2010 Midwest Eye Research Symposium
MERS 2010 took place on Friday June 25, 2010 at
The University of Iowa Seabohm Conference Room
Keynote Speaker / Session Chairs
Deborah A. Ferrington, PhD - Keynote Speaker
Neena B. Haider, PhD - Chair, Session I
Steven H. DeVries, MD, PhD - Chair, Session II
Robert F. Mullins, PhD - Chair, Session III
Session I - Session Chair Neena B. Haider, Ph.D.
Session II - Session Chair Steve DeVries, M.D., Ph.D.
Session III - Session Chair Robert F. Mullins, PhD
A Real-Time PCR Approach for the Quantification of Ganglion Cell Loss in Mouse Eyes
D. Ahram1,2, A. Cook1, M. H. Kuehn1,2
1Department of Ophthalmology and Visual Sciences, 2Interdisciplinary Graduate Program in Genetics; The University of Iowa, Iowa City, IA
Purpose: Many optic neuropathies, including glaucoma, are characterized by retinal ganglion cell (RGC) loss and visual field defects. In rodent models the estimation of RGC loss using standard histological methods is a standard approach to evaluate glaucomatous or ischemic damage. However, counting RGCs it is time consuming and often a rate limiting experimental step. We here present a quantitative real-time PCR method for estimating RGC numbers by measuring the expression of RGC specific gene transcripts relative to reference genes in an ischemia-induced mouse eye model.
Methods: Retinal ischemia was induced in C57BL6/J mice by applying hydrostatic pressure to the anterior chamber of the eye at varying pressure intensities and durations. Non-ischemic control mice were included. Total RNA was collected from mouse retinal tissue and cDNA was synthesized. A real-time quantitative PCR assay was conducted and expression of specific mouse RGC marker genes (Pou4f3, Pou4f2, Nell2 and Rbpms) was estimated by normalizing against a set of uniformly expressed reference genes (Sdha, Tbp and Ubc). Gene expression levels were assessed using comparative and absolute quantification methods. Furthermore, viable RGC counts in the ischemic retinas were quantified and validated using immunohistochemistry.
Results: Ischemia related tissue damage in the mouse eye was noted in the form of whitening of the anterior segment and blanching of the retinal arteries. All marker and reference genes were found to be expressed in the retina. Overall, the RT-PCR results showed reduced marker gene expression in the retinas of mice exposed to higher pressures and displaying greater ischemic damage relative to control mice.
Conclusions: We are currently in the process of evaluating the accuracy of gene expression results for each marker gene in order to determine the gene most suitable for the quantification of RGCs. If validated, this method can significantly improve RGC count accuracy and reduce the time and effort needed to assess retinopathy related RGC loss.
Identification of a Targeting Signal in the Synaptic Vesicle Protein, Synaptophysin
M. Brucato 1, V.Y. Arshavsky1, S.A. Baker1,2
1Department of Ophthalmology, Duke University, Durham, NC; 2Department of Biochemistry, University of Iowa, Iowa City, IA
Purpose: Photoreceptors are specialized neurons, consisting of several discrete compartments organized in a linear array. These compartments include the photosensitive outer segment, the biosynthetic inner segment, the nuclear region, and the synaptic terminal. The signals and mechanisms that ensure the proper sorting, targeting and trafficking of proteins to these various compartments are poorly understood, particularly in the case of the synaptic terminal. Synaptophysin spans the membrane four times and is found in oligomers within the synaptic vesicle membrane, where it is believed to contribute to the regulation of various aspects of the synaptic vesicle cycle. The objective was to determine the minimal sequence within synaptophysin that signals the protein to be targeted to the synaptic terminal.
Methods: Constructs consisting of a membrane-associated YFP as a reporter fused to various regions of synaptophysin were expressed in the rod photoreceptors of transgenic Xenopus laevis. The subcellular localization of these proteins in tadpole photoreceptors was assessed by confocal microscopy.
