prevention and Treatment of Visual Loss, VA Medical Center, Iowa City, IA.
Purpose: To functionally test if immune processes are both initiated and capable of causing damage in glaucoma, adoptive transfers between mouse models of glaucoma and normal recipient mice have been carried out.
Methods: B6.Sh3pxd2bnee (nee), immune-deficient B6.129S7-Rag1tm1Mom/J/nee (nee/Rag1), or C57BL/6J wild-type (B6) mice served as donors. Splenocytes (5x106) or CD19 (1.5x106) and CD3 (1x106) lymphocyte fractions were transferred to 2-month-old B6 recipients. RGC density (RGC/mm2) was evaluated via gamma-synuclein immunostaining. Cytokine profiles of recipient splenocytes after PMA/Ionomycin or CD3 stimulation were determined using a multiplex immunoassay.
Results: Transferred splenocytes from nee mice elicit significant loss of RGC in recipient mice after 4 months when compared to control or B6 recipients (RGC/mm2 in ctrl: 2290±254, B6 rec: 2284±299, nee rec: 1886±267, p<0.03). Transfer of nee/Rag1 splenocytes did not induce loss of RGC (nee/Rag1 rec: 2423±383 RGC/mm2). Animals having received nee T-cells also display significantly reduced RGC density when compared to B6 T-cell recipients (RGC/mm2 in B6 T-cell rec: 2251±221 vs. 1831±245 in nee T-cell rec, p=0.004). Transfer of nee B-cells leads to a modest reduction of RGC (RGC/mm2 in B6 B-cell rec: 2259±266 vs. 1968±317 in nee B-cell rec, p=0.07). TNF-α, IL-6 and IL-10 release of nee recipient splenocytes are significantly increased when compared to CTRL (p<0.05).
Conclusions: These data demonstrate that glaucomatous RCG loss elicits an adaptive immune response that is capable of promoting IOP-independent RGC loss following adoptive transfer. It is conceivable that once established such an autoimmune response could lead to continued RGC loss in glaucoma patients despite significant reduction in IOP.Back to top
Hsu, Y, Garrison, JE, Nishimura, DY, Bugge, KE, Searby, CC, Sheffield, VC
Department of Pediatrics, University of Iowa
Purpose: The outer segments of photoreceptors are specialized sensory cilia. The BBSome, a protein complex consisting of BBS1, 2, 4, 5, 7, 8, 9 and 18, is required for protein trafficking within the cilia, and is important for photoreceptor function. Human patients and mouse models with Bardet-Biedl syndrome (BBS) have retinal degeneration. However, it is not clear whether the BBSome plays a developmental role in the formation of the retina in addition to maintaining normal retinal function. Using a mouse model that enables the tamoxifen inducible deletion of Bbs8, we test whether the loss of the BBSome during eye development disrupts retinal function.
Methods: Deletion of Bbs8 during postnatal eye development or during adulthood was induced by tamoxifen injection. Electroretinogram was performed 1 to 9 months after tamoxifen injection. The integrity of the retina was examined by histology.
Results: The inducible deletion of Bbs8 in adult mice causes significant deterioration of retinal function. Furthermore, the deletion of Bbs8 in juvenile mice during postnatal eye development causes rapid retinal degeneration comparable to congenital Bbs8 knockout mice. The data suggest that the BBSome plays an important role in retinal development in addition to maintaining the function of photoreceptors after eye development is complete.
Conclusions: The BBSome is required both during retinal development and during adulthood for normal retinal function. The deletion of BBSome components during postnatal eye development causes accelerated retinal dysfunction compared to the deletion of BBSome components during adulthood, which causes a slower but significant rate of retinal degeneration.
1 Searby, Charles
1 Zhang, Feng,
2,3 Sheffield, Val1
1Pediatrics, Carver College of Medicine, University of Iowa, Iowa 2North Texas Eye Research Institute, UNT Health Science Center at Fort Worth, Texas 3McGovern Institute for Brain Research at Massachusetts Institute of Technology, Cambridge, Maryland
Purpose: Glaucoma is a leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) remains the most important risk factor for glaucoma. Mutations in myocilin (MYOC) have been reported in >16% of Juvenile Open Angle Glaucoma and about 4% of Primary Open Angle Glaucoma patients. Mutations in MYOC lead to myocilin misfolding and are associated with endoplasmic reticulum (ER) stress in the trabecular meshwork (TM), the tissue that maintains aqueous humor outflow and regulates IOP. ER stress and/or death of the TM cells 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 and characterized human TM cell lines overexpressing wild type and mutant human MYOC and looked for ER stress markers using quantitative PCR, Western immunoblotting and immunocytochemistry. MYOC was targeted by either transient transfection of px330-CRISPR plasmids or adenovirus Ad5- mediated transduction of guide RNAs targeting exon 1 of the MYOC gene. Human primary TM cells were treated with Ad5-crMYOC virus and evaluated for DEX-mediated MYOC accumulation and ER stress. Intravitreal injection of Ad5 viruses in transgenic (Tg)-hMYOCY437H ocular hypertension mice was performed to study the effect on IOP.
Results: The results revealed that TM cell lines overexpressing mutant MYOC have increased accumulation of myocilin in the ER and increased ER stress compared to wild type. Both transient transfection and Ad5-mediated transduction reduce levels of MYOC and ER stress in these cells. Primary TM cells have reduced levels of DEX-mediated ER stress when pretreated with Ad5-CRISPR as compared to null virus controls. Ad5-crMYOC virus significantly reduces IOP as compared to Ad5-cas9 control virus in Tg-hMYOCY437H mice model.
Conclusions: This is a proof of principle study indicating that targeting the mutant MYOC gene can relieve ER stress in TM cells, rescuing their function and health, and preventing glaucoma.Back to top
Jiao, Chunhua;1 Kucukevcilioglu, Murat;2 Jeong, Woojin;3 Lee, Kyung Moo;4 Garmager, Allison;1 Zarei, Kasra;4 Abramoff, Michael D; 1,4 Sohn, Elliott H 1
1Wynn Institute for Vision Research, Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa; 2Department of Ophthalmology, Gulhane Military Medical School, Ankara, Turkey; 3 Department of Ophthalmology, Dong-A University College of Medicine and Medical Science Research Center, Busan, Korea; 4Iowa Institute for Biomedical Imaging, University of Iowa
Purpose: To detect the early retinal neurodegeneration by comparing histological changes in the retinal nerve fiber layer and ganglion cell layer (RNFL/GCL) complex to retinal topography in streptozotocin(STZ)-induced diabetes Mellitus (DM) using the Iowa Reference Algorithm (IRA) for three-dimensional automated layer segmentation of spectral domain optical coherence tomography (OCT).
Methods: C57BL/6J mice (8-10 weeks old) were assigned into STZ-induced DM (n=21) and age-matched control cohorts (n=25). OCT imaging (Bioptigen) was performed at baseline, 6 weeks, and 20 weeks after induction of DM. Using IRA, average change in thickness of the RNFL-GCL was quantified and compared at 0, 6 weeks, and 20 week. Ganglion cell density was determined in every 200um interval on either side of central disc after SNCG antibody labeling. Cell death ELISA was performed to determine retinal cellular apoptosis.
Results: RNFL-GCL thickness in DM mice was significant thinner compared to age-matched controls at 6 weeks (8.97um vs 10.54um, p<0.05) and 20 weeks (6.45um vs 8.98um, p<0.001). Ganglion cell density was reduced significantly at 20 weeks DM group than control (44.1% vs. 50.2%, p<0.05) but not at 6 weeks. Cell death ELISA indicated significant enhanced apoptosis (1.20mU vs. 0.84mU, p<0.05) in 20 weeks DM mice compared with controls but not at week 6.
Conclusions: Inner retinal degeneration is a prominent feature of diabetic retinopathy in this mouse model. Attenuation of the RNFL-GCL complex in mice with 6 weeks of diabetes suggests that OCT may be a more sensitive measure for detecting the earliest retinal changes induced by diabetes.
Johnson, Brodie D.1, Gramlich, Oliver W.2, Kuehn, Markus H.2,3
1College of Engineering, Iowa State University; 2The University of Iowa, Department of Ophthalmology, 3VA Center of Excellence for Prevention and Treatment of Visual Loss, Iowa City, IA
Purpose: Microglia are responsible for surveillance and immune response in the central nervous system. The location and morphology of the Microglia could indicate the function within the layers of the retina.
