2011 Midwest Eye Research Symposium
Sponsored by the The Institute for Vision Research at The University of Iowa
MERS 2011 took place on Friday July 29, 2011 at
|   Randy H. Kardon:||The light induced electromyogram (EMG): A reflex pathway integrating the melanopsin retinal ganglion cell, trigeminal sensory nucleus and facial nerve|
|   Rick R. Blodi:||Photoreceptors with no outer segments can support useful vision|
|   Simran K. Brar:||A disease mutation of Bestrophin causes altered chloride channel and may provide insight into macular degeneration|
|   Aparna Lakkaraju:||Function and regulation of HDL modulating proteins apolipoprotein E, hepatic lipase and lipoprotein lipase in the RPE|
Session II - Introduction by Session Chair Michael P. Fautsch, Ph.D.
|   Maura McGrail:||A novel eye tumor line in Zebrafish that models human retinoblastoma|
|   Sharolyn Kawakami-Schulz:||Phenotypic differences between Destrin mutants are due to allelic difference and modified by genetic background|
|   Jessica E. McDonald:||Effects of latanoprost on diurnal intraocular pressure and pupil diameter in normal and glaucomatous cats|
|   Gulab Zode:||A chemical chaperone rescues glaucoma in a mouse model of primary open angle glaucoma|
Session III - Introduction by Session Chair Michael G. Anderson, Ph.D.
|   Bliss O’Bryhim:||Endothelial progenitor cell recruitment in retinal revascularization|
|   Kerstin M. Janisch:||Over-expression of +TIPs in zebrafish cone photoreceptors|
|   Yuan Pan:||Characterization of HCN1 trafficking in rod photoreceptors|
|   Budd A. Tucker:||Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene MAK as a cause of retinitis pigmentosa|
|Dina Ahram:||Clinical and Genetic Investigation of Familial Angle-Closure Glaucoma in the Basset Hound|
|Hamisha Ardalani:||Quantifying Variation in Genomic Networks of Circadian Rhythm|
|Matti P. Asuma:||Oxytocin receptor signaling in retina pigment epithelial cells|
|Sajag Bhattarai:||Clefts between RPE and photoreceptor cell outer segments in the retinal degeneration 10 (rd10) mutant mouse|
|Tryphena L. Cuffy:||Examining the LYSTbg-J mutation to extend genetic pathways of exfoliation syndrome|
|Hesam Dashti:||Systematic Detection of Retinal Endothelial Gene Functions in Multiple Organs through Design of Functional Networks|
|Heather Dehlin:||Altered response to light in mice lacking the de-ubiquitinating enzyme USP2|
|Qiong Ding:||Lack of immunoglobulins does not prevent C1q binding to RGC and does not alter the progression of experimental glaucoma|
|Arlene V. Drack:||Light reduction,neuroprotection, and gene replacement therapy in mouse models of RP|
|James Driver:||Novel Automated Image Analysis Methods for Detection of Morphological traits in Retinal Vasculature in Health and Disease|
|Joe Fogerty:||Biochemical Purification of MFRP and Evidence of Geographic Atrophy in MFRP-/- Mice|
|Jilian Goetz:||Elucidating retinal ganglion cell diversity using single-cell transcriptomics|
|Dan Gratie:||Morphological correlation between optical coherence tomography (OCT) and histology in mouse retina|
|Ralph J. Hazlewood:||Identification and Characterization of Genetic Factors Responsible for Cavitary Optic Disk Anomalies|
|Adam Hedberg-Buenz:||Genetic pathways contributing to the iris transillumination defects of exfoliation syndrome|
|Sarah Hengel:||Characterization of Synaptophysin’s Assembly and Subcellular Localization|
|Elena Hernandez-Merino:||Analysis Of Retinal Nerve Fiber Layer Thickness And Retinal Ganglion Cell Function During Acute Elevation Of Intraocular Pressure And Its Relationship With Ocular Perfusion Pressure In The Canine Eye|
|Helga Kecova:||Effect of Cholinergic Drugs on Optic Nerve Structure and Function in Glaucoma|
|Elyse Kleifgen:||Evaluation of optic nerve head morphology by SD-OCT in normal and glaucomatous cats|
|Demelza Koehn:||Mapping the Gene Underlying Central Corneal Thickness QTL 1|
|Hasti Mirkia:||Contributions to Perceptual Geometry of Space: A Learning Theoretic Approach|
|Kabhilan Mohan:||Functional Retinal Ganglion Cell PERG Deficits in an Acute and Chronic Mouse Model of Parkinson’s Disease|
|Michael A. Moriarty:||Lipoprotein Interactions in the Retinal Pigment Epithelium and Choroidal Tissue|
|Pavitra Narasimha:||RNAi therapy for Spinocerebellar ataxia 7 (SCA7)|
|Arash Sangari:||Biological Complexity in Systems Biology of Circadian Clocks|
|Leandro Teixeira:||Ultrastructural Abnormalities of the Trabecular Meshwork Associated with Cyp1b1-Deficiency|
|Katie Weihbrecht:||Identification of the interacting partners of ciliopathy-related retinal degeneration genes LCA5, SDCCAG8, and INPP5E|
|Scott Whitmore:||ARMS2 associated gene expression differences in macular RPE and choroid|
Dina Ahram1,2, Sinisa D. Grozdanic3, Helga Kecova3, Markus H. Kuehn1,2,3
1Department of Ophthalmology and Visual Sciences, 2Interdisciplinary Graduate Program in Genetics; The University of Iowa, Iowa City, Iowa. 3Department of Veterans Affairs – Center for Prevention and Treatment of Vision Loss Iowa City, Iowa
Purpose: Primary angle closure glaucoma (PACG) is an optic neuropathy marked by progressive retinal ganglion cell degeneration, cupping of the optic nerve head and subsequent loss of vision in association with a gradual increase in intraocular pressure (IOP). The condition is most commonly caused as a result of the collapse of the irido-corneal angle due to the movement of the root of the iris anteriorly towards the cornea. Several breeds of dogs develop the condition and the prognosis for affected animals is typically poor. We have identified a number of Basset Hound pedigrees with characteristic autosomal recessive PACG that in many aspects recapitulates the clinical PACG phenotype observed in human patients.
Methods: Clinical examination and tests conducted on all participating animals included IOP measurement with tonometry, retinal examination using retinoscopy and electroretinography as well as examination of anterior segment structures using gonioscopy and high frequency ultrasonography. Immunohistochemical and histological analyses were performed on affecteds and unaffecteds derived ocular tissues. Sequencing of genes implicated in other forms of glaucoma, including Cyp1b1, Myoc, Pitx2 and Adamts10 was performed in affecteds and unaffecteds. Gene expression analysis on anterior segments derived from affecteds and unaffecteds was performed using the software package Partek. SNP chip derived data was also used to conduct loss of hetergozygosity analysis using the statistical packages Plink.
Results: Progressive increase in IOP that coincided with complete angle and ciliary cleft collapse was observed in all affecteds by 20 months of age. Dramatic retinal ganglion cell dysfunction and deteriorating pERG amplitudes were noted in affecteds vs. unaffecteds. Histological analysis confirmed ciliary cleft collapse, optic nerve head cupping and retinal ganglion cell degeneration in affecteds vs. unaffecteds. Sequencing of genes previously implicated in glaucoma identified no significant variants. Gene expression and loss of heterozygosity analyses revealed a number of potentially significant genes and loci of homozygosity shared among all affecteds animals that require further analysis to be confirmed.
Conclusions: The ultimate goal of this research is to utilize the Basset Hound PACG model in order to identify the disease causing mutation. We are uniquely positioned to achieve our goal through a combination of high density mapping of genetic markers, as well as analysis of multiple quantitative traits, copy number variations and gene expression changes in PACG Basset Hound pedigrees, which may enable us to identify the gene or gene network implicated in PACG. Ultimately, we anticipate for these studies to provide valuable insight into the pathophysiology and genetics of human PACG.
Arash Sangari2, Hamisha Ardalani4 and Amir Assadi1,3
1 Department of Mathematics, 2Department of Electrical and Computer Engineering, 3Comparative Biological and Medical Sciences, 4Department of Biomedical Engineering, University of Wisconsin-Madison.
Purpose: The 24-hour periodic rotation of the earth about its axis has a profound impact on a living organism’s metabolism, physiology, and behavior. While the diurnal rhythms of these biological oscillations are readily observed, it is less obvious that measurement of time is an innate ability of most organisms. For instance, most organisms anticipate the sunrise and adjust their biology accordingly; and even when they are deprived of exogenous time cues, the diurnal rhythms persist.
Methods: We recognize circadian rhythms by several attributes besides their periodic dynamics, such as being endogenously generated and self-sustaining; thus, they retain their rhythmic character under constant environmental conditions, such as constant light (or dark) and constant temperature. Moreover, circadian rhythms tend to exhibit temperature compensation, so that over a range of ambient temperatures, the period remains relatively constant.
Results: The results for two model organisms, the plant Arabidopsis and the animal Mus musculus, compare and contrast the dynamic patterns in homologous genes in terms of their mRNA expression and the architecture of their gene regulatory networks. Tables ofsimilarities and notable contrasts are provided, and visualization of their dynamics will be demonstrated based on our new algorithms.
Conclusions: In this project, we compare the genomic underpinnings of circadian rhythms under perturbations of light and temperature in two model organisms to quantify variation in the gene regulatory networks and mRNA expressions.
M. P. Asuma1, W. Luo1,D. M. Pillers1, 2, B. R. Pattnaik1, 2, 3
Departments of 1Pediatrics, 2Eye Research Institute, 3Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI.
Purpose: Retinal Pigment Epithelial (RPE) cells are localized between choroidal vasculature and light sensitive neural retina. These RPE cells develop early during retinogenesis, secrete vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF) to regulate retinal vasculature and provide continued nourishment for retinal neurons for perceiving vision. Polarized distribution and signaling through many ion-channels and second messengers regulate most of the RPE cell function. Oxytocin, an uterotonic agent used to induce labor, binds to cell-surface receptors to activate complex intracellular signaling pathway. Since oxytocin is localized in posterior retina and the level of oxytocin in the retina is controlled through circadian and light cycle, we sought to study the oxytocin signaling mechanism in human RPE cells.