Results: The YFP reporter was found throughout the cell but strongly accumulated in the outer segment, as we have previously described for membrane proteins lacking targeting information in this cell type. We found that only the cytoplasmic C-terminus of synaptophysin, fused to the reporter was restricted to the inner segment plasma membrane and synaptic terminal. Several deletion constructs of the C-terminus revealed that the last 10 residues of synaptophysin’s C-terminus were sufficient to target the reporter to the synaptic terminal.
Conclusions: We have identified a novel synaptic targeting sequence.
Voted Outstanding Oral Presentation
Structural-Functional Analysis of cep290 in zebrafish vision
L. M. Baye1, X. Patrinostro1, J. S. Beck4, S. Swaminathan1, Y. Zhang2, E. M. Stone3,4, V. C. Sheffield2,4, D. C. Slusarski1
The University of Iowa Departments of 1Biology; 2Pediatrics; 3Ophthalmology and Visual Sciences, Iowa City, IA; 4Howard Hughes Medical Institute
Purpose: CEP290 is a large, multi-domain gene implicated in several cilia related syndromic disorders including Meckel-Gruber, Senor-Loken, Joubert and Bardet-Biedl Syndrome (BBS). Moreover, CEP290 is the most frequently mutated gene underlying the non-syndromic blinding disorder Leber's congenital amaurosis (LCA). Using the zebrafish model system, we characterized the developmental role of cep290and used a functional assay to identify regions of the CEP290 protein necessary for vision.
Methods: RT-PCR and in situ hybridization was used to examine the temporal and spatial expression of cep290during development. An antisense oligonucleotide (Morpholino) designed to generate an altered cep290splice product that models a common LCA mutation was utilized for gene knockdown. Since CEP290 has been implicated in BBS, embryos were analyzed for phenotypes associated with bbs knockdown: formation of the ciliated Kupffer’s Vesicle (KV) and melanosome transport. The startle response assay was used to asses vision function. To determine if truncated forms of human CEP290 RNA can offset the knockdown defects, RNA was co-injected with the Morpholino.
Results: Knockdown of cep290yields defects in the KV, delays in melanosome transport, a curved body axis and vision impairment. The vision defect was suppressed by expressing only a portion of the human CEP290 gene.
Conclusions: Consistent with a role in BBS, knockdown of cep290results in KV and transport defects. Moreover, we demonstrate that disruption of cep290results in vision loss and that expression of only a CEP290 fragment restores vision. These data highlight a potential treatment for LCA patients with mutations in CEP290.
Sequence Driven ENU-mutagenesis to Create New Mouse Models of Ocular Disease
S. Bushek1, D. Yu 2, T. E. Scheetz1, V. C. Sheffield2,3, M.H. Kuehn1
Departments of 1Ophthalmology and Visual Sciences, 22Pediatrics, The University of Iowa; 33Howard Hughes Medical Institute
Purpose: The identification of genetic mouse models of disease facilitates the discovery of disease genes and provides models for studies directed at developing treatment modalities. Access to new mouse models with mutations in genes of interest remains rate-limiting to progress, both of science as a whole, and for individual research programs. We are conducting a high through put genetic screen with the goal of identifying over 100 strains of mice carrying mutations in genes with ocular and cilia function.
Methods: Male mice were treated with N-ethyl-N-nitrosourea (ENU) and bred with normal females to create F1 offspring carrying numerous random heterozygous mutations. Using bioinformatics approaches we selected 400 genes including those with suspected roles in cilia development or maintenance, photoreceptor, retinal ganglion cell, and trabecular meshwork function. Two exons were chosen from each gene for sequence analysis (800 exons total). Using next-generation sequencing technology, all 800 exons will be sequenced in over 2,000 F1 mice. Mice carrying desirable mutations will be identified and bred. Theoretically, this number of animals is sufficient to ensure that a non-synonymous mutation will be detected at least once in every screened exon. In addition, all F1 mice are monitored for ocular and systemic abnormalities, intraocular pressure elevation, and pupil light reflex deficiencies.
Results: To date approximately 600 F1 mice have been generated and we are in the process of sequencing all exons the first 400 individuals. In addition, a number of F1 mice have been detected with presumably dominant mutations resulting in microophthalmia, hydrocephalus, IOP elevation, and developmental abnormalities.