Methods: Immunohistochemistry was used to determine the location of the microglia in the human retina, as well as levels of activation. Retinas of healthy subjects and subjects with glaucoma were examined. The retinas were stained using antibodies against Iba1, γ-synuclein, and a UEA lectin. Location was determined by analysis of a 3-dimensional reconstruction. Activation was determined by counting the number of processes, as well as qualitative evaluation based on body size.
Results: Microglia are located in three different layers of the retina. Microglia in the nerve fiber layer (NFL), are more activated than in other layers as evidenced by fewer average number of processes. In contrast microglia are found to be ramified along blood vessels in the retinal ganglion cell layer (RGCL), and ramified in the inner plexiform layer (IPL). In the glaucomatous samples examined, activated microglia are predominantly detected in the RGCL instead of the NFL.
Conclusions: Differences in the morphology of microglia exist within the cell layers of the human retina that are more pronounced than those observed in lower mammals. The location of the activated microglia also may differ between normal subjects and subjects with glaucoma.
Karumanchi, Devi Kalyan; Gaillard, Elizabeth
Northern Illinois University
Purpose: To investigate the physical and chemical biomarkers for the glycolytic modifications in the lens in order to develop a diagnostic tool for the early diagnosis of diabetes related eye diseases.
Methods: Lens protein, α-crystallin was glycated and the formation of advanced glycation end-products (AGEs) was screened using steady state and time resolved fluorescence measurements. This procedure was further extended to measure the fluorescence lifetimes of the intact lenses of diabetic and non-diabetic donors.
Results: Emission spectra were recorded from unmodified and glycated alpha crystallin for the excitation wavelengths set at 340 and 435 nm. By excitation at 340 nm, emission spectra showed one distinct peak located at about 440 and 460 nm compatible with the absorption caused by AGEs. A broad band was observed at 500-510 nm by increasing the excitation wavelength to 435 nm. Time-resolved ﬂuorescence measurements were measured in triplicate and then the fluorescence decay profiles were analyzed assuming that the model followed a multiple exponential fit. . In the visible region, unmodified α-crystallin showed no fluorescence and therefore, no lifetimes. However, for glycated α-crystallin, we could see new shorter and longer lifetimes with increase in the time of glycation.
Conclusions: With excitation wavelength at 435 nm and an emission maxima between 500-510 nm, we do not see the influence of tryptophan on the spectral profiles. However, we see a characteristic difference in the fluorescence intensity between non-diabetic and diabetic lenses which follows the trend observed in unmodified and glycated α-crystallin. By exciting the diabetic lenses at 435 nm, we have observed that there is a significant difference in contribution and also an occurrence of new lifetimes. The appearance of new lifetimes with increase in age or disease progression in diabetic lens might be due to the formation of extensive AGE cross-links along with other post-translational modifications. Characterization of the AGE fluorophores in this particular spectral region is in progress.
Khan, Jamil, Goldberg, Mara, Rusillo, Madia, Linderman, Rachel, Carroll, Joseph
Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, WI 53226, USA
Purpose: Optical coherence tomography angiography (OCTA) enables in vivo, quantitative evaluation of retinal vasculature, including the foveal capillary network that defines the foveal avascular zone (FAZ). The purpose of this pilot study was to assess the repeatability, measurement error, and inter-subject variability of FAZ area measurements obtained with OCTA in healthy subjects.
Methods: OCTA was performed using the RTVue XR Avanti in 184 eyes from 92 subjects (ages 8-71) with no known ocular pathology. Two foveally-centered 3x3 mm scans were acquired per eye. The lateral dimension of each scan was corrected for individual differences in axial length. Measurements of the FAZ were derived from images of the superficial capillary plexus using the manufacturer's ReVue software. Repeatability measurements were calculated based on within-subject standard deviation (Sw) as outlined by Bland & Altman (1996).
Results: Consistent with previous reports, there was individual variation in both the size and shape of the FAZ. The average FAZ area across all eyes was 0.2380 mm2, with a standard deviation of 0.0126 mm2. Repeatability was estimated to be 0.0575 mm2 (95% confidence = 0.0545-0.0605 mm2), while the measurement error was 0.0407 mm2 (17.1%).
Conclusions: While our data were collected in a healthy population, we observed substantial variation in image quality that likely contributed to the relatively high measurement error. Manual correction of the FAZ area may improve the accuracy and repeatability of the FAZ measurements. Nevertheless, the data provided here offer a useful foundation for constructing a normative database of FAZ morphology.
Laboissonniere, Lauren1, Goetz, Jillian1, Mani, Adam2, Schwartz, Greg2, Trimarchi, Jeffrey1
1Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa 2Feinberg School of Medicine, Northwestern University, Chicago, Illinois
Purpose: Retinal ganglion cells comprise a small portion of the vertebrate retina, yet they are the sole communication between retina and brain. This population of neurons is composed of more than 25 different types responsible for responding to varying light stimuli and coordinating with visual reflex centers. Despite studies characterizing the distinct morphologies of RGC subtypes, the transcriptomic profiles of many of these cells have remained elusive. Specifically, there has not been a definitive study that links morphological and functional properties of ganglion cells to their transcriptome. Our project aims to uncover the genetic networks operating within subsets of retinal ganglion cells identified by different characteristics, including their physiology and morphology.
Methods: Individual retinal ganglion cells were isolated from adult mouse retinas based upon morphology and physiology. After isolating the RNA and amplifying the cDNA for each individual cell, the resulting cDNA was hybridized to microarrays and analyzed.
Results: Gene clusters have been identified that correlate with specific morphologies/electrophysiological properties of ganglion cells. This method has also revealed transcriptomic variation between individual cells, which we have validated using in situ hybridization. In particular, through our analysis of melanopsin positive On Alpha cells and On Direction Selective cells we have identified potential specific marker genes for each subset.
Conclusions: The analysis of single cells allows for in-depth studies regarding the genetic profile responsible for a particular cell?s function. Our studies have yielded multiple genes that correlate with cell function that we will utilize in the future to better understand the unique roles of each subtype.
Ophthalmology and Visual Sciences, McPherson Eye Research Institute, University of Wisconsin - Madison, Madison, WI, United States.
Cellular clearance mediated by the autophagy-lysosomal pathway is an evolutionarily conserved mechanism for removal of damaged proteins and organelles. Autophagy is especially important in the post-mitotic retinal pigment epithelium (RPE), a highly metabolically active tissue that is a primary site of damage in age-related macular degeneration (AMD). A primary function of the RPE is the phagocytosis and digestion of shed photoreceptor outer segment tips. Over a lifetime, undegraded outer segment components including vitamin A metabolites called bisretinoids accumulate within RPE lysosomes. We recently reported that lipofuscin bisretinoids interfere with autophagy in polarized primary RPE monolayers and in a mouse model of Stargardt inherited macular degeneration. High-speed live imaging showed that bisretinoids interfere with multiple steps of canonical autophagy in the RPE, including autophagosome biogenesis and trafficking and autophagosome-lysosome fusion. The mechanistic target of rapamycin (mTOR) and the transcription factor EB (TFEB) are master regulators of autophagy and lysosome biogenesis. mTOR phosphorylates TFEB and retains it in the cytosol. Upon dephosphorylation, TFEB translocates to the nucleus and induces transcription of target lysosomal and autophagy genes. We now show that outer segment phagocytosis induces nuclear translocation of TFEB, which is inhibited in RPE with bisretinoids. Exogenous expression of TFEB in the RPE increases the activity of lysosomal hydrolases. These data suggest that age-related decline in cellular clearance could contribute to RPE dysfunction and promote vision loss in diseases like AMD.
Laird, Joseph G.1, Pan, Yuan1, Modestou, Modestos1, Yamaguchi, David M.1, Song, Hongman2, Sokolov, Maxim2 Baker, Sheila A.1
1Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA 2Department of Ophthalmology, West Virginia University School of Medicine and West Virginia University Eye Institute, Morgantown, West Virginia 26506, USA
Purpose: In this study we investigated the trafficking of the sodium pump, Na+/K+-ATPase (NKA), in photoreceptors. NKA is essential for maintaining electrochemical gradients. Distinct isoforms of NKA are found in different tissues and subcellular localization patterns. For example, NKA a1 is widely expressed, NKA a3 is enriched in neurons, and NKA a4 is a testes specific isoform found in sperm flagella. In some tissues, ankyrin, a key component of the membrane cytoskeleton, can regulate the trafficking of NKA.
Methods: Subcellular protein localization of NKA, NKA chimeras/mutants, and ankyrin-B was determined using immunohistochemistry of mammalian and transgenic X. laevis retinas.