Methods: We cultured fetal human RPE cells using optimized growth medium. Single cell RT-PCR was performed by harvesting cellular content after each electrophysiology experiment. Transcripts for Oxytocin receptor (OTR) and RPE specific markers were amplified. Oxytocin mediated mobilization of intracellular Ca2+ was carried out using standard ratio metric Fura-2 measurements.
Results: RPE cells in culture showed Kir7.1 current and the presence of Kir7.1 transcript. Results of single cell RT-PCR amplification showed presence of an appropriate size band for OTR. Further confirmation of RPE cells were established by amplification of RPE65, bestrophin, Na-K-ATPase and ezrin. Cells treated with oxytocin through perfusion solution resulted in an increase in intracellular Ca2+.
Conclusions: We have showed that oxytocin receptors are present in RPE cells and activation of these receptors result in an increase of intracellular Ca2+. Many of the RPE cell functions like fluid transport are directly modulated by changes in intracellular Ca2+. It is perceived that RPE cell might use this signaling mechanism as a paracrine signaling pathway to respond to light and circadian changes which are yet to be identified.
Sajag Bhattarai1, Dan Gratie1, Alina Dumitrescu1, Michael G. Anderson1,2, Demelza Koehn2, Edwin M. Stone1, Robert F. Mullins1, Arlene V. Drack1
1Department of Ophthalmology and Visual Sciences, 2Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA.
Purpose: Rd10 mice represent a model of human retinitis pigmentosa (RP) due to mutation in the β-subunit of rod cGMP-phosphodiesterase (Pde6β). Outer nuclear layer (ONL) degeneration starts at P16 and by P40 to P50, no ONL remains. Numerous treatments have been tested using this mouse model. Use of the in vivo imaging technique optical coherence tomography (OCT) recently led to the observation by us and others that a “cleft” beneath the retina can be seen on OCT around P30. Our purpose is to further characterize these clefts.
Methods:Electroretinograms (ERG), OCT, and histology were performed to evaluate the morphology and functional effect of these clefts. ERG (Diagnosys) was performed using gold ring electrodes. Retinal OCT (Bioptigen) using rectangular volume scan was performed. Fixed retinal sections were processed for histochemistry using H and E stain.
Results: Rd10 mice from P30 to P120 included 5 reared in 580-740 nm light, 7 reared in darkness, 6 reared in 12 hour light/dark cycles, and 6 treated with Tauroursodeoxycholic Acid (TUDCA). Rd10 mice developed a cleft between the RPE and photoreceptor layer around P30. All of the rd10 mice (n= 24) regardless of their treatment status demonstrated clefts. Other strains (rd12, Cln3WT, rd16,C57BL/6J-Tyrc-2J, Bbs1M390R/M390R, n=21) with and without RP, older than P60 were investigated and none had clefts. Histology was evaluated. ERG demonstrated a difference between treated and untreated rd10 mice with decline in ERG related to photoreceptor loss, not the cleft. Photographs of retinas with clefts on OCT showed no retinal detachment or anomalies.
Conclusions: Rd10 mice develop a subretinal cleft around P30. This is a new phenotypic feature that was not previously described and was not noticeable until the advent of OCT. As all rd10 mice develop the cleft, it is uncertain whether this affects the ERG. It appears more similar to retinoschisis than to retinal detachment, and may be magnified by the enhanced Y axis of the OCT.
Rick R. Blodi1AB, Austin Barclay1AB, Megan Riker1AB, Robert F. Mullins1AB, Steven F. Stasheff1BC, Arlene Drack1AB, Markus H. Kuehn1AB, Alisdair R. Philp2, Val C. Sheffield1BCD, Edwin M. Stone1ABD, and Stewart Thompson1AB
1ADepartment of Ophthalmology and Visual Sciences, 1B The Institute for Vision Research, 1C Department of Pediatrics, 1D The Howard Hughes Medical Institute, University of Iowa, Iowa City, IA; 2 Department of Biochemistry and Nutrition, Des Moines University, Des Moines, IA.
Purpose: Stem cells introduced to the sub-retinal space do not assume a photoreceptor-like structure unless a relatively intact outer-retinal architecture is present. This seriously limits the utility of stem cells in treatment of photoreceptor degenerations, where treatment will realistically only be given when the outer retina is severely degenerate. The purpose of this study was to test whether photoreceptors with no outer segments were capable of supporting useful vision.
Methods: Mice tested had photoreceptors with no outer segments (P90 Rd2/Rd2), no photoreceptors (P360 Rd2/Rd2), or a normal retina (wild-type). Vision-augmented-locomotion was measured as increased running wheel activity in dim light when compared to activity in complete darkness. Acuity and contrast sensitivity were tested using optokinetic responses. Light evoked retinal responses were tested by electroretinography. Photoreceptor state was checked using histology.
Results: P90 Rd2/Rd2 mice had vision-augmented-locomotion, but only at luminance levels higher than those supporting vision in wild-type mice (lowest luminance for P>0.01 significant response: WT 0.01 cdm-2; P90 Rd2/Rd2 1.0 cdm-2). P90 Rd2/Rd2 also had a measurable but reduced acuity (WT 0.407 c/d; P90 Rd2/Rd2 0.269 c/d) and contrast sensitivity (WT 35.4%; P90 Rd2/Rd2 61.2%). A basis for visual function in P90 Rd2/Rd2 mice was demonstrated by a severely reduced but measurable electroretinogram (b-wave amplitude: WT 755 μV; P90 Rd2/Rd2 125 μV). No visual or electroretinogram responses were measurable in P360 Rd2/Rd2 mice.
Conclusions: Photoreceptors with no outer segments can support measurable retinal function and useful vision. Absence of outer segments did result in reduced acuity, contrast sensitivity and loss of vision in very dim luminances. This means that, despite reduced function, stem cell introduction of photoreceptor precursors may be able to restore useful vision even where photoreceptor morphology and organization is not fully restored.
S K Brar1, P J Halbach1, S A Tokarz1, B R Pattnaik2, D M Pillers3
1Pediatrics; 2Pediatrics, Ophthal & Visual Sci, Eye Research Institute; 3Pediatrics, Eye Research Institute; University of Wisconsin, Madison, WI
Purpose: Macular degeneration impairs the vision of countless adults and is commonly understood to be a consequence of aging. In reality, macular degeneration, specifically Best Vitelliform Macular Dystrophy (BVMD), is also a serious pediatric concern. BVMD is caused by dominantly-inherited mutations of bestrophin, a protein localized on the basolateral membrane of retinal pigment epithelial (RPE) cells. In functional RPE cells, bestrophin complexes form the Ca2+-activated Cl- channels responsible for the flux of Cl- anions across the membrane. Since Cl- regulates essential cellular events, mutations in bestrophin may be detrimental to RPE cell function. Using a simplified cell model, we sought to determine the influence of one BVMD-associated bestrophin mutation on cell function. We hypothesized that altered Cl- transport, resulting from this bestrophin mutation, will cause cellular dysfunction similar to that seen in BVMD patients.
Methods: Using site-directed mutagenesis, we introduced the N296H genetic mutation associated with BVMD into a vector containing PIRES, GFP, and human bestrophin 1 (hBest1). The vector was sequenced to verify the mutation was present. The hBest1 wild-type and hBest1 N296H mutant were introduced to cultured Chinese Hamster Ovary (CHO) cells via electroporation. The effectiveness of the electroporation was evaluated using live-cell fluorescence imaging. Ionic flux due to the two clones was measured as current-amplitude using the whole-cell mode of patch-clamp electrophysiology.
Results: GFP-positive cells confirmed the ectopic expression of hBest1and hBest1 N296H in CHO cells. Under conditions of high intracellular Ca2+, recorded Cl- current due to hBest1 exhibited both inward and outward current. The corresponding current-amplitude plot was linear with resting membrane potential close to 0mV. Substitution of bath Cl- with other anions matched previously published characteristic bestrophin Cl- channel current. In comparison, mutant hBest1 N296H transfected cells had significantly altered Cl- current magnitude and preference for other anions.
Conclusions: Our results suggest that hBest1 is part of a complex Ca2+-regulated Cl- channel. We expect the current magnitude and anion preference of BVMD RPE cells to be similar to that of the hBest1 N296H mutant CHO cells, suggesting that this is an underlying etiology for BVMD.
Tryphena L. Cuffy1, Colleen M. Trantow2, Michael G. Anderson1,2,3
1Interdisciplinary Graduate Program in Genetics, 2Molecular Physiology and Biophysics, 3Ophthalmology and Visual Sciences, 1University of Iowa, Iowa City, IA.
Purpose: Exfoliation syndrome (XFS) is a common age-related disorder characterized by the pathological accumulations of fibrillar exfoliative material in the anterior chamber of the eye. Patients with XFS can go on to develop exfoliative glaucoma; potentially as a result of an accumulation of exfoliative material at the drainage structures of the eye. Human eyes with XFS exhibit a striking pattern of Marcel-like iris transillumination defects. The same pattern is recapitulated in mice containing the Lystbg-J mutation. In yeast two-hybrid assays others have identified a small number of proteins capable of interacting with the LYST WD40 motif – one of these being CSNK2B.
Methods: We have found that the molecular basis of the bg-J mutation is a three base pair deletion in the WD40 encoding region of the Lyst gene. Testing CSNK2B, as a candidate interacting protein, previous GST-pulldown experiments confirmed that wild-type LYST can bind to CSNK2B whereas LYSTbg-J cannot. In addition, microarray analysis of mice irides was performed to identify possible candidate genes involved in XFS.
Results: Results from these experiments suggest that LYST may play a role in regulating activity or localization of CSNK2B. In testing this hypothesis, experiments with primary fibroblasts expressing a GFP-tagged CSNK2B fusion protein suggest that LYST regulates the subcellular localization of CSNK2B. Furthermore, two CSNK2B substrates identified from the microarray analysis, (PER2 and CDH1), exhibit functional deficits in the presence of the Lystbg-J mutation.