Conclusions: The advantages of this approach are: 1) recessive mutations can easily be identified, 2) both null alleles and hypomorphs will be detected, and 3) significantly more cost effective than traditional mutagenesis screening or targeted deletion protocols. We anticipate that that the establishment of this repository of mouse strains with mutations affecting ocular function will have a significant impact on vision research.
Involvement of the Complement Cascade in Glaucoma
A. Cook1, Q. Ding1, M. H. Kuehn1
1Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, IA
Purpose: Glaucoma is a group of diseases that lead to progressive retinal ganglion cell loss with optic nerve damage that may result in blindness. Our previous studies have demonstrated that the degeneration of the optic nerve and retina is associated with increased synthesis and deposition of components of the complement cascade. Our current studies are designed to determine if the complement system response in glaucoma modulates the development or progression of the disease.
Methods: Using immunohistochemistry, complement deposition was determined in human donor eyes and optic nerves with and without glaucoma. Functionally, we determined the role of complement by inducing elevated intraocular pressure in mice with targeted deletions in either the for complement component 3 (C3) or the complement component 1, subunit A (C1qa) gene. The degree of damage was determined by counting the number of surviving RGC in flat-mounted retinas and through evaluation of optic nerve crossections following staining with paraphenylene-diamene.
Results: Complement components C1qa and C3 are locally synthesized in the glaucomatous and ischemic retina. Complement components accumulate specifically in association with cellular structures in the nerve fiber layer, the retinal ganglion cell layer, and in the optic nerve. Functionally, mice with a genetic disruption of the complement cascade display reduced RGC and axon loss one week after an ischemic insult. However, RGC and axon loss continues and three weeks after ischemia, knock outs display levels of optic nerve damage similar to normal mice.
Conclusions: Our data demonstrate that while other mechanisms are ultimately responsible for RGC death, complement actively destroys damaged RGC in the retina in an effort to minimize the period of retinal inflammation. Complement activation appears to accelerate RGC damage either through direct lysis induced by the formation of the membrane attack complex, and/or through C3a and C5a anaphylatoxin mediated glial cell activation resulting in the release of neurotoxic intermediates.
Voted Outstanding Poster Presentation
Utilizing B6-Lystbg-J Mice to Extend Genetic Pathways of Exfoliation Syndrome
T. L. Cuffy1, C. M. Trantow2, M. G. Anderson1,2,3
1Interdisciplinary Graduate Program in Genetics, 2Molecular Physiology and Biophysics, 3Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA.
Abstract Exfoliation syndrome (XFS) is a common age-related disorder involving pathological accumulations of a fibrillar exfoliative material in the anterior chamber of the eye, pigment dispersion, and a striking pattern of Marcel-like iris transillumination defects. In parallel with these anterior chamber abnormalities, intraocular pressure can become elevated and glaucoma often ensues. Mice containing the B6-Lystbg-Jmutation recapitulate many aspects of XFS and represent one of the only known mouse models of this disease. The goal of these experiments is to extend knowledge of genetic pathways relevant to XFS by studying the molecular basis of the bg-J mutation. We have previously found the bg-J mutation is caused by a three base pair deletion in the WD40 encoding region of the Lyst gene. WD40 motifs frequently mediate protein-protein interactions and a small number of proteins capable of interacting with the LYST WD40 motif in yeast two-hybrid assays have been identified by others. Testing one of these, CSNK2B, as a candidate interacting protein, GST-pulldown experiments confirmed that wild-type LYST can bind to CSNK2B whereas -LYSTbg-J can not. This result suggests that LYST may play a role in regulating activity or localization of CSNK2B. In ongoing work, this hypothesis is being tested by studying the localization of CSNK2B and studying the influence of the B6-Lystbg-JJ mutation on various CSNK2B substrates. Preliminary experiments with primary fibroblasts expressing a GFP-tagged CSNK2B fusion protein suggest that LYST indeed regulates the subcellular localization of CSNK2B. Furthermore, two CSNK2B substrates (PER2 and CDH1) exhibit functional deficits in the presence of the B6-Lystbg-J mutation. Combined, these results implicate CSNK2B and its substrates in the pathogenesis of XFS.