Results: Ankyrin-B localized to synapses of multiple neurons and Muller glia but did not colocalize with NKA in photoreceptors. In transgenic tadpoles, GFP-NKA a3 and a4 localized to photoreceptor inner or outer segments respectively in accordance with the endogenous localization of a3 in photoreceptor inner segments and a4 in sperm flagella. Examining the subcellular localization of a series of a3/a4 chimeras and mutants revealed a VxP trafficking motif necessary for the outer segment localization of a4. However, this motif was insufficient to overcome the signals retaining a3 in the inner segment.
Conclusions: Ankyrin-B does not regulate the trafficking of NKA in photoreceptors. Ankyrin-B may have an important role in synaptic biology which can be explored in future studies. A search for novel trafficking signals in NKA revealed a VxP motif found to be required for the ciliary targeting of NKAa4. This motif is similar to the trafficking signal in rhodopsin, highlighting the conservation of trafficking pathways. Future studies will focus on identifying how NKA a3 is retained in the inner segment.
Lewis, Carly1, Hedberg-Buenz, Adam1, Anderson, Michael1,2
1Departments of Molecular Physiology and Biophysics and 2Ophthalmology and Visual Sciences, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA
Multiple lines of evidence support vascular influences to glaucomatous neurodegeneration. The purpose of these experiments was to test whether proximity to the major superficial retinal vessels influences cellularity in the retinal ganglion cell layer (RGCL) in the nee mouse model of glaucoma versus strain-matched WT controls. H&E stained retinal flat mounts were collected from 3- to 5-month-old B6 congenic mice homozygous for the nee mutation and healthy C57BL/6J controls, and cellularity was quantified in three zones parallel to each vessel (1, closest to vessel; 2, middle; 3 furthest). Quantified cells were classified using a customized command-based software tool that sorts nuclei into four distinct cell categories based on a variety of measured morphological parameters including size, shape and color intensity. As expected for mice with glaucoma, retinal cellularity was decreased in nee mice compared to healthy controls (7184+1000 vs 9254+652 cells/mm2, P=0.002). Across all areas, zone 1 had a significantly higher density of cells than either zone 2 or 3 in both nee (9488+1030 cells/mm2, P=0.001) and control mice (10961+744 cells/mm2, P=0.0004). When these cells were classified, nee mice exhibited a high density of cells in the displaced amacrine cell (DAC+) category in zone 1 compared to zone 2, while control mice showed no difference in DAC+ density between these zones. These results indicate that morphologically distinct (non-RGC) cells are enriched adjacent to retinal vasculature in healthy and to a greater extent in glaucomatous mice.
Linderman, Rachel; Goldberg, Mara; Khan, Jamil; Russillo, Madia; Carroll, Joe
Medical College of Wisconsin, Department of Ophthalmology
Purpose: OCT Angiography is a non-invasive approach that allows visualization of the retinal vasculature with high resolution. As the previous methodology (fluorescein angiography) was high-risk, there is a relative paucity of data regarding the normal retinal vasculature. Thus, the purpose of this pilot study was to examine interocular symmetry of vascular density in the normal retina.
Methods: One hundred twenty-four eyes in 62 patients with no known eye disease were imaged using RTVue XR Avanti to obtain OCT angiograms. Scan size was 3x3mm centered on the fovea with two scans acquired per eye. Percent vascular density was exported using the RTVue XR Avanti Software and the average of the two scans from each eye was used for comparison.
Results: Across all eyes, percent vascular ranged from 38.43 to 54.69. Percent vascular density in the right eye was 49.24, while in the left eye it was 50.27 (p=0.007). These differences, while small, appeared uniformly distributed throughout the macula. Substantial variability in the image quality was observed across subjects and was noted to dramatically affect percent vascular density measures.
Conclusions: Given the sensitivity of these measurements to image quality, further work is needed to determine whether the observed differences are real. Regardless the data provided here provide a useful starting point to a normative database of retinal vasculature.
Mavlyutov Timur, Epstein, Miles, Guo, Lian-Wang
University of Wisconsin, Madison
Purpose: The Sigma-1 receptor (S1R) is known to play a protective role in the central nervous system including the retina. A major barrier for understanding the underlying mechanism is an ambiguity of S1R subcellular localizations.
Methods: We thus conducted the first electron microscopy (EM) study of S1R subcellular distribution in the mouse retina.
Results: Immuno-EM imaging showed previously under-appreciated S1R presence in photoreceptor cells. Unlike in other cell types in previous reports, in photoreceptor cells S1R was found in the nuclear envelope but not localized in the endoplasmic reticulum (ER), raising a possibility of S1R-mediated modulatory mechanisms different than conventionally thought. While in bipolar cells S1R was detected only in the nuclear envelope, in ganglion cells S1R was identified predominantly in the nuclear envelope and found in the ER as well.
Conclusions: A predominant localization of S1R in the nuclear envelope in all three retinal neurons implicates a potential role of S1R in modulating nuclear activities. Moreover, its absence in the plasma membrane and presence in the subsurface ER cisternae that are juxtaposed to the plasma membrane in ganglion cells may lend mechanistic insights generally important for frequently reported S1R modulations of ion channels in neurons.
Meyer, Kacie J.1,2, Whitmore, S. Scott2, Scheetz, Todd E.2, Anderson, Michael G.1,2
1Department of Molecular Physiology and Biophysics and 2Department of Ophthalmology and Visual Sciences, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA
Purpose: Epigenetics likely contributes to genetically complex ocular diseases with missing heritability and the variable expressivity observed in monogenic ocular disease. Genomic imprinting is an epigenetic phenomenon resulting in differential allelic gene expression that is dependent on the parent-of-origin. The goal of this study is to identify imprinted genes expressed in the mouse retina.
Methods: Using RNA-Seq to determine parent-of-origin expression, we analyzed the retinal transcriptome of two genetically diverse inbred mouse strains and their resulting F1 progeny from reciprocal crosses.
Results: To date, the data identify 22 known imprinted genes that are expressed and imprinted in the retina of mice. Of these, 5 genes are paternally imprinted and 17 genes are maternally imprinted. Initial analysis has not identified novel imprinted retinal genes.
Conclusions: This work provides proof-of-principle evidence of a role for imprinted genes in the retina. Future studies will investigate the potential contribution of imprinted genes to the pathogenesis of retinal disease.
Department of Biology, Department of Medical and Molecular Genetics, and Stark Neurosciences Research Institute, Indiana University, Indianapolis IN 46202
Purpose: Human pluripotent stem cells (hPSCs) have the ability to serve as comprehensive models of retinogenesis as well as effective tools for the study of retinal disease. In order for this potential to be realized, however, hPSCs must be directed to differentiate in a stepwise manner that recapitulates the major stages of retinal development. Thus, efforts were undertaken to guide the differentiation of hPSCs to a retinal lineage in a manner that allowed for the three-dimensional differentiation and organization of retinal cells from hPSCs. Furthermore, induced pluripotent stem cells (iPSCs) were generated from a glaucoma patient exhibiting an E50K mutation in the Optineurin (OPTN) gene, associated with degeneration of RGCs leading to normal tension primary open angle glaucoma, with these cells differentiated to an RGC fate and assayed for phenotypic characteristics of glaucomatous neurodegeneration.
Methods: hPSCs were induced to differentiate towards a retinal fate and assayed via immunocytochemistry and qRT-PCR analysis. The three dimensional organization of retinal cells derived from hPSCs was analyzed, including the ability of these cells to form discrete retinal-like layers. Furthermore, fibroblasts were obtained from a glaucoma patient exhibiting an E50K mutation in the OPTN gene, and these fibroblasts were used to generate patient-specific OPTN iPSCs. OPTN iPSCs were differentiated to an RGC fate and phenotypic differences between wild type and OPTN iPSC-derived RGCs were explored.
Results: Within 40 days of differentiation, retinal cells including RGCs could be readily identified based upon phenotypic characteristics as well as their three-dimensional organization into retinal-like structures. Analysis of hPSC-derived RGCs revealed that these cells expressed numerous morphological, phenotypical, and physiological characteristics of RGCs. In addition, OPTN iPSC-derived RGCs were found to display striking phenotypic differences compared to wild type cells.
Conclusions: The data presented demonstrates the ability of hPSCs to serve as a reliable in vitro model of human retinogenesis and retinal disease, as seen by their ability to yield a population of cells possessing a full complement of retinal-associated characteristics. These results will facilitate future studies into the disease-related degeneration of RGCs and as such, will be instrumental as a tool for the study of optic neuropathies, as well as the development of therapeutic approaches.