Conclusions: Combined, these results implicate CSNK2B and its substrates in the pathogenesis of XFS. It also identifies candidate genes that may also play a role in the genetic pathway of XFS.
Hesam Dashti1, James Driver1, Nasim Jamali2, Nader Sheibani2, Amir Assadi1,3
1Departments of Mathematics, 2Ophthalmology and Visual Sciences, 3Comparative Biological and Medical Sciences, University of Wisconsin-Madison.
Purpose: Determining organ-specific genes, the effects of genes on other genes, and their modes of action, forms an important class of problems in understanding the pathogenesis of diseases in various organs and designing target specific therapies. An important consideration of these side effects is the alteration of genes’ functionalities. Changing gene functionalities under genotypic stimuli emphasizes the critical need to identify organ-specific genes. Despite many unknown gene functionalities, genes with a fundamental role in angiogenesis have been well studied. Identifying phenotypes associated with mutant-gene expression patterns related to vascular endothelial growth factor (VEGF) attracted much attention over the past few decades. CD31/PECAM-1 is a member of the immunoglobulin (Ig) gene super family that promotes endothelial cell migration and has been implicated in angiogenesis and inflammation in vivo.
Methods: To identify organ-specific genes, we examined gene expression profiles of endothelial cells prepared from retina, kidney, and lung of wild type and PECAM-1-deficient mice. Using DNA microarray analysis, we identified retinal organ-specific genes and those specifically impacted by lack of PECAM-1. Next, we designed and implemented an approach to confirm and predict the functionalities of genes of both known and unknown functionality. The functionalities of known genes are provided by The Database for Annotation, Visualization and Integrated Discovery (DAVID).
Results: As a result, we identified 2,000 genes whose functionalities are related to angiogenesis and whose expression was impacted in an organ specific manner.
Conclusions: We are determining how these organ specific gene changes are influenced by the status of PECAM-1 in an organ specific manner.
Heather Dehlin, Alison Lewis, Joseph C. Besharse
Medical College of Wisconsin, Dept of Cell Biology, Neurobiogy, and Anatomy (CBNA), Milwaukee WI
Purpose: To ask whether the circadian de-ubiquitinating enzyme, USP2, is required for normal circadian behavior. USP2 is a clock-controlled DUB highly expressed in brain and retina. We are interested in understanding whether USP2 is a component of the molecular clock, a clock output gene, or has a role in setting the clock.
Methods: We have generated mice lacking USP2 and characterized their behavior under various lighting paradigms. USP2-/- and wild type littermates were housed under constant light (LL), LL at increasing irradiances, and were exposed to extended light periods (ZT12-ZT16) at increasing irradiances and allowed to free-run in constant dark (DD). To ask whether loss of USP2 effects core clock expression in retina, mice lacking USP2 were crossed to PER2::LUC mice and PER2 expression was measured in retina by luminescence detection.
Results: USP2-/- housed in low LL conditions acutely delay their circadian phase, whereas USP2-/- mice housed in LL remain significantly more rhythmic when compared to wild type littermates. Additionally, USP2-/- mice phase delay significantly more than USP2+/+ when exposed to low irradiance light at the day-to-night transition. Periods are longer in USP2-/-/PER2::LUC retina compared to USP2+/+/PER2::LUC retina. However, we observed more variability and arrythmicity in PER2::LUC expression in USP2 knockouts consistent with the transcript expression of mPer2 in USP2-/- retina.
Conclusions: USP2 is required for the molecular clock to reset appropriately in response to changing light schedules indicating that USP2 may function as a clock input signal. Additionally, periods measured in USP2 knockout retina are altered suggesting a potential role for USP2 in normal clock function. USP2 could have multiple targets resulting in a number of roles relating to the clock.
Qiong Ding1, Alina V. Dumitrescu1, Amy C. Cook1, Susan M. Bushek1, Markus H. Kuehn1
Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, IA
Purpose: We previously reported deposition of complement components 1q (C1q) and 3 (C3) in association with retinal ganglion cells (RGC) in the glaucomatous retina. C1q is not known to bind directly to cells, relying instead on adapter molecules. Traditionally these were assumed to be immunoglobulins (Ig) but it has now become clear that a number of molecules can fulfill this role. The purpose of this study was to evaluate whether C1q binding and activation of the complement cascade in the glaucomatous retina is dependent upon the presence of Ig.
Methods: Experimental glaucoma was induced in C57BL/6J and B6.129S7-Rag1tm1Mom/J (RAG1-/-) mice using the microbead occlusion model (N=20/group). Damage to the optic nerve as well as the fraction of surviving RGC were determined using an established grading scheme and through counting of gamma synuclein positive cells in whole mounted retinas, respectively. Binding of C1q and C3 to the RGC was visualized using immunohistochemistry of retinal cryosections. Complement activation was also evaluated in vitro using dissociated retinal cell cultures.
Results: Injection of microbeads into the anterior chamber of mice induces mild IOP elevation in the majority of treated eyes (average = 4.9 mmHg above baseline). Elevated intraocular pressure was maintained for up to 4 weeks. This experimental glaucoma results in RGC loss, progressive optic nerve damage, and concomitant C1q binding to RGC in both normal and RAG1 -/- mice. The absence of Ig does not affect the rate of axonal damage or RGC loss (p=0.39 and 0.12 after 2 and 4 weeks, respectively). C1q and C3 immunoreactivity associated with RGC was observed both in normal and RAG1-/- mice. RGC in retinal cultures maintained in serum-free media are also C1q immunoreactive, demonstrating that Ig is not required for C1q binding to damaged RGC.
Conclusions: Our data demonstrate that lack of immunoglobulins and mature T/B cells does not influence the progression of glaucoma. Furthermore, immunoglobulins do not appear to be required for C1q binding and complement cascade activation on damaged RGC. These findings suggest that C1q recognizes an alternative binding partner expressed by stressed RGC.
Arlene V. Drack1, Alina Dumitrescu1, Sajag Bhattarai1, Dan Gratie1, Seongjin Seo1, Stewart Thompson1, Megan Riker1, Darryl Nishimura2, Edwin M. Stone1,3, Val C. Sheffield2,3, Robert F. Mullins1
Departments of 1Ophthalmology and Visual Sciences, and 2Pediatrics, University of Iowa, Iowa City, IA 3Howard Hughes Medical Institute
Purpose: To evaluate diverse treatment modalities in mouse models of cilia-related retinal degeneration.
Methods: Mouse models of Bardet Biedl syndrome (BBS) types 1, 2, 3, 4 and 6 were generated using homologous recombination. Rd10 mice were used as a control non-cilia related retinopathy. Pupillometry, electroretinography, indirect ophthalmoscopy, fundus photography, and histology were performed to evaluate whether treatment modalities altered the course of the retinopathy. Treatment modalities included dark rearing, delivery of neuroprotective molecules, and subretinal injection of AAV-WTBbs1.
Results: Mice homozygous for the most common human BBS mutation, M390R, demonstrated improved pupillary responses after being reared in the dark (F-test comparison of curves P < 0.0001). Bbs1 mice reared in the dark have some preservation of ERG amplitudes at 6 mos. Bbs1 M390R homozygous mice (N=14) that received unilateral subretinal injection of AAV-WTBbs1 demonstrated expression of Bbs1 protein in photoreceptors. After initial small decrease in ERG amplitudes, ERG appeared to stabilize in treated eyes relative to control, uninjected eyes. Ongoing studies include evaluation of treatment with the neuroprotective agent, TUDCA. Rd10 mice demonstrate less attenuation of ERG amplitudes when systemic TUDCA is administered every 3 days. This protocol is currently being applied to Bbs1 M390R/M390R mice.
Conclusions: Ciliopathies such as BBS cause severe early onset retinal degeneration in humans. In a mouse model of BBS, light reduction and subretinal gene therapy show potential as treatment modalities, however longer follow up is required in this slowly evolving model. Neuroprotective molecules may also have a role.
Hesam Dashti1, James Driver1, Nader Sheibani2, Amir Assadi1,3
1Departments of Mathematics, 2Ophthalmology and Visual Sciences, 3Comparative Biological and Medical Sciences, University of Wisconsin-Madison
Purpose: Analyzing and quantifying phenotypic traits forms an essential step in biomedical research. A great deal of research is currently devoted to genotype-phenotype mapping. In this vein, recognizing biologically significant features in medical images, and their accurate assessment, remains a challenging problem between the interface of genomic and biomedical sciences and its clinical applications. Research on phenotyping diseases of the eye using fundus images presents highly complex data with rewarding outcomes. Fundus images provide the ability to noninvasively study in vivo vasculature integrity, function, and a gateway to the central nervous system via the optic disc.
Methods: We focused on quantifying phenotypic features of retinal fundus images by extracting vascular structures and identifying the optic disc. To this end, we applied a combination of advanced analytical methods, including deterministic and heuristic algorithms based on partial differential equations to identify, extract, and study phenotypic features.
Results: We report on the relevance of these algorithms towards quantifying complex phenotypic traits. The results of the analysis of data sets of retina images from the STARE and DRIVE public databases, and those obtained in our laboratory, are presented.
Conclusions: These algorithms are useful as initial steps in phenotyping retinal blood vessel abnormalities associated with a disease state and distinguishing pathological morphologies from normal patterns of vasculature as they develop in many eye diseases such as age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity.
Joseph Fogerty and Joseph C. Besharse
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin
Purpose: MFRP is a type II transmembrane protein that is expressed specifically in the RPE and ciliary body epithelium. In humans, mutations in MFRP cause nanophthalmos, foveoschisis, and retinitis pigmentosa, as well as choroidal transmission hyperflourescence. We have identified a novel mouse mutation, Mfrp174delG, which results in retinal degeneration. The purpose of the current study is to describe the mutant pathology and to discover novel properties of MFRP by biochemical purification.
Methods: Standard histology and electron microscopy were used to examine eyes of mutant and control mice. MDCK cells that stably express epitope-tagged full-length MFRP or only its cytosolic domain were generated, and candidate interactions were identified by tandem affinity purification and mass spectrometry.