Poster Board #5
In-vivoCharacterization of Sustained Release Timolol Microspheres
A.V. Dumitrescu1, J. Bertram2, L.R. Olson3, J.K. Jens3, N.M. Ellinwood3, Y.H. Kwon1, E. Lavik4, M.H. Kuehn1
1Ophthalmology and Visual Science, University of Iowa; 2Biomedical Engineering, Yale University; 3Department of Animal Science, Iowa State University; 4Biomedical Engineering, Case Western Reserve University
Purpose: Although several clinically effective intraocular pressure (IOP) lowering drugs are available for the treatment of glaucoma, patient compliance is often poor leading to suboptimal IOP control and avoidable loss of vision. To address this problem, we recently fabricated microspheres that can deliver timolol maleate as they slowly dissolve and can be delivered by subconjunctival injection. Here we evaluate the in vivo release characteristics and tissue compatibility of the microspheres in rabbit and cat eyes.
Methods: Timolol microspheres were fabricated using a double emulsion technique a blend of poly(lactic-co-glycolic acid) and poly(D,L-lactic acid). Microspheres were delivered subconjunctivally by a single injection into the eyes of either normal New Zealand White rabbits or cats. Aqueous humor was repeatedly collected for up to three months (rabbits) or 32 days (cats), and vitreous humor was obtained from rabbits after each animal was sacrificed. The concentration of timolol was determined using HPLC/MS at 294 nm. Histochemical and immunohistochemical methods were used to evaluate whether injection of the spheres elicited an inflammatory response in the anterior segment of rabbit eyes.
Results: The fabricated microspheres have an average size of 16.1 uM (+/- 5.1 uM) and are capable of delivering 21.8 ug of timolol per mg of microspheres over 107 days in vitro. In vivo we were able to detect timolol in the aqueous humor (150 ng/ml) of cats until the end of the experiment (32 days after injection). In rabbits timolol could be detected at clinically effective levels for thus far for up to 55 days after injection in the aqueous humor and up to 62 days in the vitreous humor. Gross observation of the eyes at the injection sites did not reveal signs of inflammation, infection, or other side effects in rabbits (0/60 eyes) or cats (0/12 eyes). Histochemical and immunohistochemical examination of rabbit eyes 10 and 21 days after injection did not reveal signs of inflammation. Timolol was undetectable in the serum of all animals and at all times assayed (detection limit = 0.05 ng/ml).
Conclusions: Our data demonstrate that the sustained delivery of Timolol from a single injection for significant periods of time is feasible. In addition, thus far this delivery method has proven to be safe and well tolerated.
Stem Cells for Treatment of Blinding Diseases
J. P. Fernandez de Castro1, E.M. Stone1,2, R. F. Mulllins1, M. H. Kuehn1
1Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA; 2Howard Hughes Medical Institute
Purpose: Stem cells have the potential to restore vision or prevent vision loss in many ocular diseases. Here we evaluated the use of stem cells to functionally replace retinal pigmentary epithelium (RPE) cells in a mouse model of Leber congenital amaurosis.
Methods: The pluripotency of embryonic murine stem cells expressing early markers of stem cells like alkaline phosphatase, SSEA-1 and SSEA-4 were assessed by the formation of a teratoma in vivo by injecting the induced pluripotent stem cells into hind limb muscles of 6-week-old immunocompromised SCID mice. The confirmed pluripotent cells were allowed to grow into embryoid bodies and then committed to differentiate into retinal cells by stepwise treatment with the small molecule factors CKI-7 and SB-431542. RPE defects were induced in albino mice through sodium iodate injections and differentiated cells were subsequently transplanted via subretinal injection. The transplanted eyes were analyzed in vivo using fundus photography and ERG and histologically using light microscopy.