Ohlemacher, Sarah1, Sridhar, Akshayalakshmi1, Xiao, Yucheng2, Hochstetler, Alexandra1, Sarfarazi, Mansoor5, Cummins, Theodore2,4, Meyer, Jason1,3,4
Departments of 1Biology, 2Pharmacology and Toxicology, 3Medical and Molecular Genetics and the 4Stark Neurosciences Research Institute, Indiana University Purdue University Indianapolis, Indianapolis IN 46202 5Department of Human Molecular Genetics, University of Connecticut, Farmington CT 06030
Purpose: The ability to derive retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) provides an unlimited supply of cells for the study of RGC development and disease, with important implications for drug screening, disease modeling, and cell replacement strategies. However, the ability to derive RGCs from hPSCs has been largely overlooked to date. Thus, the ability of hPSCs to yield a population of RGCs was explored. Additionally, induced pluripotent stem cells (iPSCs) were generated from a glaucoma patient exhibiting an E50K mutation in the Optineurin (OPTN) gene, associated with degeneration of RGCs leading to normal tension primary open angle glaucoma. These cells were differentiated to an RGC fate and assayed for phenotypic differences compared to a wild type line of iPSCs.
Methods: hPSCs were induced to differentiate towards a retinal fate following previously established protocols and RGCs were subsequently characterized for the expression of RGC related features. Skin fibroblasts were obtained from a glaucoma patient exhibiting an E50K mutation in the OPTN gene, and these fibroblasts were used to generate patient-specific OPTN iPSCs using mRNA reprogramming. OPTN iPSCs were differentiated to a retinal lineage in a stepwise fashion that allowed for the definitive identification of RGCs based upon gene expression patterns. Phenotypic differences between wild type and OPTN iPSC-derived RGCs were explored, including changes in golgi fragmentation, apoptosis, and autophagy.
Results: Within 40 days of differentiation, RGCs could be readily identified due to the expression of RGC-associated markers as well as their documented transition through a highly enriched retinal progenitor stage. Analysis of hPSC-derived RGCs revealed that these cells expressed numerous morphological, phenotypical, and physiological characteristics of RGCs. In addition, OPTN iPSC-derived RGCs were found to display striking phenotypic differences compared to wild type cells.
Conclusions: The data presented demonstrates the ability of hPSCs to serve as a reliable source of RGCs, including those derived from glaucoma patient sources, as seen by their ability to yield a population of cells possessing a full complement of RGC-associated characteristics. These results will facilitate future studies into the disease-related degeneration of RGCs and as such, will be instrumental as a tool for the study of optic neuropathies, as well as the development of therapeutic approaches.
Oikawa, Kazuya1,2, JN VerHoeve2, CA Rasmussen2, JA Kiland2, EA Hennes-Beean2, S Splinter-Bondurant3, X-Y Liu3, A Ikeda4, NM Ellinwood5, GJ McLellan1,2,3
1Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, WI 2Department of Opthalmology & Visual Sciences, School of Medicine and Public Health, Madison,WI 3University of Wisconsin-Madison Biotechnology Center, Madison, WI 4McPherson Eye Research Institute, Madison, WI 5Department of Animal Science, Iowa State University, Ames, IA
Purpose: To identify early changes in structure, function and gene expression in the optic nerve head (ONH) in feline congenital glaucoma (FCG).
Methods: Nineteen 10-12 week-old cats (11 FCG and 8 control) were studied. IOP was measured three times weekly by rebound tonometry. ONH cube scans were acquired by spectral domain optical coherence tomography (OCT) for analysis of cup depth, posterior laminar displacement, width of neural canal opening and pre-laminar tissue thickness. Visual evoked cortical potentials (VEPs) were recorded, from which root mean square (RMS) of the early wavelets and peak amplitudes of the late positive component (?P2?) were calculated. Following euthanasia, RNA was extracted and purified from ONHs for sequencing. Optic nerve axons were quantified using a semi-automated targeted sampling method. Data from one eye per cat were compared by unpaired t-tests.
Results: In FCG cats, mean [SD] IOP (18.7 [5.0] mmHg) was significantly higher (12.8 [0.8] mmHg; P=0.0014) and VEP P2 amplitude and RMS were significantly lower than normal (p=0.045, and 0.024, respectively). Compared to normal cats, the ONH in FCG had a significantly increased optic cup depth and reduced pre-laminar tissue thickness (p=0.028 and 0.007, respectively). Mean axon count was not significantly different. Analysis of ONH differentially expressed genes is in progress.
Conclusions: Cats with FCG show IOP elevation, ONH remodeling, and optic nerve functional impairment early in life prior to significant axon loss. Ongoing analysis of differentially expressed genes in the ONH of these cats may provide insight into early pathophysiology of optic neuropathy in glaucoma.
Patterson, Emily J, Langlo, Christopher, Carroll, Joseph
Medical College of Wisconsin
Congenital achromatopsia (ACHM) is an autosomal recessive disorder characterized by impaired visual acuity, absent color vision, nystagmus and photophobia. Crucial to the success of emerging gene therapy trials for ACHM is the development of accurate outcome measures to monitor treatment efficacy. Although color vision loss may be one of the least troublesome manifestations of ACHM, its measurement has potential as a valuable assessment tool in ACHM clinical trials. We recruited 11 patients with confirmed mutations in the CNGB3 gene, 2 patients with unconfirmed mutations (aged 14-56), and 13 normal subjects (aged 23-49). ACHM patients completed 2 trials of the Color Assessment and Diagnosis (CAD) test, modified in order to compensate for reduced visual acuity and to mask the detection of rod-mediated signals. Normal subjects completed the test using both the standard and modified parameters. ACHM patients had higher thresholds (29.2 to 38.3 CAD units for red-green and 12.1 to 15.2 for yellow-blue) than normal subjects (1.16 to 2.04 and 0.91 to 1.73), using the modified protocol. Whether the lower thresholds observed in some ACHM subjects were due to ineffective luminance masking or in fact represented genuine residual color vision is yet to be determined. There was a small learning effect between trials for red-green thresholds (p <.05), however the ACHM patients with the lowest thresholds showed excellent repeatability. Despite the challenges that ACHM poses for measurement of color vision, the current findings support the continued exploration of the CAD test in this patient group.
Rea, Brandon J.; Bittle, Jada; Russo, Andrew F.
Dept. of Molecular Physiology and Biophysics, Interdisciplinary Graduate Program in Neuroscience, Dept. of Neurology, University of Iowa, Iowa City, Iowa 52242; Center for the Prevention and Treatment of Visual Loss, Iowa VA Health Care System, Iowa City, Iowa, 52246
Purpose: Migraine is a neurological disorder that plagues over 18% of women and 6% of men in the United States. More than just a headache, migraines frequently have debilitating pain, last for up to 72 hours, and at least one of two symptoms: nausea and/or vomiting, or photophobia and phonophobia. Photophobia is a condition where low to normal levels of light are painful and cause the perceiver to be light aversive. The neuropeptide Calcitonin Gene-Related Peptide (CGRP) is elevated during migraine. Intravenous injection of CGRP has also been shown to induce migraine-like headache in migraineurs only with non-migraineurs experiencing a less severe headache or fullness-of-head. This clinical evidence suggests that CGRP plays a key role in migraine etiology. Currently we have an established mouse model for CGRP-induced photophobic behavior. However, we have yet to establish a link between CGRP-induced photophobia and associated pain due to migraine-like symptoms in this model. We hypothesized that after administration of CGRP our mice would exhibit increased expression of discomfort or pain when exposed to light.
Methods: Mice were acclimated to a restraint and recorded via camera during dark and light conditions. Mice were then given an intraperitoneal injection of CGRP (0.5 mg/kg) and recorded during dark and light conditions. Using the Mouse Grimace Scale, mice were independently scored by two blinded observers for pain expression.
Results: CGRP caused a significant increase in pain expression compared to a saline control in both dark and light conditions. No significant difference was observed between dark and light.
Conclusions: CGRP does cause increased pain expression in our mouse model compared to control.
Russillo, Madia; Goldberg, Mara; Khan, Jamil; Linderman, Rachel; Connor, Thomas; Han, Dennis; Kim, Judy; Carroll, Joseph
Medical College of Wisconsin, Department of Ophthalmology
Purpose: OCT-Angiography is a novel imaging technique that employs motion contrast image processing to generate noninvasive angiography-like images of the living human retina. The technique offers a number of advantages over conventional fluorescein angiography, however there is minimal data directly comparing the two techniques. Here we compare findings from FA and OCT angiography in patients with retinal vascular diseases.