Results: Histological examination of Mfrp174delG eyes showed progressive retinal degeneration and RPE atrophy, as well as upregulated RPE microvilli. Biochemical purification of tagged full-length, but not cytosolic MFRP yielded a list of 21 candidate interacting proteins, with roles including actin dynamics, mTor and MAPK signaling, apoptosis, and cell adhesion.
Conclusions: MFRP is required for RPE maintenance, and photoreceptor loss in mice lacking MFRP is likely non-autonomous. Candidate interacting proteins suggest potential roles for MFRP that are critical for proper cell function. Furthermore, we believe that Mfrp174delG animals may prove useful in the study of atrophic macular degeneration, an RPE degenerative disease that is a significant cause of human blindness and which has no effective treatments or animal models.
Jillian Goetz, Gregory M. Martin, Laura R. Ellson, and Jeffrey M. Trimarchi
Department of Genetics, Cellular, and Developmental Biology, Iowa State University, Ames, IA
Purpose: An understanding of the gene networks responsible for the production of specific cell-types is essential to generate a large number of any distinct retinal cell-type from stem cell populations. The lab aims to understand the gene expression programs responsible for generating neuronal diversity in the developing retina, specifically among the retinal ganglion cells. I am using the information gleaned from these experiments to attempt to enhance the efficiency with which ganglion cells are generated from retinal progenitor cells and induced stem cell (iPSC) populations.
Methods: Single cell transcriptomics in conjunction with overexpression experiments are being utilized to examine the gene networks that produce distinct retinal ganglion cells. Single cells were isolated from dissociated adult retinas and retinas from an early developmental stage (E14.5). The cDNAs resulting from these individual retinal cells were hybridized to mouse Affymetrix arrays.
Results: Using the Parv-Cre X TdTomato reporter mouse, individual fluorescent ganglion cells have been isolated and profiled. We are currently identifying gene clusters that correlate with different ganglion cell subsets. We are validating these array results by in situ hybridization. In addition, we have been overexpressing specific transcription factors found in our analysis of E14.5 developing ganglion cells and assessing the subsequent cell fate and gene network changes.
Conclusions: Single-cell transcript profiling has begun to reveal the signatures of distinct ganglion cells. Heterogeneously expressed genes include transcription factors, signaling molecules and ion channel genes. Our initial overexpression results indicate several of the identified transcription factors play important roles in controlling cell fate.
Dan Gratie1, Sajag Bhattarai1, Alina Dumitrescu1, Demelza Koehn, Rob Mullins1, Ed Stone1,4, Val Sheffield2,4, Michael Abramoff1, Arlene Drack1
Departments of 1Ophthalmology and Visual Sciences, and 2Pediatrics, 3Physiology, University of Iowa, Iowa City, IA 4Howard Hughes Medical Institute
Purpose: Optical coherence tomography (OCT) is a technique for obtaining sub-surface images of translucent or opaque materials at a resolution equivalent to a low-power microscope. It is effectively ‘optical ultrasound’, imaging reflections from within tissue to provide cross-sectional images. The purpose of this poster is to characterize the correlation between OCT and histology for mice models of human disease commonly used in the research laboratory.
Methods: Laboratory beagles (n=20) with experimentally induced laser Mice of different ages, from P60 to 6 months, were used. Bioptigen OCT and histology using H and E stain were performed to evaluate and compare the morphology.
Results: Two albino mice without retinal degeneration (aged 180 days), and two albino mice (aged 168 days) with PDE6beta mutations and one Bbs1 mouse aged 4 months were studied. Visualization of the retinal layers with OCT was excellent, and correlation between OCT layers and histologic layers of the retina was performed. The image quality of OCT in an albino animal was compared with that of a pigmented mouse of similar age.
Conclusions: : OCT is a useful method for in vivo evaluation of retinal morphology. It is reproducible and feasible. Correlation between OCT and histology is required. OCT may allow us to follow treated and control mice for long time periods without the need to sacrifice animals at numerous time points. Pigmentation does play some role in the quality of the image.
R.J. Hazlewood1, B.R. Roos1, R.A.Honkanen2, L.M. Jampol3, W.L. Alward1, J.H. Fingert1
Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA; 2Department of Ophthalmology, State University of New York at Stony Brook, Stony Brook, NY; and 3Department of Ophthalmology, Feinberg School of Medicine, Northwestern University, Chicago, IL
Purpose:Glaucoma is the second leading cause of blindness in America. The chief feature is progressive degeneration of the optic nerve seen as an excavation of the optic nerve head. C avitary optic disc anomalies (CODA) is a rare congenital disease also characterized by excavation of the optic nerve head. Consequently, we have studied CODA as a model of the optic nerve disease in glaucoma. We are searching for the gene that causes autosomal dominant inheritance of CODA in a large family using positional cloning.
Methods:Prior linkage studies mapped the CODA genes to a 13.5 Mbp segment of chromosome 12q14. We have examined the linked region for the gene that causes CODA using DNA sequencing and comparative genome hybridization (CGH).
Results:Study of affected family members with CODA using CGH identified a copy number variation (CNV) within the previously linked locus. All family members with CODA had two extra copies of a 6kb segment of DNA upstream of the matrix metallopeptidase 19 (MMP19) gene. Using a quantitative PCR assay, this CNV was not detected in unaffected family members or in normal controls.
Conclusions:We report a CNV upstream of MMP19 in the previously linked region that is co-inherited with CODA in our family. We hypothesize that this CNV leads to dysregulation of the expression of the MMP19 gene and ultimately to the development of CODA. Future studies to characterize this CNV include FISH, southern blotting, experiments with reporter gene constructs to examine MMP19 expression, and the creation of transgenic animals.
Adam Hedberg-Buenz1, Colleen McDowell1, and Michael G. Anderson1,2
Departments of 1Physiology and Biophysics and 2Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA.
Purpose:Exfoliation syndrome (XFS) is the most commonly identified cause of secondary open-angle glaucoma. C57BL/6J mice with the Lystbg-J mutation recapitulate aspects of XFS, including a unique pattern of iris transillumination defects, referred to as “Marcel-TID.” Studies of Marcel-TID offer an opportunity to examine molecular pathways contributing to XFS. The purpose of these experiments was to identify and test the role of additional genes contributing to XFS by performing phenotype- and candidate-driven screens for genes influencing Marcel-TID in mice.
Methods: Marcel-TID were assayed by slit lamp photography. Defect severities were quantified and averaged among mice of the same genotype. In a phenotype-driven approach, two inbred strains of mice carrying the Lystbg-J mutation were utilized in mapping crosses to screen for genetic modifiers. In a candidate-driven approach, gene expression profiling was performed to identify contributing pathways.
Results: From the phenotype-driven approach, Lystbg-J iris phenotypes are more severe on the DBA/2J vs. C57BL/6J genetic background. Genetic crosses mapped this modifier to the Tyrp1b mutation present in DBA/2J mice. Subsequent studies illustrated that Tyrp1b mutation may influence Marcel-TID by increasing levels of lipid hydroperoxides in the iris. From the candidate-driven approach, microarray analysis identified 460 statistically significant changes in gene expression between Lystbg-J mutant and control mice.
Conclusions: These results identify Tyrp1 as a modifier of Lystbg-J iris phenotypes and suggest oxidative damage to lipid membranes contributes to XFS pathophysiology. A large number of expression changes were detected in comparative transcriptional profiling; ongoing experiments continue to test the extent these candidates, and others, may impact XFS phenotypes.
Sarah R. Hengel and Sheila Baker
Department of Biochemistry, University of Iowa, Iowa City, IA
Purpose: Synaptic vesicles mediate communication between neurons. Within synaptic vesicles, one of the most abundant proteins is synaptophysin (SYP). SYP has been proposed to participate in the formation of synaptic vesicles, regulation of endocytosis and regulation of fusion. In humans, SYP is an essential protein as mutations have been implicated in X-linked mental retardation. The mechanism by which it is trafficked to synaptic vesicles is unknown. An assay using GFP reporter constructs expressed in transgenic frog photoreceptors has revealed that SYP contains a signal directing its localization to the synapse. It is known that targeting signals often function by directly interacting with a component of the cell’s trafficking machinery, such as a regulatory or molecular motor protein. Therefore, to understand the mechanism by which SYP’s targeting occurs, in one aim we will first identify and characterize the proteins that interact with its targeting sequence. Our second aim is to determine if conserved cysteine residues form inter and/or intra-molecular disulfide bridges that are important for SYP’s oligomerization state and targeting.
Methods:The first aim will be tested using a tandem affinity purification assay to pull out interacting prey proteins from retinal lysates. The second aim will be tested by expressing SYP and a SYP mutant in neurons in order to characterize their subcellular localization as well as analytical ultracentrifugation sedimentation velocity experiments to determine their oligomerization state. The SYP mutant contains its four conserved intravesicular cysteine residues mutated to serine in order to inhibit disulfide bridging.
Results: I have successfully cloned a TAP tag and am currently optimizing protein expression. I have successfully cloned SYP-RFP and SYP-RFP mutant cDNA for characterization of subcellular localization in neurons.
Conclusions: These results are expected to reveal how SYP is trafficked to the synapse.
Elena Hernandez-Merino1,2, Helga Kecova1,2, Samantha J. Jacobson1,2, Matthew M. Harper1,2, Tatjana Lazic1,2, Randy H. Kardon2,3, Sinisa D. Grozdanic1,2
1 Veterinary Clinical Sciences, Iowa State University, Ames, IA. 2 Veterans Administration Center for Prevention and Treatment of Vision Loss, Iowa City, IA. 3 Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA
Purpose: Determine the effect of acute episodes of elevation of intraocular pressure (AEIOP) on retinal nerve fiber layer (NFL) thickness and retinal ganglion cell (RGC) function and its relationship with ocular perfusion pressure (OPP) using Spectral-Domain Optical Coherence Tomography (SD-OCT) and pattern electroretinography (pERG) in healthy canine eyes.
Methods: AEIOP was induced in 4 healthy beagles. IOP was elevated to 30, 40 and 50 mmHg by anterior chamber cannulation. SD-OCT was used to evaluate NFL thickness in the peripapillary region, temporal-superior (corresponding to the human macula) and temporal-inferior retina before and during elevation of IOP. pERG and blood pressure data were collected during AEIOP to evaluate RGC function and OPP (systolic, mean and diastolic perfusion pressure; SPP, MOPP and DPP, respectively).