Results: The mouse embryonic stem were capable of teratoma induction and could subsequently be differentiated to retinal progenitor cells expressing RPE markers and synthesizing pigment. Albino mice with provoked RPE defects that received subretinal injections of these cells showed subretinal pigment scattered throughout the posterior pole. While the ERG is flat after sodium iodate exposure in untreated mice, animals that received an injection of RPE progeniyor cells show a recovery of both the a-wave and the b-wave after the transplant. Light microscopy shows scattered pigmented cells on Bruch’s membrane closely associated with the location of surviving photoreceptors.
Conclusions: The differentiated retinal cells resemble RPE cells in the marker expression, pigment production, and location within the retina. Furthermore, these cells appear to preserve or restore some photoreceptor function in a mouse model of Leber congenital amaurosis. This study helps understand the potential of stem cells and allows the development of transplantation therapies for retinal diseases, the discovery of therapeutic drugs and investigation of the retinal development and disease mechanisms.
Visual System Damage in a Rodent Model of Blast-Induced Traumatic Brain Injury
M. M. Harper1,2, K. Mohan1,2, S. D. Grozdanic1,2, A. Ghosh3, V. Anantharam3, A. Kanthasamy3, A. G. Kanthasamy3, R. H. Kardon1,4 , T. Lazic1,2
1Veterans Administration Center for Prevention and Treatment of Visual Loss – Iowa City; 2Department of Veterinary Clinical Sciences-Iowa State University; 3Department of Biomedical Sciences – Iowa State University; Department of Ophthalmology and Visual Sciences – University of Iowa4
Purpose: To characterize effects of blast-mediated injury on the structure and function of the visual system in order to improve diagnostic and treatment modalities for soldiers exposed to blast-mediated traumatic brain injury (TBI).
Methods: Healthy adult C57/Bl6 mice were exposed to a blast wave (137 kPa) using a custom built blast chamber. Visual function and structure were evaluated using chromatic pupillography (cPLR), electroretinography (ERG) and spectral-domain optical coherence tomography (SD-OCT). Retina and brain histological analysis were performed to evaluate changes associated with blast injury.
Results: Assessment of the pupil light reflex using cPLR demonstrated decreased maximum pupil constriction diameter in blast injured mice (n=20) when using red light stimuli of 630 nm wavelength (Ctrl=1.12 ± 0.05 mm; TBI = 1.38 ± mm; p = 0.0063, Student’s t-test) or blue light stimuli of 480 nm wavelength (Ctrl = 0.56 ± 0.02 mm; TBI = 0.71 ± 0.02 mm; p = 0.0008) 24h after injury. Baseline pupil diameters before illumination were not significantly different between control and blast injured animals (Ctrl = 2.11 ± 0.05 mm; TBI = 2.11 ± 0.04 mm; p = 0.9914). Analysis of the scotopic maximum combined response revealed no significant difference between control animals (b-wave, 274.5 ± 6.31), and blast-injured animals (427.4 ± 45.60, p = 0.0694) 24h after injury. Three months following injury the significant thinning of the superior retinal nerve fiber layer was present in blast injured animals when compared to controls (Ctrl = 45+4µm, TBI = 32+1µm; p = 0.009). Histological analysis showed loss of retinal ganglion cells and recipient thalamic neurons.
Conclusions: Blast-induced traumatic brain injury is associated with severe functional and structural retina and optic nerve deficits in the experimental mouse model. Utilization of non-invasive functional (cPLR) and structural (OCT) diagnostic tests may be useful approach for early detection of visual system abnormalities in blast-injured subjects.
Support: Department of Veterans Affairs – Center for Prevention and Treatment of Vision Loss Iowa City
Replication of an Association Study of Pigment Dispersion Syndrome
R. J. Hazlewood1, B. R. Roos1, W. L. M Alward1, Y. H. Kwon1, E. M. Stone1,3, V. C. Sheffield2,3, T. E. Scheetz1, J. H. Fingert1
Departments of 1 Ophthalmology and Visual Sciences, and 2Pediatrics, The University of Iowa, Iowa City, IA; 3Howard Hughes Medical Institute
Purpose: To identify genetic risk factors for pigment dispersion syndrome (PDS) by conducting a two stage genome-wide association study (GWAS).