Methods: Twelve eyes in 9 patients with type II diabetes and 3 eyes in 2 patients with retinal vein occlusion were imaged with RTVue XR Avanti to obtain OCT angiograms. All subjects had FA done within one year of OCT-A session. The scanning area captured 3x3mm and 6x6mm sections centered on the fovea.
Results: Compared to FA, the OCT-A images showed improved visualization of retinal vasculature over in all cases. Visualization of the foveal capillary network and foveal avascular zone (FAZ) by OCT-A is time independent and not obscured by dye leakage or wash out. Our case of BRVO showed that OCT-A is capable of visualizing vasculature through hemorrhage and possible cotton wool spots. In diabetic patients, microaneurysms visualized on FA were frequently detectable on the corresponding OCT-A image, usually arising from the deep capillary layer.
Conclusions: Imaging by OCT-A technology is comparable to FA findings with enhanced visualization of vascular details. However the ability of OCT-A to segment various vascular layers has significant potential for examining disease progression and monitor vascular changes in response to various treatments.
Scheetz, Todd E., DeLuca, Adam P., Stone, Edwin M.
Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Department of Ophthalmology, Iowa City, IA
Purpose: In recent years, clinical genetic testing for inherited eye diseases has moved to genome-wide approaches like whole exome sequencing (WES) that have the power to detect unexpected phenotype-genotype correlations. We have discovered many cases of non-syndromic disease caused by genes usually associated with syndromic disease.
Methods: To date, WES has been obtained for 759 probands with inherited eye diseases. All putative disease-causing mutations in genes relevant to a patientís disease are confirmed with bidirectional sequencing and phase is established using samples from family when available.
Results: 4/570 photoreceptor-disease exome patients were homozygous for BBS1 M390R, yet escaped clinical diagnosis as BBS, including a case of a patient with apparent Usher Syndrome caused by the confluence of BBS1-associated Retinitis Pigmentosa (RP) and SLC26A4-associated deafness. An additional screen of 219 patients revealed three non-syndromic RP patients with homozygous M390R. Similarly, in FLVCR1, a gene reported to cause PCARP, we discovered plausible disease-causing genotypes in 8/570 patients that escaped the PCARP diagnosis. Interestingly, six of these patients have c.1092+5G>A heterozygous with more severe mutations, suggesting it could be hypomorphic allele. In both cases, upon careful reexamination some patients had subtle syndromic features, and some had non-syndromic disease.
Conclusions: Genome-scale experiments like WES in patients with non-syndromic photoreceptor diseases can lead to discoveries of genotypes in genes only known to cause syndromic disease. These genotype/phenotype mismatches need to be resolved either by a careful clinical reevaluation that discovers subtle syndromic stigmata that escaped previous detection, or by gathering enough non-syndromic patients to expand the phenotypic scope of the disease. If the genotype-phenotype mismatch is not resolved between the clinic and the laboratory, the likelihood of false positives increases, and syndromic features that could be relevant to patient care will be overlooked.
Schwarz, Chaid1, Aldrich, Benjamin T.2,3, Burckart, Kimberlee3, Schmidt, Gregory A.3, Reed, Cynthia R.3, Greiner, Mark A.2,3, Sander,Edward A.3
1Department of Biomedical Engineering, University of Iowa, Iowa City, IA 2Department of Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, Iowa City, IA 3Iowa Lions Eye Bank, Coralville, IA
Purpose: Descemet's membrane endothelial keratoplasty (DMEK) is an increasingly popular surgical procedure for treating corneal diseases. Anecdotal evidence suggests that the force required to peel the membrane, in preparation for surgical implantation, is significantly higher for diabetic donor tissue compared to non-diabetic and may lead to higher preparation failures (Greiner et al., 2014). Here, we report on the development of a testing method for quantifying the peel strength of human donor corneas, and our progress on quantitatively assessing the putative differences in diabetic and healthy tissue.
Methods: Sourced healthy (H), ungraded diabetic (D), and severe diabetic (SD) donor corneas were selected for efficacy as surgical implants. To reflect loading conditions used in surgical preparation, we applied cantilever bending to rectangular sectioned samples. Here, a load was applied to the end of a partially peeled endothelial-Descemet's membrane by a thin Nitinol bar calibrated to a known force-displacement relationship. Matlab was used to analyze video tracking bar displacement and therefore the peel force.
Results: Ongoing results show general trends with no difference between healthy and ungraded diabetic donor tissue. Contrary trends appear between healthy and severe diabetic tissue [force/width (g/mm) prior to peel propagation, mean std.dev., 0.080 0.008 (H), 0.0810.010 (D), 0.108 0.029 (SD)].
Conclusions: Results indicate that our test method is sufficient in measuring very low forces (>0.01 gf). Additionally, severe diabetes mellitus may be a risk factor influencing donor preparation failures.
Scott, Anthony, Slusarski, Diane
Department of Biology, University of Iowa, Iowa City, IA
Purpose: The zebrafish is an excellent model for in vivo analysis of gene function. The larval zebrafish displays a fully laminated retina by 72 hours-post-fertilization (hpf) and behavior responses to visual stimuli can be reliably tested by 5 days-post-fertilization (dpf). While acquiring hundreds of larval zebrafish for analysis of visual function is simple, individual testing of visual competence is slow, tedious, and prone to human error and bias. I aimed to develop a method of high-throughput analysis of the zebrafish visual response while minimizing error.
Methods: Using the ViewPoint ZebraBox hardware we simultaneously captured the motion of 96 larval zebrafish as we subject them to visual-startle stimuli. I developed software, Visual Interrogation of Zebrafish Manipulations (VIZN), to automatically analyze the response of every individual fish at each stimulus, group by genotype, plot the data, and perform statistics.
Results: To validate the system, I knocked-down genes associated with human retinitis pigmentosa (RP) in the zebrafish. VIZN successfully analyzed the data showing a significant decrease in visual responses in the experimental fish compared to controls, and these results are consistent with manually collected and analyzed data.
Conclusions: VIZN combined with the ZebraBox successfully automates the visual startle response assay and can test and analyze 96 zebrafish in approximately 30 minutes, in contrast the manual analysis for the same number of fish which would take over 8 hours. This allows for rapid, high-power, analysis of rare RP candidate genes and for testing of small-molecule reagents that suppress the vision defects.
Snyder, K.C.1,2, Kim, C.B.Y.1, Nork, T.M.1,3, Hennes-Beean, E.A.1, Teixeira L.B.C.3,4, Ellinwood, N.M.5, Verhoeve, J.N.1,3, McLellan, G.J.1,2,3
1Dept. of Ophthalmology and Visual Sciences, School of Medicine and Public Health; 22Dept. of Surgical Sciences, School of Veterinary Medicine; 3McPherson Eye Research Institute, and 4Dept. of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI; 5Dept. of Animal Sciences, Iowa State University, Ames, IA.
Purpose: To establish and validate measures of optic nerve (ON) axon and retinal ganglion cell (RGC) soma function in FCG.
Methods: Glaucomatous cats were derived from a colony that represents the ortholog of human PCG (GLC3D) caused by an LTBP2 mutation. Visual Evoked Cortical Potentials (VEPs) and Pattern Electroretinograms (PERGs) were recorded in 39 sedated cats at 6, 12, 18, and 24 months of age. VEP stimuli were 4.1 Hz white flashes (2.7 cd-s/m2 on a photopic background of 30 cd/m2), presented monocularly. PERG stimuli were high contrast (>0.99) black and white horizontal stripes (mean luminance 150 cd/m2), presented monocularly at 5 spatial frequencies, reversing at rates of 4 reversals/sec. Responses were averaged per temporal frequency, per testing session; with two testing sessions conducted 7-21 days apart. In cats euthanized at different ages, ONs were processed for axon quantification using a semi-automated targeted sampling method. Data were analyzed using custom MatLab routines with averaged responses compared by two-tailed student's t-test with Bonferroni correction for multiple pair-wise comparisons and by two-way ANOVA.
Results: VEP amplitudes were significantly lower than normal in 6-month-old PCG cats and strongly correlated with ON axon counts in cats less than one year old. The relationship between VEP P2 amplitude and ON axon count was linear in cats <1 year-old. PERG deficits suggested RGC dysfunction, but differences were not consistently significant across all time points.
Conclusions: Our findings support the use of VEPs as a non-invasive, quantitative measure of ON structural damage in young cats with PCG.