Results: There was a significant increase in NFL thickness in the superior quadrant of the peripapillary scan at 40 mmHg (p<0.05) and 50 mmHg (p<0.01) when compared to pre-stress testing values. The thickness of the NFL before stress testing and at IOP 30, 40 and 50 mmHg for the inferior, temporal and nasal portions of the circle scan did not show significant change. Correlation analyses showed a significance between the pERG amplitude in microvolts and the IOP (p=0.002; r2=0.3566), the MOPP (p<0.0001; r2=0.4763), the SPP (p=0.0014; r2=0.3281) and the DPP (p=0.0002; r2=0.4819).
Conclusions: An acute episode of elevated IOP produces an increase in NFL thickness that correlates to a decrease in RGC function. We demonstrate a significant correlation between OPP and RGC function evidenced by a decrease in pERG amplitude with a decrease in perfusion pressure.
Kerstin M. Janisch and Joseph C. Besharse
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin
Purpose: The maintenance of photoreceptor (PR) size is vital for retinal health, since diseases such as chorioretinopathy exhibit altered PR size. We studied the mechanisms of PR length regulation through microtubule plus-end-tracking proteins (+TIPs) EB1, EB3 and DCX, which stabilise the plus-end of microtubules (MTs). To understand how these +TIPs influence PR size, we used over-expression in zebrafish PR cells. Our hypothesis is, that these +TIPs play a role in size regulation of PR.
Methods: Zebrafish eggs were injected at one-cell stage with either EB1-mCherry, EB3-mCherry or mCherry-DCX constructs under the transducina cone promoter. Embryos are screened for expression levels (++ and +) at 5 days post fertilisation (dpf). Structural microscopy studies were done at 5dpf and 10dpf eyes. For biochemical analysis, 10dpf eyes were used for subcellular fractionation or immunoprecipitation (IP).
Results: There was no significant difference in any of the 5dpf eyes for eye diameter, length and PR outer segment area. At 10dpf, eyes had significantly increased PR outer segment area, and eye size. Interestingly, high expression of EB1-mCherry and EB3-mCherry resulted in a severe phenotype with swollen RPE and choroid, disorganised PR layering and a spaceous cell distribution; whereas mCherry-DCX eyes did not show this phenotype. Biochemical analysis showed that b-catenin is upregulated in nuclear extracts of 10dpf eyes. IPs confirmed the EB-mCherry proteins being in complex with APC.
Conclusions: Overexpression of +TIPs in cones not only increases the size of PR outer segments, but has also effects on eye size. Biochemical data suggests activation of b-catenin/TGF-dependent genes.
Randy Kardon M.D. Ph.D.1,2, Pieter Poolman Ph.D.1,2, Susan Anderson B.S.1,2, Jan Full BSN1,2, Ana Recober M.D.3, Andy Russo Ph.D4
Departments of 1Veterans Affairs, 2Ophthalmology and Visual Sciences, 3Neurology, and 4Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA.
Purpose: The Photo-Blink Reflex protects the eye after a bright flash of light. Melanopsin containing retinal ganglion cells may comprise the afferent arm of this reflex through projections to the trigeminal nucleus and may explain the paradoxical photosensitivity in patients blinded by photoreceptor loss. To characterize this reflex, the EMG, pupil movement and skin conductance were recorded to increasing red and blue light stimuli in human subjects
Methods: Five subjects were tested under both scotopic and photopic conditions using red (640nm) and blue (485nm) Ganzfeld light one second in duration over a 6 log unit range of intensity (0.5 log unit steps). Time-stamped, computerizedrecording of the pupil, orbicularis and procerus muscle EMG, and skin conductance were measured simultaneously.
Results: The EMG showed a linear increase with log unit light intensity in parallel with pupil responses, but with a threshold approximately 3 log units less sensitive. At the brightest light intensities, an involuntary reflex blink was also triggered. Similar to pupil responses, the EMG showed a sustained, prolonged response to bright blue light stimuli, consistent with intrinsic activation of melanopsin containing retinal ganglion cells. Subjects with the largest light-induced EMG responses also had the largest changes in skin conductance.
Conclusions: We provide the first physiological evidence in humans that the light induced EMG response is mediated by melanopsin containing retinal ganglion cells, providing input to the trigeminal sensory nucleus, which then stimulates efferent output from the facial nucleus (as evidenced by EMG responses of orbicularis and procerus muscles) and sympathetic nervous system (as evidenced by increases in skin conductance). This unique light reflex may be useful in monitoring afferent light input affected by retinal and optic nerve disorders and also in diagnosing photosensitivity due to either ocular or central causes.
Sharolyn V. Kawakami-Schulz1, Angela M. Verdoni1, Sakae Ikeda1,2, Akihiro Ikeda1,2
Department of 1Medical Genetics, 2Eye Research Institute, University of Wisconsin, Madison
Purpose: Mutations in the gene Destrin (Dstn), an actin depolymerizing factor, cause murine corneal abnormalities. A null mutation, termed Dstncorn1, results in corneal hyperproliferation, inflammation and neovascularization in the A.BY background (A.BY Dstncorn1). Mice with a point mutation, termed Dstncorn1-2J, display mild hyperproliferation in the corneal epithelium but no neovascularization in a C57BL/6(B6) background (B6 Dstncorn1-2J). The goal of this study is to determine whether phenotypic differences in the cornea of these two mutants are due to the allelic differences between Dstncorn1 and Dstncorn1-2J, or the result of genetic background effects from A.BY or B6.
Methods: We generated two congenic mouse lines, B6.Cg-Dstncorn1 and A.BY.Cg-Dstncorn1-2J, to compare to the original lines. We performed immunohistochemistry to assay differences in neovascularization, inflammation and hyperproliferation.
Results: Phenotypic differences are due to allelic differences between Dstncorn1 and Dstncorn1-2J. Additionally, our results indicate that the phenotype is also modified by genetic background. Most notably, the Dstncorn1 mutation leads to neovascularization that is not seen in Dstncorn1-2J mice in either background. Therefore, neovascularization can be attributed to allelic differences between Dstncorn1 and Dstncorn1-2J. Neovascularization in the cornea of Dstncorn1 mice is significantly reduced in the B6 background, indicating the existence of genetic modifier(s).
Conclusions: By comparing the original mutants to congenic lines with the Dstncorn1 or Dstncorn1-2J mutations, we found that abnormal phenotypes result from the allelic difference between Dstncorn1 and Dstncorn1-2J and are also modified by genetic factors. Our results suggest that natural genetic variation affects phenotypic severity in Dstncorn1 mice.
H. Kecova1,2, E. Hernandez-Merino1,2, S.J. Jacobson1,2, A. Crumpton1,2,M. Pryde1,2, K.N. Hamouche1,2, R.H. Kardon2,3, M.M. Harper1,2, S.D. Grozdanic1,2
Department of Veterinary Clinical Sciences - Iowa State University, Ames, IA; 2Veterans Administration Center of Excellence for Prevention and Treatment of Vision Loss, Iowa City, IA; 3Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA.
Purpose: To characterize effects of topical (demecarium bromide-DB) and systemic (citicoline-citi) cholinergic drugs on optic nerve (ON) function and structure in canine model of primary glaucoma.
Methods: Nerve fiber layer (NFL) thickness was evaluated by optical coherence tomography (OCT) in healthy (HB) and glaucomatous (GB) Bassets. Retinal vessel diameter (OCT) and ON function (pattern electroretinography - pERG) was evaluated in GB before and after DB/citi treatment. GB treated with DB/citi and non-treated GB were subjected to provocative test of ON function by acute elevation of intraocular pressure with concurrent pERG recording.
Results: OCT analysis showed early thickening of NFL in GB. DB and citicoline did not affect NFL thickness (p>0.5, Paired t-test), but caused significant retinal vessel dilatation (DB: p=0.046, citi: p<0.0001). Baseline pERG amplitudes significantly improved in DB-treated dogs (baseline = 3.6+0.3μV; post treatment = 4+0.4μV; p=0.0004) but not in citi-treated dogs. However, both DB and citi had significant protective effect on ON function during acute IOP stress challenge: at IOP of 50 mmHg, pERG amplitude in non-treated GB declined to 22.7+6% of pre-elevation value, while citi-treated GB retained 47.1+7% and DB-treated GB retained 57.6+7% of baseline pERG amplitude (p<0.0001, ANOVA).
Conclusions: Early glaucoma in dogs results in structural (NFL thickening) and functional (pERG deficits) changes. Cholinergic drugs reversed functional optic nerve deficits and/or improved optic nerve resistance to acute IOP stress. Both citi and DB caused dilation of retinal vessels. Cholinergic agents should be considered as neuroprotective therapy in glaucoma treatment.
Elyse Kleifgen1, Ashley Finch1, Carol A Rasmussen1 and Gillian J McLellan1,2,3
Departments of 1Ophthalmology and Visual Sciences and 2Surgical Sciences and 3Eye Research Institute, University of Wisconsin-Madison
Purpose: To determine the nature of early changes in optic nerve head (ONH) morphology that occur in cats with primary congenital glaucoma (PCG).
Methods: Cirrus SD-OCT (Carl Zeiss Meditec Inc., Dublin, CA) was used to acquire 5-Line Raster Scans centered on the ONH of 11 cats (6 normal, 5 PCG) at 6-month intervals (ages 0.5-1.5 years). Each scan had a signal strength of 10/10. Intraocular pressures (IOPs) were measured at time of scan acquisition. Manual measurements of width of the neural canal opening (NCO); maximal cup depth (CD); posterior displacement of the lamina cribrosa (PDL), and pre-laminar tissue thickness (PLT) were obtained by a consistent masked observer using Image J (NIH). One scan for each eye was analyzed at each time-point and results for right and left eyes averaged for each cat. Comparisons between groups were made by non-parametric ANOVA and multiple comparisons post-test, with p<0.05 considered significant.