Methods: DNA samples from an initial cohort of 98 subjects with PDS and 200 ethnically matched controls were previously genotyped with 262,000 SNPs distributed across the genome in the first study. Here a second cohort of 100 PDS patients and controls will be genotyped at 500,000 SNPs using Affymetrix microarrays. Genotypes from individual SNPs will be analyzed using ANOVA. P-values will be adjusted for multiple measures using the Bonferroni correction. SNP data will also be analyzed to search for clusters of 4 or more neighboring SNPs (less than 200 Kbp apart) that all have p-values = 0.001. The results of genotyping the second cohort will be compared to the first cohort to search for associations that are replicated.
Results: Following Bonferroni correction for multiple measures, study of the first cohort identified 320 individual SNPs had p-values of = 0.05. Twelve clusters of SNPs with p-values = 0.001 were identified. No significant association was identified at a previously reported PDS locus (GPDS1). Genotyping of the second cohort is underway.
Conclusions: A genome-wide association study of a small cohort of PDS patients and controls identified twelve chromosomal loci that may contain risk alleles for PDS. Follow-up studies with a second cohort are underway to search for clusters associated with PDS in both cohorts. Studies of the second cohort of patients and controls may confirm associations identified with studies of the first cohort and facilitate identification of the specific DNA sequences that are the source of these potential PDS risk alleles.
Genetic Modifiers of Lyst-Mediated Ocular Disease
A. Hedberg-Buenz1, C. M. Trantow1, M.G. Anderson1,2
Departments of 1Physiology and Biophysics, and 2Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA
Purpose: Exfoliation syndrome (XFS) is a common age-related disorder involving pathological accumulations of a fibrillar exfoliative material in the anterior chamber of the eye, iris pigment dispersion (IPD), and a striking pattern of Marcel-like iris transillumination defects. In parallel with these anterior chamber abnormalities, intraocular pressure can become elevated and glaucoma often follows. Mice having the LystbgJ mutation on the C57BL/6J genetic background recapitulate many aspects of XFS and represent one of the only known mouse models of this disease.
Methods: Here, Lyst-mutant iris phenotypes were used to uncover genetic modifiers of Lystpathways. In a genetic background driven approach, a congenic strain of mice with the Lystbg-J mutation were created to examine iris phenotypes associated with Lystpathways in the context of the DBA/2J (D2) genetic background, which is known to exhibit IPD.
Results: LystbgJ-mediated iris phenotypes were enhanced in D2 mice, implicating genetic modifiers of Lyston this background. A mapping cross revealed that LystbgJ iris phenotypes were intensified in mice homozygous for the Tyrp1b mutation.
Conclusions: These results suggest Tyrp1 as a likely modifier of Lyst pathways. In addition, the change to the D2 genetic background also exposed late-onset neurodegenerative phenotypes involving cerebellar Purkinje cell degeneration and elevated levels of lipid hydroperoxides in iris. This suggests an association between oxidative damage to lipid membranes and severity of Lyst-mediated phenotypes, uncovering a new mechanism contributing to pathophysiology involving LYST.
Ocular Phenotype of Fibulin-2 Deficient Mice
J. Hernandez1, E. M. Stone1,2, R. F. Mullins1
1Department of Ophthalmology and Visual Sciences, The University of Iowa; 2Howard Hughes Medical Institute
Purpose: Fibulin-2 belongs to the fibulin family of extracellular matrix proteins. Fibulin-2 and fibulin-5 mutations have been associated with age-related macular degeneration (AMD), a leading cause of blindness in the elderly characterized by vascular changes and deposits beneath the retinal pigment epithelium (RPE). In this study we looked to determine the effects of a targeted Fbln2 mutation on mouse choriocapillaris and RPE.
Methods: Eyes from 4 Fbln2-/- mice and age and strain matched mice were taken at 2 time points: 12 months and 20 months. 90nm sections of the eyes were photographed, focusing on the choriocapillaris/RPE area of the eye. Extracellular matrix was characterized and vessel heights were measured.
Results: There was no significant difference in capillary height between 12 month Fbln2-/- and control mice. However, in 20 month mice, the capillary height of Fbln2-/- was found to be 41% lower than age-matched controls (p< 0.001). In addition, 20 month Fbln2-/- eyes were found to have subRPE deposits when compared to controls.