Songstad, Allison E., Luke A. Wiley, Cathryn M. Cranston, Miles J. Flamme-Wiese, Jeremy M. Hoffmann, Christine M. Haas, Edwin M. Stone,Robert F. Mullins, Budd A. Tucker
Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Department of Ophthalmology, Iowa City, IA
Purpose: Age-related macular degeneration (AMD) involves the dysfunction and eventual death of choroidal endothelial, retinal pigment epithelial (RPE), and photoreceptor cells. Induced pluripotent stem cell (iPSC)-based strategies designed to replace both photoreceptor and RPE cells are currently a major scientific focus. For some patients, success of these approaches may also require replacement of choroidal endothelial cells (CECs). The purpose of this study was to generate CECs from two Tie2 GFP iPSC reporter lines to develop differentiation and transplantation protocols.
Methods: Dermal fibroblasts from the Tie2 GFP mouse were isolated and reprogrammed into two iPSC lines via viral transduction of the Yamanaka factors. iPSC potency was characterized via RT-PCR, immunocytochemistry (ICC), and teratoma formation assays. The iPSCs were differentiated into CECs using a co-culture method with either the RF6A CEC line or primary mouse CECs. The iPSC-CECs were characterized via RT-PCR and ICC for various EC- and CEC-specific markers.
Results: Both iPSC lines were co-cultured with either the RF6A cell line or primary mouse CECs and differentiated into CECs. The two iPSC-CEC lines expressed the EC markers CD31, CD34, eNOS, FOXA2, ICAM1, PLVAP, TIE2, VE-Cadherin, and VWF, as determined by RT-PCR, expressed the CEC-specific markers CA4 and TTR, as determined by RT-PCR and ICC, and were morphologically indistinguishable from native CECs that expressed ZO-1 and VE-Cadherin in cell-cell adhesions.
Conclusions: We have successfully generated Tie2 GFP iPSCs that can be differentiated into choroidal-like ECs. This work has paved the way for future studies focused on investigating AMD pathophysiology and CEC replacement.
Sowers Levi1,2,3, Rae, Brandon1, Kuburas, Adisa1, Teran, Leonel1, Ma, Shawn1, Russo, Andrew1,2,3,4
1The Department of Physiology and Biophysics, The University of Iowa, Iowa City, IA 2Pain Research Program, The University of Iowa, Iowa City, IA 3The VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA 4Department of Neurology, The University of Iowa, Iowa City, IA
Purpose: Veterans returning from active duty are at an increased risk for post-traumatic headache (PTH) and migraine1-4. Migraine alone affects 10% of men and 25% of women, with the lifetime risk increasing to 18% and 43% respectively. Sensory abnormalities are present both in individuals with PTH and migraine including extreme light and sound sensitivity. Light sensitivity in patients with PTH or migraine can be quite debilitating and treatments are lacking. One reason interventions and treatments continue to fall short is that there is a poor understanding of the relevant neuroanatomical correlates that underlie sensory changes in migraine. In this study we attempt to identify anatomical regions associated with the development of photophobia associated with migraine and PTH. We hypothesize that posterior thalamus (Po) is a critical brain region for the development of migraine and PTH induced light-aversion.
Methods: We used an optogenetic approach to probe areas of the brain thought to be important in the development of photophobia. Channelrhodopsin-GFP driven by the CaMKII promoter was used to stimulate neurons at either 5 or 20 Hz. Direct injection of CGRP (2μg) into brain regions was done using stereotaxic methods. To assess light-aversive behaviors, the modified light-dark box assay was used.
Results: Preliminary evidence suggests that direct optical stimulation of the Po thalamic nuclei increased light-aversive behavior in mice. Moreover, direct bilateral CGRP injection into the Po also led to significant light-aversive behavior in mice.
Conclusions: Our preliminary evidence suggests that the Po could be a critical brain region in the development of light-aversive behaviors during migraine and PTH.
Sridhar, Akshayalakshmi; Ohlemacher, Sarah K.; Langer, Kirstin B.; Meyer, Jason S.
Department of Biology, Indiana University, Indianapolis IN 46202
Purpose: The derivation of retinal neurons from a patient-specific source enables studies of human development and disease. Reprogramming of somatic cells via mRNA-based approaches eliminates risks associated with viral-vectors and may provide a safer alternative, but the efficient retinal differentiation of such cells has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of hiPSCs generated via mRNA reprogramming to effectively yield retinal phenotypes.
Methods: Multiple lines of hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retrovirus delivery of reprogramming factors. New lines of hiPSCs were established from each reprogramming method, and hiPSCs were then differentiated to a retinal fate following established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation was compared in hiPSCs derived by both methods at various stages of differentiation.
Results: Both mRNA and retroviral methods of reprogramming yielded stable lines of hiPSCs expressing numerous pluripotency-related characteristics as assessed by immunocytochemistry and RT-PCR. Upon differentiation, mRNA reprogrammed hiPSCs were capable of robust differentiation toward a retinal phenotype as assessed by the expression of retinal-associated genes. Within two months of differentiation, many types of retinal cells could be derived from both sources, including retinal pigment epithelium, photoreceptors, and retinal ganglion cells.
Conclusions: The data presented demonstrates the feasibility of utilizing mRNA-based reprogramming strategies to derive lines of patient-specific hiPSCs for purposes of retinal differentiation. Such methods represent a promising new approach for hiPSC-based translational studies of the retina.
Street, Aaron; Brazel, Zach; Karumanchi, Devi Kalyan; Gaillard, Elizabeth
Northern Illinois University
Purpose: To develop liposomal formulations for the sustained and controlled release of anti-VEGF antibodies to treat neovascularization associated with diabetic retinopathy and wet AMD.
Methods: Stable liposomal formulations were made using a modified lipid hydration and extrusion method. A model fluorescent protein was encapsulated in the liposomes for preliminary studies and evaluation of the vehicle. The liposomes were evaluated based on particle size, surface morphology, percentage drug encapsulation and time of release using dynamic light scattering, transmission electron microscopy, fluorescence spectroscopy and USP4 SOTAX dissolution apparatus, respectively. Biophysics of liposomes was determined using small angle X-ray scattering and differential scanning calorimetry.
Results: The liposomal formulations after extrusion exhibited a narrow size distribution of approximately 100-150 nm in diameter with around 85-92% encapsulation efficiency. From the in vitro drug release studies, we observed a timed release over a period of 6-8 months depending on the composition of the formulation.
Conclusions: Liposomes are non-toxic, biodegradable artificial vesicles composed of phospholipids and cholesterol. Abrishami et al have been able to obtain a sustained release of the anti-VEGF drugs up to a period of 42 days. Currently, we have been successful in encapsulating a model protein into our stable liposomal formulations and attain a controlled release over a period of 6 months in vitro. In the future, we are interested in encapsulating the protein drugs- Avastin and Lucentis to show the efficiency of our drug delivery vehicle. With this study, our efforts would be to decrease the frequency of intravitreal injections from 12 to 2 per year, thereby effectively making the treatment more economical.
Tan, Li Xuan; Toops, Kimberly; Lakkaraju, Aparna
University of Wisconsin-Madison
Purpose: Maintenance of membrane integrity is critical for cell survival. Assembly of sub-lytic membrane attack complexes (MAC) on the plasma membrane of retinal pigment epithelial (RPE) cells damages membrane integrity and contributes to the pathogenesis of age-related macular degeneration (AMD), a major cause of irreversible blindness in older adults. Nucleated cells have multiple protective mechanisms to deal with sub-lytic MAC assembly. Here, we aim to: (a) identify complement regulatory mechanisms in the RPE and (b) determine how these mechanisms are affected by pathological insults.
Methods: Live-cell imaging, TIRF, immunofluorescence staining, biochemical assay
Results: Our data show that membrane integrity of the RPE after sub-lytic MAC attack is maintained by the concerted actions of complement regulatory proteins and lysosome exocytosis. In cells with lipofuscin bisretinoids, cholesterol-mediated activation of acid sphingomyelinase (ASMase) interferes with the delivery of the GPI-anchored CD59 to the plasma membrane and inhibits lysosome exocytosis. Pharmacological removal of cholesterol as well as inhibition of ASMase restores these protective mechanisms.
Conclusions: Our studies provide insights into how membrane integrity can be compromised and restored in the face of multiple pathological insults in the RPE and suggest that precise organelle trafficking is critical for preventing chronic inflammation in the RPE and the retina.
Tatro, Nicole J., DeLuca, Adam P., Christopher, Mark A., Stone, Edwin M., Scheetz, Todd E.
Wynn Institute for Vision Research, University of Iowa
Purpose: Goldmann perimetry has long been the standard for assessing visual fields in patients with inherited retinal disease. While new systems like the Octopus 900 allow digital collection of these data, there exists a wealth of historic Goldmann visual field (GVF) data including patients followed for several decades. We implemented a system that allows the capture of historic GVF data, and extraction of relevant phenotypic information including the volume of the hill of vision. We compare this system to a previous system by Weleber et.al.