Results: Compared to normal cats, the ONH of PCG cats had a significantly narrower NCO and reduced PLT, and increased CD and PDL. When examining the effects of age on these parameters, CD was significantly increased and PLT significantly reduced in PCG cats by 6m of age. The NCO was significantly narrower in 1y-old PCG cats. PDL tended to increase with age in PCG cats.
Conclusions: Initial studies indicate that significant changes in optic nerve head morphology occur in cats with PCG by 6m of age.
Demelza Koehn1, Maurisa Aimable1, and Michael G. Anderson1,2
Departments of 1Molecular Physiology & Biophysics and 2Ophthalmology & Visual Sciences, The University of Iowa, Iowa City, IA
Purpose: Glaucoma is a leading cause of blindness in the United States. Important risk factors contributing to glaucoma include elevated IOP, positive family history, age, race, and thin central corneal thickness (CCT). Our goal is to identify genes that regulate the magnitude of CCT, using it as an entry point for studying the etiology of glaucoma. Using a quantitative approach with intercrosses between mice with thin corneas (C57BLKS/J; KS) and thick corneas (SJL/J; SJL), we identified Central corneal thickness QTL 1 ( Cctq1), which spans a ~15 cM region on mouse chromosome 7. The purpose of these experiments is to fine-map the position of Cctq1, identify known DNA variations within the Cctq1 interval using bioinformatics, and to begin testing candidate genes.
Methods: To map the Cctq1 gene, the genomic interval was divided into fourths (i.e., Intervals I, II, III, and IV) by intercrossing KS. Cctq1KS/SJL N4F2 congenic mice. Mice harboring recombinant chromosomes were identified by following genotypes at 5 polymorphic markers (D7Mit318, D7Mit31, D7Mit321, D7Mit220, and D7Mit238). These recombinant mice (and controls) were phenotyped for CCT, and differences in CCT were statistically analyzed using ANOVA. To sequence candidate genes (Pak1 and Aqp11), exons were PCR amplified and sequenced.
Results: Congenic mice harboring either Cctq1 interval I, II or III all confer a significant increase in CCT compared to control mice (P < 0.01). Mice harboring Cctq1 interval IV have CCT values not statistically different than control mice. Using bioinformatics, we identified 8 known nonsynonymous polymorphisms within the Cctq1 interval, the most interesting of which is within Adamtsl3. Of candidates sequenced, none of the exons contained any DNA variations between KS and SJL parental strains of mice.
Conclusions: Our results suggest at least two possibilities: 1) more than one CCT-regulating gene exists within Cctq1; or 2) there is too much genetic heterogeneity in our N4 mice to be able to uncover the specific allele. To differentiate between the two possibilities, we are continuing the backcross to N10 to reduce the genetic heterogeneity. We are also continuing candidate gene sequencing.
Arash Sangari 2,4, Hasti Mirkia4, Amir Assadi1,3,4
1Departments of Mathematics, 2Electrical and Computer Engineering, 3Comparative Biological and Medical Sciences, University of Wisconsin~Madison,4Persepolis Research Group
Purpose: Visual perception is the result of collective function of the eyes and the brain. The eyes collect images and the brain learns the perceptual geometry of objects, surfaces, lines and shapes. These elements are like symbolic grammars to the vision language. Vision as an entrance gate of human emotion has a significant role in analytical psychology, to the degree that these visual elements can make different emotional stimulus for the witnesses. The aim of this research is to provide a computational model for formation of the perceptual space concept and analyze the psychological role of these elements in human emotions.
Methods: The model applies statistical learning theory to data that is highly correlated with the intelligent system’s representation of memories of visual stimuli of prototypes of space, and infers statistical correlates and probabilistic structures within them.
Results: Our research provides the first steps of a computational model for a theory of perceptual geometry of space; namely, formation of the concept of spatial form from perspective. Our research on analysis of behavioral data is in progress.
Conclusions: We anticipate that the completed version of our theory will interpolate between ideas of Helmholtz, Henri Poincare', and Einstein on the conceptual level, will provide a computational explanation for modern psychophysical results such as the multi-scale theory of Koenderink, and could potentially contribute to a better understanding of modern neurobiological correlates of space such as the recent FMRI experiments of Nancy Kanwisher.
Aparna Lakkaraju and Jin Xu
Department of Ophthalmology & Visual Sciences, University of Wisconsin-Madison, Madison, WI
Purpose: To investigate the roles of apolipoprotein E, hepatic lipase and lipoprotein lipase in retinal pigment epithelial (RPE) cells. Disturbed cholesterol homeostasis contributes to age-related macular degeneration (AMD): cholesterol is enriched in drusen in the Bruch’s membrane and we have shown that the lipofuscin fluorophore A2E causes RPE cholesterol storage. ApoE, hepatic and lipoprotein lipases regulate serum HDL levels and are associated with AMD. However, there is no correlation between serum HDL levels and AMD risk.
Methods: Immunostaining and immunoblotting were used to analyze the sub-cellular localization and expression of ApoE, hepatic and lipoprotein lipases. Live imaging of GFP-tagged ApoE2/E3/E4 in RPE cells were imaged by spinning disk microscopy. Cholesterol and lipid droplets were visualized by filipin and bodipy 493/503.
Results: Endogenous ApoE is in late endosomes and the Golgi. ApoE2/E3/E4 vesicles moved at ~3µm/second and ApoE4 vesicles are larger than ApoE2 or ApoE3 vesicles. Outer segment phagocytosis, lipofuscin accumulation and LXR/PPARg agonists modulate protein expression, cholesterol levels and lipid droplet stores in RPE cells.
Conclusions: Serum HDL levels are poor predictors of AMD risk because retinal cholesterol homeostasis is controlled by the RPE, although little is known about how this occurs. RPE cells express hepatic and lipoprotein lipases and ApoE isoforms modulate the ligand-binding and lipolytic activities of these enzymes. Understanding the function, regulation and interplay between ApoE2/3/4, hepatic and lipoprotein lipases in the RPE and retina is important because these proteins influence dietary delivery of carotenoids and the response to statins, which may be beneficial in AMD.
J.E. McDonald1,2, J.A. Kiland1, P.L. Kaufman1, E. Bentley2, and G.J. McLellan1,2
1Department of Ophthalmology & Visual Sciences, and 2Department of Surgical Sciences, University of Wisconsin-Madison, WI
Purpose: To determine effects of latanoprost on intraocular pressure (IOP) and pupil diameter (PD) in normal and primary congenital glaucoma (PCG) cats.
Methods: IOP (TonoVet®) and PD were measured OU in 12 adult cats (6 normal, 6 PCG), 3x/wk for 3wks prior to; for 3wks during, and for 3wks following BID treatment with latanoprost 0.005% OD, with OS serving as a vehicle-treated control. One day prior to and on the first and last days of the treatment phase, IOP and PD were measured hourly for 8h. Fluorophotometry was carried out prior to initiation of and conclusion of the treatment phase. Peak IOP, maximal IOP fluctuation and area under the IOP versus time curve (cIOP) were calculated for each week of the study. Comparisons were made by Student t-test, or ANOVA with Tukey-Kramer post-hoc test, with p <0.05 considered significant.
Results: Latanoprost had no effect on IOP or aqueous humor flow rates in normal cats. Mean IOP was significantly lower in treated vs. control eyes of PCG cats up to 8h following a single application of latanoprost. Maximum IOP-lowering effect (mean =63%) was observed at 3h. Latanoprost caused miosis OD in all cats, with rebound mydriasis observed 24h post-treatment. Over the treatment phase, weekly cIOP significantly increased in both normal and PCG cats.
Conclusions: Latanoprost lowered IOP acutely in PCG cats, but this effect was not sustained. Our findings do not support latanoprost treatment for long-term IOP-lowering in cats. The mechanism for acute IOP reduction in PCG cats is unknown.
Maura McGrail, Ying Wang, Pavel A. Brodskiy, Elizabeth M. Whitley, Jeffrey J. Essner, Donald S. Sakaguchi
Department of Genetics Development and Cell Biology, Iowa State University, Ames, IA
Purpose: We have isolated a novel eye tumor model in a transgenic zebrafish line that is genetically linked to the transgene insertion site. The dominant pattern of inheritance and histopathological features of the tumors suggest similarities with the human childhood cancer retinoblastoma. The purpose of this study is to examine the origin of neoplastic cells in the retina and identify the molecular lesion linked to the tumor phenotype.
Methods: The tumor phenotype has been transmitted through three generations by outcrossing tumor positive fish to wild type and separating progeny classes based on inheritance of the RFP expressing Tg(RFP)is18 transgene. To examine proliferating cells in the retina adult fish are exposed to a BrdU pulse and sacrificed. Heads are processed and sectioned for histopathological and immunocytochemical analyses with antibodies to BrdU, retinal cell type markers, and retinal progenitor markers. Directed sequencing and candidate gene approaches are being used to identify the molecular lesion responsible for the tumor phenotype.
Results: Genetic analyses reveal the tumor phenotype appears in fish that have inherited the Tg(RFP)is18 transgene with >50% penetrance by 1 year of age. Histological and cytological analyses of adult Tg(RFP)is18 fish indicate the ocular tumors are located in the neural retina of the eye and show architectural features present in human retinoblastoma. The tumor mass contains proliferating cells, disorganized retinal cell layers, putative synaptic regions, and rosettes containing photoreceptor-like cells.
Conclusions: Our analyses indicate the zebrafish Tg(RFP)is18 line is a model of human retinoblastoma. An update of our results will be presented.
K. Mohan1,2, M.M. Harper1,3, K. Schipull1, A. Ghosh2, A.G Kanthasamy2, A. Kanthasamy2, R.H. Kardon3, S.D. Grozdanic1,2
1Veterinary Clinical Sciences Iowa State University, Ames, IA; 2Department of Biomedical Sciences, Iowa State University, Ames, IA, 3Veterans Administration Medical Center – Iowa City, IA
Purpose: To characterize the acute and chronic functional changes of the retina in a rodent model of Parkinson’s disease (PD) using pattern electroretinography (pERG).