Conclusions: Fbln2-/- mice were found to have reduced choriocapillaris vascular density when compared to age-matched mice. Additionally, the Fbln2-/- eyes showed marked subRPE deposits resembling basal laminar deposits seen in human eyes with AMD. This suggests that the vasculature may play a role in the maintenance of the RPE, or the subRPE deposits may lead to vascular attenuation. Alternatively, events independent of vasculature and subRPE deposits may be causing both. These data may help elucidate the role of fibulin genes in AMD.
Immunological characterization of non-paraneoplastic auto-immune retinopathy (npAIR) and cancer associated retinopathy (CAR)
E. Hernandez-Merino1,2, S. Jacobson1,2, T. Lazic1,2, M.M. Harper1,2,H. Kecova1,2 S. Soltek3, V. Umansky3, S. Eichmüller3, R.H. Kardon2, S.D. Grozdanic1,2
1Veterinary Clinical Sciences Iowa State University, Ames, IA; 2 Veterans Administration Center for Prevention and Treatment of Visual Loss, Iowa City, IA; 3Deutsches Krebsforschungszentrum (DKFZ) and University Hospital Mannheim, Heidelberg, Germany
Purpose: To evaluate the immunological profile of npAIR and CAR in dogs with spontaneously occurring disease and in ret transgenic melanoma bearing mice.
Methods: Immunohistochemical analysis was used to detect CD3+ T-cells, CD79+ B-cells, and CD11b+ microglia in the retina of dogs with spontaneously occurring npAIR (n=8), CAR (n=3) and healthy control dogs (n=6). Additionally, Ret mice (n=6), healthy control mice (n=6) or healthy mice that received an adoptive transfer (AT) of Ret mouse serum (n=7) were analyzed. Diagnosis of npAIR or CAR in dogs was established based on the results of visual function testing, electroretinography, chromatic pupillography and retinal histology.
Results: All dogs with CAR (100%, 3/3) and npAIR (100%, 8/8) had a significantly higher presence of retinal CD3+ T-cells, compared to healthy controls (0%, 0/6), whereas CD79+ B-cells were observed in 37.5% of npAIR dogs (3/8) and none in dogs with CAR (0%, 0/3) and healthy control dogs (0%, 0/6). Activated microglia (CD11b+ cells with short thick bushy processes) were present in 37.5% of npAIR dogs (3/8) and 66% of CAR dogs (2/3) compared to 16% in healthy controls (1/6). Adoptive transfer of ret mouse serum to healthy mice induced npAIR, which was characterized by cystic changes in retinal pigment epithelium and destruction of photoreceptor outer segments. CD3+ T-cells were found in the retina of AT-npAIR mice (100% 7/7) and CAR mice (33% 2/6), but were absent in control mouse retinas (0% 0/6). CD79+ B-cells and CD11b+ microglia were present in the retinas of AT-npAIR mice (42% 3/7), and absent in the retinas of CAR (0%, 0/6) and control (0%, 0/6) mice.
Conclusions: Immunological characterization of spontaneously occurring npAIR and CAR in canine patients revealed cystic RPE changes and photoreceptor destruction coupled with CD3+T-cell, CD79+ B-cell and CD11b+ microglial infiltration and activation in the retina. Adoptive transfer of CAR serum to healthy mice induced npAIR in healthy mouse eyes, which resulted in a pathophysiology similar to dogs with npAIR and CAR. Utilization of adoptive transfer induction methods may provide a reliable, reproducible laboratory model for studying early changes in relatively rare syndromes (npAIR, CAR) that do not present clinically until a dramatic loss of vision is detected. Developing therapeutic strategies targeting different components of the immune system may be an effective approach for treatment of npAIR and CAR induced visual loss.