Methods: A custom profile was developed for TruthMarker to allow the tracing and registration of isoptors from scanned GVFs. A post processing program has been implemented to calculate the area of each isoptor while correcting for the cartographic distortion inherit in the planometric GVF projection and to calculate the total volume of the hill of vision.
Results: GVFs were digitized by two readers on both systems for a set of 17 fields. A comparison between the platforms, showed excellent reproducibility with a Pearson's correlation of r2 = 0.9997. This compares well with the inter-reader variability of r2 = 0.9998 on the TruthMarker-based system and r2 = 0.9993 on the Weleber et al. system. All of these values are well within the test-retest variability of Goldmann perimetry.
Conclusions: Our method showed excellent correlation with the existing methodology. TruthMarker also allows flexible capture of custom phenotypic data via an XML-based configuration file. The support of modern technologies and mobile devices will allow larger-scale digitization studies to proceed.
Trotter, Alana 1, Langlo, Christopher2, Carroll, Joseph1,2,3, Connor Jr., Thomas1
1Department of Ophthalmology, Medical College of Wisconsin, 2Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 3Department of Biophysics, Medical College of Wisconsin
Purpose: To understand cellular level changes in a case of juvenile pigmentary retinopathy using adaptive optics scanning light ophthalmoscopy (AOSLO).
Methods: One 10-year-old asymptomatic subject with pigmentary retinopathy was recruited. Clinical evaluation included fundus exam, visual field testing and electroretinography (ERG); imaging included optical coherence tomography (OCT), fundus autofluorescence and confocal and split-detector AOSLO. Examinations were repeated at 7, 18, 30 and 40 months. Cone density was assessed at eight locations: at the foveal center, four locations at 0.65 degrees from the foveal center and at 1, 5 and 10 degrees temporal to the fovea at each annual visit.
Results: The subject's pigmentary retinopathy appeared stable throughout the follow-up period. Visual acuity and night vision were unaffected, and visual fields revealed no defects or changes over time. OCT imaging showed no obvious disruption. There was an overall reduction in the number of waveguiding cones on confocal AOSLO consistent with mild to moderate photoreceptor abnormalities on ERG, though there was no cone loss between time points. Split detection AOSLO revealed remnant cone inner segment structure within dark areas on confocal AOSLO.
Conclusions: While juvenile pigmentary retinopathy may result from a range of disorders, this case appears non-progressive. There was minimal functional loss observed in this patient and no evidence of phenotypic progression during a 40-month period. Pairing follow-up with clinical examination with high-resolution imaging allows for monitoring disease progression or stability and can assist in diagnosis.
Toops, Kimberly A.; Tan, Li Xuan; Lakkaraju, Aparna
Ophthalmology and Visual Sciences, University of Wisconsin - Madison, Madison, WI, United States. McPherson Eye Research Institute, University of Wisconsin - Madison, Madison, WI, United States.
Purpose: The retinal pigment epithelium (RPE) depends on its endo-lysosomal network for degrading and recycling phagocytosed photoreceptor outer segments. With age, declining efficiency of this system leads to the formation of aggregates within (lipofuscin bisretinoids) and around (drusen) the RPE. Bisretinoids, visual cycle byproducts that accumulate in RPE lysosomes, increase lysosomal pH and trap excess cholesterol. Late endosomes and lysosomes are transported along microtubules within the cell. We recently showed that bisretinoids increase tubulin acetylation on stable microtubules, which interferes with autophagosome trafficking and autophagy. Here, we investigated how bisretinoids and cholesterol affect the organization and microtubule-mediated transport of late endosomes and lysosomes.
Methods: Adult primary RPE were genetically or chemically labeled with markers for late endosomes, lysosomes, microtubules and cholesterol. Control RPE and cells treated with the bisretinoid A2E were imaged live using highspeed spinning disk confocal microscopy (Andor). 4D image analysis (Imaris) was performed to quantify organelle numbers, morphology and trafficking. RPE lysates from wild type and Stargardt disease (Abca4-/-) mice were immunoblotted for endo-lysosomal proteins.
Results: Analysis of live imaging data showed intriguing differences between late endosomes and lysosomes in the RPE. We observed fewer late endosomes, with limited long-range movements in cells with A2E, compared to control RPE. These data were confirmed in vivo: Abca4-/- RPE had less CD63 (a marker for late endosomes), compared to age-matched wild types. Lysosome numbers were unchanged in A2E-treated cells, but lysosome volume increased. Lysosomes in cells with A2E exhibited trafficking anomalies in the population with displacements > 2 μm: these moved faster with altered microtubule association and frequent directional switches.
Conclusions: Our data indicate that subtle defects in morphology and transport could contribute to a slow loss of endolysosomal degradative and signaling functions in RPE with bisretinoids. Importantly, these anomalies are deviations from normal trafficking patterns, as opposed to a total cessation of organelle trafficking. Live imaging can provide valuable insight into organelle traffic and function in the RPE and help identify key vulnerabilities that can promote diseases like macular degenerations.
Whitmore, S Scott; DeLuca, Adam P; Zeng, Shemin; Affatigato, Louisa M; East, Jade S; Tucker, Budd A; Stone, Edwin M; Mullins, Robert F; Scheetz, Todd E
Stephen A. Wynn Institute for Vision Research, The University of Iowa
Purpose: Retina-specific alternative splicing contributes to the proper function of the retina, and aberrant splicing of critical genes can lead to retinal degeneration (e.g., MAK, SNRNP200, ABCA4, BBS8). To investigate splicing differences across temporal, macular, and nasal regions of the neural retina, we reanalyzed our previously published RNA-Seq dataset for isoform-level expression differences.
Methods: We used the Tophat2 software to map over 560 million paired-end RNA-Seq reads from three regions of the retina and RPE/choroid of four human donors (24 total samples), and we identified known and novel transcript structures as described in (Whitmore et al. Exp Eye Res 2014). We considered individual splicing isoforms to be differentially expressed if the expression between regions was at least two-fold different and the q-value < 0.01.
Results: Sixty-seven transcripts were differentially expressed between macular retina and temporal retina, while 148 transcripts were differentially expressed between macular and temporal retina. These genes were enriched for ontology terms associated with locomotor activity and neurological signaling. As we observed previously for gene-level comparisons, no alternative isoforms were differentially expressed between nasal and temporal regions of the retina. Additionally, we identified several transcriptional events that are specific to the neural retina.
Conclusions: Alternatively spliced transcripts do not appear to be differentially transcribed between nasal and temporal retina at our criteria for differential expression. This result suggests that alternative splicing may not strongly shape the nasal and temporal differences observed in some retinal degenerations, such as MAK-associated retinitis pigmentosa.
Wiley L.A.1, Anfinson K.R.1, Cranston C.M. 1, Kaalberg E.1, Ochoa D. 1, Shrestha S. 1, Owens L.A. 1, Mullins R.F.1, Stone E.M.1, and Tucker B.A.1
1Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology, Carver College of Medicine, University of Iowa, Iowa City, IA
Purpose: Juvenile neuronal ceroid lipofuscinosis, or Batten disease, is a lysosomal storage disorder with an estimated prevalence of 1 in 100,000 live births. This disease is characterized by early onset blindness, subsequent motor deficits, seizures and premature death. The most common disease causing mutation observed is a 1 kb genomic deletion, which spans exons 7-8, in the gene CLN3. The purpose of this study was to develop gene augmentation and genome editing based approaches for the treatment of CLN3-associated disease.
Methods: Skin biopsies were obtained from patients with CLN3-associated Batten disease and iPSCs generated from cultured keratinocytes via viral transduction of the transcription factors OCT4, SOX2, KLF4 and c-Myc. Lentiviral (LV) and adeno-associated viral (AAV) constructs carrying full-length CLN3 under the control of the cytomegalovirus promotor were cloned, packaged and transduced into iPSC-derived neurons. CLN3 expression was assessed via rt-PCR, Western blotting and immunofluorescence. CRISPR/Cas9 constructs harboring specific small guide RNAs targeting the 1 kb deletion in CLN3 were designed, cloned and tested. For correction of the CLN3 locus we also generated a donor construct carrying wild-type CLN3 spanning exons 7-8 to be co-delivered with CRISPR/Cas9 plasmids to induce homology directed repair.