Methods: Pattern evoked electroretinography was used to objectively measure retinal ganglion cell function. To elicit a pERG response, a black and white vertical stimuli was used (9° full field 1 Hz frequency, 1-30 Hz filter, 200 stimuli repetitions, photopic conditions). Baseline amplitudes (N35-P50 and P50-N95) and latencies (N35, P50, and N95) were evaluated in all mice prior to induction of PD (n=20). A PD phenotype was created by injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) acutely or chronically after evaluation of baseline pERG parameters.
Results: Analysis of the acute MPTP model of PD revealed a significant decrease in the P50-N95 amplitude 1 day post induction (2.89±1.05 µV, mean±SEM) when compared to baseline recordings (8.02±1.18 µV; p=0.0151, Paired t-test). Analysis of P50-N95 amplitude in the chronic model of PD (0.89±0.48 µV p<0.05, ANOVA) revealed a significant decrease from baseline values (9.36±1.68µV) at 16 days after induction of the model, however there were no significant deficits detected at 1, 4 and 8 days post induction. The pERG latencies were not significantly different from the pre-recorded values in both groups.
Conclusions: Acute and chronic models of PD show significant loss in retinal ganglion cell function as demonstrated by deficits in pERG amplitudes.
Michael A. Moriarty and Robert F. Mullins
Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA
Purpose: Age-related Macular Degeneration (AMD) is the leading causes of blindness in the Western World. One of the hallmarks of AMD pathogenesis is the deposition of lipid containing drusen in Bruch’s membrane. We investigated the involvement of lipoproteins in this deposition through identifying the presence of lipid binding moieties present in the retinal pigment epithelium (RPE), Bruch’s membrane, and choroid.
Methods: Histochemistry was utilized to identify lipoprotein binding sites within human choroid and RPE tissue. Furthermore, RT-PCR was used in order to identify expressed ECM and receptor proteins with the capacity to bind LDL in this ocular tissue. Organ culture experiments on porcine RPE/choroid incubated with acetylated LDL (AcLDL) and LDL were also performed.
Results: Immunohistochemistry revealed binding of AcLDL and LDL on CD45 positive cells in choroid. Staining with anti-Iba-1 showed binding of LDL occurred on macrophages. Binding to Bruch’s membrane was unclear due to autofluorescence. Organ culture of porcine RPE/Choroid showed uptake of AcLDL by RPE and some punctate staining in choroidal cells.
Conclusions: LDL and its modified variants such as AcLDL are shown to be capable of binding in regions of the RPE/choroid, especially to white blood cells; however, determination of binding to Bruch’s membrane was less clear. Ongoing studies will compare LDL metabolism in normal and AMD donors.
Pavitra Narasimha1, Ryan L. Boudreau2, Megan J. Riker3, Sajag Bhattarai3, Arlene V. Drack3 and Beverly L. Davidson2,4,5
1Interdisciplinary Program in Genetics; Departments of 2Internal Medicine, 3Ophthalmology and Visual Sciences 4Molecular Physiology and Biophysics, and 5Neurology, University of Iowa, Iowa City, IA
Abstract: Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disease and one of nine polyglutamine (polyQ) expansion diseases. SCA7 is unique among the polyQ diseases in that there is a moderate to profound vision loss due to retinal degeneration, with vision loss often being the first reported symptom. Normal individuals have ~10 CAG repeats in ATXN7. An expansion of 37-59 CAG repeats results in initial cerebellar neurodegeneration, while an expansion of >59 CAG repeats causes retinal as well as cerebellar degeneration. Anticipation is a common feature in the disease and early onset SCA7 is severe and progresses rapidly, causing death early in life. The National Ataxia Foundation has reported the incidence of SCA7 to be <1 in 100,000 with SCA7 representing 2% of all SCAs. In certain populations there is a higher incidence of the disease. In South African populations it is the second most prevalent ataxia.
Currently, there are no effective treatment strategies for this disease. Previously, Boudreau et al., have shown that RNA interference (RNAi) therapy by non-allele specific silencing of Huntington mRNA provides therapeutic benefit in mice with Huntington’s disease. Based on these prior studies in my lab, I hypothesize that SCA7 retinal degeneration can be alleviated using allele-specific or non-allele specific RNAi-based gene silencing therapies.
Three specific aims will address this hypothesis: a) design and test RNAi vectors to knockdown ATXN7 in vitro by allele-specific and non-allele specific silencing, b) characterize the retinal degeneration phenotype in SCA7-92Q transgenic mice and identify retinal cells that express Atxn7 mRNA, c) determine if in vivo knockdown of ATXN7 by allele-specific and non-allele specific RNAi-mediated gene silencing improves retinal phenotypes in SCA7-92Q mice and perform transcriptional profiling to address the effects of allele-specific versus non-allele specific silencing.
These studies will address the possibility of RNAi therapy in SCA7 retinal degeneration and will also assess the feasibility of allele-specific versus non-allele specific silencing for SCA7.
B.E.O’Bryhim1, R.S. White2, and R.C.A. Symons1,2
Departments of 1Molecular and Integrative Physiology and 2Ophthalmology, University of Kansas Medical Center
Purpose: Vasoproliferative retinopathies, including retinopathy of prematurity, diabetic retinopathy, and neovascular age-related macular degeneration, are the leading cause of blindness in the US. Emerging evidence demonstrates that endothelial progenitor cells (EPCs) play an important role in angiogenesis, and their recruitment to the retina is likely to be essential in neovascularization. The purpose of this study is to characterize differences in EPC recruitment to the retina during revascularization between mice that are differentially susceptible to the murine model of retinopathy of prematurity, oxygen-induced retinopathy (OIR).
Methods: C57BL/6J and B6(Cg)-Tyr2cJ/J ("B6(Cg)") were exposed to the OIR protocol to induce vaso-obliteration of the retinal vasculature. Mice were anesthetized, perfused with fluorescein-labeled dextran, and enucleated. The area of central avascularity was measured immediately upon return to normoxia (P12) and 96 hours after return (P16). Additional retinae were stained for the presence of AC133+ progenitor cells, which were counted by an observer blind to strain.
Results: Examination of avascular area at P12 and P16 demonstrated that the B6(Cg) mice and C57BL/6J mice exhibit similar levels of vaso-obliteration, but that the B6(Cg) mice have smaller avascular areas by the later time point. B6(Cg) also have more EPCs in perivascular areas of the retina during this process.
Conclusions: Examination of avascular area at both P12 and P16 demonstrated that the B6(Cg)-Tyr2cJ/J mice revascularize more quickly than C57BL/6ByJ mice, and they have greater numbers of EPCs. This suggests that EPC recruitment plays a role in revascularization and may explain the differential response to OIR that these strains exhibit.
Yuan Pan and Sheila Baker
Department of Biochemistry, University of Iowa, Iowa City, IA.
Purpose: Hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) is a non-selective cation channel that is expressed in retina, hippocampal neurons and cardiomyocytes. In photoreceptors, HCN1 functions in regulating the speed of light responses, which has been demonstrated by prolonged light responses in HCN1 homozygous deletion mice. Within a rod photoreceptor, HCN1 is localized in the membrane of inner segment and synapse. Failure to target HCN1 to its proper sub-cellular compartments would prevent it from functioning. Here in this study, our goal is to identify the mechanism controlling HCN1 trafficking to the inner segment and synapse of a photoreceptor. One aim of this study is to identify the sequence that is necessary and sufficient for HCN1 targeting in rods. The second aim is to test whether targeting of HCN1 is dependent on ankyrin B, an important membrane adaptor protein that also resides specifically in the inner segment of a photoreceptor.
Methods: Several human HCN1 truncation mutants will be integrated into the genome of Xenopus laevis and exhibit rod-specific expression. Localization of these constructs can be visualized with confocal microscopy. The interaction between HCN1 and ankyrin B will be tested by the membrane recruitment assay in HEK293 cells, co-immunoprecipitation and GST pull-down.
Results: Our preliminary data obtained from transgenic frogs show that the carboxyl terminus following the cyclic nucleotide binding domain of HCN1 (590-714) is sufficient for its targeting. In addition, our membrane recruitment assay and co-immunoprecipitation results do not support interaction between HCN1 590-910 and ankyrin B.
Conclusions: HCN1 C-terminus containing residues 590-714 is sufficient for its targeting in rods and it is not very likely to be regulated by ankyrin B.
Arash Sangari 2, Mohammad Khabbazian2, Mona Jalal2, Majid Arabgol1, Amir Assadi1,3
1Departments of Mathematics, 2Electrical and Computer Engineering, 3Comparative Biological and Medical Sciences, University of Wisconsin-Madison
Purpose: Computational complexity has been the subject of intensive research with rewarding theoretical and practical accomplishments. In biology, the analog nature of signals and the system observables pose a great challenge to draw parallels between the digital information theory and the analog theory of biological information. Research on quantifying biocomplexity could have a great impact in understanding myriad phenomena in the biomedical sciences and applications to public health.
Methods: Quantifying biocomplexity from DNA-level data must overcome a number of analytic and numerical problems that are posed for operations on dense matrices of very large-size, and correlations among ultra-massive data sets. Novel clustering, feature extraction and pattern recognition algorithms using methods of Empirical Topology are developed to overcome the numerical challenges. The computational implementation requires novel GPU-based strategies for massively parallel and distributed computation that are also developed within this framework.
Results: We provide a report of preliminary success in development of a quantitative theory of genomic biocomplexity. In parallel to the Kolmogrov’s theory of computational complexity, genomic biocomplexity is developed through a multi-scale multi-resolution model of the DNA-level processes that underlie stages of development and growth, or a biological behavior. Informally, biocomplexity of a phenotype is an ideal measure of the most efficient description of computations initiated at the genome level, and output the targeted phenotypic observables.
Conclusions: The Arabidopsis Biocomplexity theory and concrete case studies inspire questions regarding Biocomplexity in other organisms, in organogenesis, and myriad other open areas of research. We comment on how the discoveries of the Arabidopsis circadian rhythms could guide us to design experiments that study oscillations and waves in development of the rodent retina.