Results: IPSCs from three patients with CLN3-associated Batten disease have been generated. LV-CLN3 and AAV-CLN3 viral vectors both restored full-length CLN3 transcript and protein expression in iPSC-derived neurons. CRISPR/Cas9 constructs targeting CLN3 induced specific modification of the CLN3 locus. Patient-specific iPSCs, when transduced with CRISPR constructs along with the wild-type CLN3 donor construct, expressed restored full-length CLN3 following homology directed repair.
Conclusions: We have demonstrated that viral-mediated delivery of CLN3 is sufficient to restore full-length CLN3 protein in iPSC-derived neurons from patients with Batten disease. Furthermore, we have genomically corrected diseased iPSCs using a CRISPR/Cas9-mediated homology directed repair strategy. The in vitro success of these treatment modalities pave the road for generation and testing of clinical-grade AAV-CLN3 and CRISPR-corrected iPSCs that will be produced in a clinical GMP facility.
Williams, Brittany 1,2, Wang, Shiyi 1, Hagen, Jussara 1, Scheetz, Todd 3, Haeseleer,Francoise 4, Lee, Amy 1
1Depts. of Molecular Physiology and Biophysics, Otolaryngology Head-Neck Surgery, and Neurology, University of Iowa 2Interdisciplinary Graduate Program in Neuroscience, University of Iowa 3Depts. of Ophthalmology and Visual Sciences and Biomedical Engineering, University of Iowa 4Dept. of Physiology and Biophysics, University of Washington
Purpose: In photoreceptors, presynaptic Cav1.4 (L-type) channels mediate Ca2+ influx that promotes neurotransmitter release. These channels are comprised of a pore-forming α1 and auxiliary β2 and α2δ4 subunits. The purpose of this study was to show that alternative splicing of both α1 and β2 could give rise to Cav1.4 channels with dramatically different properties.
Methods: We identified splice variants of β2 (+ exon 7b, β2X13) and α1 (- exon 47, Cav1.4Δex47) that are expressed in human retina, by RT-PCR. Electrophysiological recordings in transfected HEK 293T cells were used to characterize the biophysical properties of the identified variants.
Results: In electrophysiological recordings of transfected HEK 293T cells, β2X13-containing channels exhibit greater voltage-dependent inactivation than those containing β2a. Cav1.4Δex47 lacks 66 amino acids of a C-terminal modulatory domain (CTM) that regulates Ca2+-dependent inactivation and voltage-dependent activation. This variant was detected in retinal samples from humans but not mice, and is expressed at lower levels than channels containing exon 47 (Cav1.4+ex47). Compared to Cav1.4+ex47, Cav1.4Δex47 activates at more negative voltages (-20 ? 5, n= 6, for Cav1.4Δex47 vs -10 ? 3, n= 6, for Cav1.4+ex47, p<0.01) and displays stronger Ca2+-dependent inactivation. Ca2+ currents for Cav1.4Δex47 inactivated ~48% during a 1-s pulse compared to ~1% for Cav1.4+ex47.
Conclusions: We conclude that alternative splicing diversifies Cav1.4 signaling properties, which may be important for the role of this channel in the development and/or function of the photoreceptor synapse.
Yang, Huan; Mavlyutov, Timur; Guo, Lian-Wang
Department of Surgery, Wisconsin Institute for Medical Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA
Purpose: Mitophagy is emerging as a critical protective mechanism for neuronal health. The Sigma-1 receptor (Sig1R) is a unique ligand-operated chaperone recently recognized to be neuroprotective. Whether the Sig1R plays an important role in mitophagy is not known.
Methods: Retina explants and mouse embryonic fibroblasts (MEFs) were isolated from Sigma-1 knockout (SigKO) and wild type (WT) mice. SigKO cell lines were created using the CRISPR/Cas9 system with NSC34 (a neuronal cell line) and HEK293 cells. The markers for mitochondria (VDAC1 and Tim23) and autophagy (LC3II and SQSTM1/P62) were quantified through Western blotting. Caspase-3/7 activity assay was used to measure apoptosis.
Results: Mitophagy was induced by CCCP, as indicated by mitochondria clearance (decrease of VDAC1 and Tim23) and Parkin recruitment to mitochondria. Mitochondria clearance was substantially impaired in SigKO retina explants, NSC34 cells, HEK293 cells, as well as MEFs compared to their respective WT control. Impaired mitophagy was associated with exacerbated apoptosis. Whereas Parkin recruitment was not affected in the absence of the Sig1R, the turnover of LC3II and SQSTM1 was significantly impeded, as measured using bafilomycin to block lysosomal degradation of autophagic cargo. Expressing the Sig1R in SigKO MEFs rescued impaired mitochondria clearance.
Conclusions: Sig1R ablation impairs lysosomal clearance of dysfunctional mitochondria promoting apoptosis. Thus the Sig1R plays an important role in mitophagy in neuronal as well as non-neuronal cells.
Zarei, Kasra; Johnson, Chris; Christopher, Mark; Scheetz, Todd; Abramoff, Michael
Institute of Vision Research
Purpose: To develop and validate an iOS application that serves as an accessible flicker fusion test. Flicker tests (both critical and contrast modulation flicker fusion) have been used for a variety of purposes in research surrounding eye diseases and neurological disorders, such as detecting glaucomatous visual field loss.
Methods: A novel flicker fusion protocol was implemented as an iOS mobile-phone application, eyeFusion (which can be downloaded for free on the iOS app store), to quantify flicker fusion threshold. Throughout the testing protocol, the central region of the device's screen is divided between a static half (median grayscale level) and a flickering half, consisting of rectangular bars oscilating between two varying grayscale values, combined with a sinusoidal transform. A fixation target is displayed for one second before each test presentation, and each test presentation lasts for one second, during which the user has unlimited time to provide a forced-choice response by selecting the flickering half. eyeFusion tests four different temporal frequencies: 3, 10, 15, and 30 Hz (in order). For each temporal frequency, the contrast was adjusted using a standard two-up one-down test paradigm. The threshold was measured as the average frequency/contrast combinations for four response reversals. Participants were tested in a dimly lit room, with the device a constant one-third of a meter away from the eye. Different test paradigms and test presentation strategies were also developed as alternative versions of eyeFusion.
Results: eyeFusion was validated by measuring individual differences and determining test repeatability in voluntary participants (with IRB approval from the University of Iowa). Concurrent test validity was determined by comparing the results of eyeFusion to the MAIA Flicker test.
Conclusions: We have presented eyeFusion as a novel, valid, robust way of quantifying flicker fusion threshold in humans. As a freely accessible iOS application, this vision test can be distributed on iOS devices all around the world. Upon testing further cohorts of patients, the goal is to eventually improve screening of eye diseases and neurological disorders.
Wei Zhu1, Qiong J. Ding1, Budd A. Tucker1, Markus H. Kuehn1,2
1The University of Iowa Department of Ophthalmology and Visual Sciences and 2Center for the Prevention and Treatment of Visual Loss, Veterans Affairs Medical Center, Iowa City, IA.
Purpose: Dysfunction and loss of trabecular meshwork (TM) cells is associated with the pathology of primary open-angle glaucoma (POAG). The overall goal of this study is to determine if the TM in glaucomatous eyes can be functionally restored through stem cell transplantation.
Methods: Mouse iPSC were differentiated into a TM-like cell type (iPSC-TM) and cells with remaining SSEA-1 immunoreactivity were removed by magnetic separation. 50,000 iPSC-TM cells were injected into the anterior chamber of 4-mon-old Tg-MYOCY437H mice (N=13). Controls included age-matched wild type littermates (N=7), Tg-MYOCY437H receiving vehicle-only injections (N=9), or injections of fibroblasts (N=4). IOP and outflow facility were measured, and the distribution of transplanted cells was assessed using immunohistochemistry.
Results: Within three weeks after transplantation of iPSC-TM the IOP in Tg-MYOCY437H declined from 14.95 mmHg to 13.08 mmHg (p=0.032), similar to that observed in wild-type mice (12.67 mmHg). In contrast, IOP of Tg-MYOCY437H having received either PBS or fibroblasts did not decrease (IOP 15.25 mmHg and 15.48 mmHg, respectively). The outflow facility in iPSC-TM mice recipients was 0.034 µl/min/mmHg, significantly higher than that in the PBS group (0.019 µl/min/mmHg, p<0.05). Immunohistochemical analysis revealed transplanted iPSC-TM in the TM of recipient mice, although off-target implantation was also commonly observed.
Conclusions: Transplantation of mouse iPSC-TM cells enhances outflow of aqueous humor and decreases IOP in this glaucoma model. These findings are encouraging and may point toward new approaches of stem cell-based therapy.