L.B.C. Teixeira 1, Y. Zhao 2, R.R. Dubielzig 1, C.M. Sorenson3, N. Sheibani2
1Comparative Ocular Pathology Laboratory of Wisconsin , 2Ophthalmology and Visual Sciences, and 3Pediatrics, University of Wisconsin, Madison, WI
Purpose: To determine the ultra structural morphology of the iridocorneal drainage angle of Cyp1b1-deficient (Cyp1b1-/-) mice.
Methods: Eyes from wild type (Cyp1b1+/+) and Cyp1b1-/- mice were enucleated and processed for transmission electron microscopy. The iridocorneal angle, trabecular meshwork (TM), Schlemm’s canal and aqueous veins were analyzed.
Results: The 7-month-old C57BL/6 Cyp1b1-/- mice presented marked disruption of the anterior and posterior TM, multifocal atrophy of the trabecular beams with accentuation of the intertrabecular spaces. Collagen of the beam cores was markedly fragmented and multifocal accumulations of nodular elastic tissue were observed. The number of endothelial cells covering trabecular beams was diminished in Cyp1b1-/- mice, and the basement membranes were multifocally disrupted. An atrophic solitary trabecular beam extending from the anterior to mid-meshwork contacting a collapsed posterior meshwork with fragmented collagen and elastic fibers surrounded by few typical endothelial cells was observed in 8.5-month-old Cyp1b1-/- mice. Schlemm’s canal and the aqueous veins were normal.
Conclusions: The Cyp1b1-/- mice presented ocular drainage ultrastructural abnormalities at 7-months and became more severe with age suggesting an age related evolution. Thus, Cyp1b1 expression/activity significantly contributes to the integrity and function of the TM. We hypothesize based on the known interactions of Cyp1b1, its deficiency interferes with the expression/function of TM matricellular proteins impairing endothelial cells attachment and extracellular matrix turnover.
Budd A. Tucker1A, Todd E. Scheetz1AC, Robert F. Mullins1A, Adam P. DeLuca1AC, Samuel G. Jacobson2, Val C. Sheffield1C and, Edwin. M. Stone1A
1ADepartment of Ophthalmology and Visual Sciences, 1BCenter for Bioinformatics, 1C Department of Pediatrics, University of Iowa, Iowa City, IA; 2Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA.
Purpose: Retinitis pigmentosa (RP) is a genetically heterogeneous heritable disease characterized by apoptotic death of photoreceptor cells. Although it is estimated that mutations in more than 100 genes will eventually be shown to cause RP, at present fewer than half of these genes have been identified. The purpose of this study was to utilize next generation sequencing and iPSC technologies to identify potential new disease causing genes in families that remained undiagnosed by previous technologies.
Methods: Next generation exome sequencing and induced pluripotent stem cells technologies were used to identify new RP causing genes and evaluate the pathogenicity of specific mutations in human retinal cells.
Results: A homozygous Alu insertion in exon 9 of MAK was identified as the cause of disease in an isolated individual with RP. Screening of 1798 unrelated RP patients identified 20 additional probands homozygous for this insertion (1.2%). All 21 affected probands were of Jewish ancestry. MAK encodes a kinase involved in the regulation of photoreceptor connecting cilium length. Immunohistochemistry of human donor tissue revealed that MAK is expressed in the inner segments, cell bodies and axons of rod and cone photoreceptors. Several isoforms of MAK that result from alternative splicing were identified. By using induced pluripotent stem cells (iPSCs) derived from the skin of the proband, a patient with non-MAK-associated RP (RP control), and a normal control, we found that a transcript lacking exon 9 was predominant in undifferentiated cells while a transcript bearing exon 9 and a previously unrecognized exon 12 predominated in cells that were differentiated into retinal precursors. However, in the proband with the Alu insertion, the developmental switch to the MAK transcript bearing exons 9 and 12 did not occur.
Conclusions: This study demonstrates the utility of exome sequencing and iPSCs technologies to efficiently identify and evaluate gene specific mutations in individuals with molecularly unconfirmed retinal disease.
Katie A. Weihbrecht1,2, Melissa C. Humbert2, Val C. Sheffield2,4, Seongjin Seo3
1Interdepartmental Genetics Program, 2Department of Pediatrics, 3Department of Ophthalmology, 4Howard Hughes Medical Institute, University of Iowa, Iowa City, IA
Purpose: While more than 150 genes have been identified as involved in retinal diseases, the function of many has not yet been determined. The goal of this project is to determine the molecular function of ciliopathy-related, uncharacterized genes by determining their interacting partners. Current genes include Leber congenital amaurosis 5 (LCA5), serologically defined colon cancer antigen 8 (SDCCAG8), and inositol polyphosphate-5-phosphatase (INPP5E).
Methods: Interacting partners will be isolated by tandem affinity purification (TAP) using HEK293T cells stably expressing each of these proteins tagged with FLAG- and S-tags. TAP will be conducted by using anti-FLAG affinity gel followed by FLAG peptide elution, then S-peptide affinity gel. Isolated proteins will be resolved in an SDS-PAGE gel, silver-stained, and submitted for mass spectrometry for protein identification. Western blotting will be used to confirm mass spectrometry results.
Results: So far, we have isolated three prominent LCA5 interacting proteins, seven for SDCCAG8, and four for INPP5E, visualized by silver staining. 14-3-3 proteins are known to interact with both LCA5 and INPP5E and we confirmed these interactions by Western blotting, verifying the validity of our approach. All of the remaining samples will be submitted for mass spectrometry for protein identification.
Conclusions: Tandem affinity purification combined with mass spectrometric protein identification is a powerful approach to elucidate the molecular functions of uncharacterized proteins and will contribute to the understanding of disease mechanisms associated with these proteins. We will investigate the relationship between 14-3-3 proteins, LCA5 and INPP5E and the biological significance of these interactions in photoreceptor cells.
S. Scott Whitmore1, Terry A. Braun1,2,3, Todd E. Scheetz1,2, and Robert F. Mullins1
Departments of 1Ophthalmology and Visual Sciences, 2Biomedical Engineering, and 3Center for Bioinformatics and Computational Biology, University of Iowa, Iowa City, IA
Purpose: The ARMS2 A69S allele is a risk factor for age-related macular degeneration; however, the impact of this allele on downstream gene expression is unknown.
Methods: To explore the consequences of the A69S allele on gene expression, we assessed gene expression levels in macular RPE and choroid from 20 human eyes using Affymetrix GeneChip Human Exon 1.0 ST Arrays. Results were analyzed using Partek Genomics Suite (6.110321) and R (2.11.1). Sixteen samples (9 AA; 4 AS; 3 SS) met our criteria for quality control. Differential expression was assessed by calculating ANOVA p-values and fold change between genotype classes. Genes from this analysis were filtered on: (a) a minimum ANOVA p-value of 0.01; (b) minimum fold change of 50%, and (c) evidence of expression in at least one genotype class (RMA expression >= 6).
Results: Filtering identified 165 genes, of which 116 (70.3%) were upregulated with increased presence of the risk allele (AS and SS). Functional classification of these genes identified seven annotated groups: ribosomal proteins or pseudogenes (7 genes); nuclear proteins primarily involved in mRNA splicing (11 genes); proteins sharing 14 repeat domains (4 genes); proteins involved in cation binding and/or transcriptional regulation (32 genes); ribonucleotide binding proteins (9 genes); signaling proteins (12 genes); and integral membrane proteins (32 genes).
Conclusions: Metabolic activity is upregulated with increased presence of the ARMS2 risk allele. However, the pathways responsible for this change are unclear. We are continuing to analyze this dataset for potential interactions between these genes.
G.S. Zode1,2, M.H. Kuehn3, D.Y. Nishimura1,2, C.C. Searby1,2, K. Mohan5, S.D. Grozdanic5, K. Bugge1,2, M.G. Anderson3,4, A.F. Clark6, E.M. Stone1,3 and V.C. Sheffield1,2,3
1Howard Hughes Medical Institute, Departments of 2Pediatrics, 3Ophthalmology and Visual Sciences, and 4Molecular Physiology and Biophysics. University of Iowa, Iowa, IA, 5Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA, and 6 Dept.Cell Biology & Anatomy and the North Texas Eye Research Institute, University of North Texas Health Science Center at Fort Worth, TX
Purpose: Myocilin (MYOC) mutations are the most common genetic cause of primary open angle glaucoma (POAG). However, mechanisms underlying MYOC-associated glaucoma are not fully understood, and a mouse genetic model that closely mimics human POAG has not been developed. The aims of this study were to a) develop a mouse POAG model b) utilize this model to investigate underlying pathogenic mechanisms, and c) evaluate therapies for treatment of MYOC-associated glaucoma.
Methods: We report the development of a transgenic mouse model (Tg-MYOCY437H), which expresses human MYOC containing the Y437H mutation within relevant eye tissues. Glaucoma phenotypes were examined by measuring intraocular pressure (IOP), retinal ganglion cell (RGC) death, and optic nerve degeneration in Tg-MYOCY437H mice (44 WT, 80 Tg-MYOCY437H mice). Western blotting and immunostaining were used to examine myocilin expression, as well as markers of endoplasmic reticulum (ER) stress in the trabecular meshwork (TM) of Tg-MYOCY437H mice.
Results: Tg-MYOCY437H mice displayed ocular manifestations of glaucoma including elevated IOP (7mm Hg increase vs WT;p<0.0001) at 3 months of age, progressive structural and functional loss of RGCs (40% loss by 12months;p<0.001), and optic nerve degeneration (45% reduction in axons count by 12months;p<0.001). We demonstrate that mutant myocilin is not secreted into the aqueous humor and accumulates in the ER of the TM, thereby inducing ER stress in the TM of Tg-MYOCY437H mice. Such chronic and persistent ER stress is associated with TM cell death and elevation of IOP in Tg-MYOCY437H mice. Reduction of ER stress by topical treatment with the chemical chaperone, phenyl butyric acid (PBA) prevents glaucoma in Tg-MYOCY437H mice by promoting the secretion of mutant myocilin in the aqueous humor, and by decreasing intracellular accumulation of myocilin in the ER, thus preventing TM cell death.
Conclusions: These results demonstrate that ER stress is linked to the pathogenesis of POAG and is a target for treatment in human patients.
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