Voted Outstanding Poster Presentation
Decreased Retinal Function in Immunoproteasome-deficient Mice
S. A. Hussong1,2, R. J. Kapphahn1, H. Roehrich3, L. M. Maldonado1, M. T. Pardue4,5, D. A. Ferrington1,2
1Department of Ophthalmology, 2Graduate program in Biochemistry, Molecular Biology, and Biophysics 3Histology Core for Vision Research, University of Minnesota, Minneapolis, MN 4 Rehabilitation Research and Development Center, Atlanta VA Medical Center, Decatur, GA;5Department of Ophthalmology, Emory University, Atlanta, GA
Purpose: Immunoproteasome produces antigenic peptides for MHC class I presentation. In the immune-privileged retina, immunoproteasome is present in high concentrations under normal conditions and is upregulated with injury that does not involve inflammation in the immune-privileged retina (Ferrington et al., J Neurochem 2008). These data suggest immunoproteasome performs functions unrelated to its role in the immune system. To directly test this hypothesis, the current study examines retinal function and morphology in immunoproteasome-deficient mice.
Methods: Two strains of immunoproteasome-deficient mice lacking either one (lmp7-/- (L7)) or two (lmp7-/-mecl-1-/- (L7M1)) immunoproteasome subunits were compared with wild-type (WT) to investigate the potential function of immunoproteasome in the retina. Retinal function was assessed using electroretinography under dark-and light-adapted conditions. Rod and cone bipolar cell composition was determined by counting the cells stained with antibodies specific to bipolar cells (Chx10) and rod bipolar cells (PKCα). Relative abundance of retinal proteins was measured by Western and slot blot immunoassays.
Results: Both strains of immunoproteasome-deficient mice exhibited significant 20-25% decrease in ERG amplitudes in response to light (p<0.01). These functional defects in immunoproteasome-deficient mice were not due to changes in retinal morphology as no difference in photoreceptor or bipolar cell content was observed. However, in the KO mice had a 30-40% decrease of mGluR6 protein content.
Conclusions: Immunoproteasome is essential for maintaining normal retinal function but does not affect overall retinal morphology. These results suggest immunoproteasome may be involved in transmission of the visual signal through the retina.
The Influence of MicroRNAs on Nr2e3 Associated Retinal Disease in the rd7 Mouse Model
A.S. Jelcick1, J. Reinecke1, Y. Yuan1, N.B. Haider1
Department of 1Genetics, Cell Biology, and Anatomy, University of Nebraska Medical Ctr, Omaha, NE.
Purpose: The variability of expression genes can contribute to the observed phenotype both a positive and negative manner. Micro RNAs (miRNA) influence gene expression by negative regulation. The purpose of this study is to examine the effect of microRNAs on Nr2e3 associated retinal disease. Nr2e3 is a crucial factor for proper photoreceptor development and function. Lack of Nr2e3 in patients is associated with enhanced S-cone syndrome and retinitis pigmentosa and in a superfluous production of blue opsin expressing cone cells and a slow progressive retinal degeneration as observed in the rd7 mouse model. We examine the influence of miRNAs on Nr2e3 associated retinal degeneration by comprehensive gene expression profile in the developing and mature rd7 retina.
Methods: microRNA was collected from embryonic (E) 18, P2, P6, P14, and P30 B6 (control) and rd7 retinas. microRNA samples were hybridized to mirVana miRNA chips. Initial normalization and analysis of miRNA expression data was performed using BRB ArrayTools. Subsequent analysis to determine statistically significant miRNAs was performed using Stanford Array Tools SAM software. microRNA gene expression differences were confirmed by quantitative real time PCR.
Results: Initial filtering, normalization and organization of miRNAs expressed revealed 1981 miRNAs to be expressed across B6 and rd7 samples. A subset of over 200 miRNAs were entered into the SAM analysis software and statistically significant miRNAs found to be differentially expressed across time points were also evaluated. Of the genes confirmed to be differentially expressed, it was observed that the target genes belonged to multiple families with multiple functions.
Conclusions: Differential expression of microRNAs were observed in B6 versus rd7 retinas and thus may contribute to retinal degeneration in these mice. Further pathway analysis will shed additional light on the possible functions of the genes regulated by these miRNAs, but initial analysis shows their involvement in regulation of genes encoding f-box proteins, nuclear receptors, and genes encoding for various types of ECM related fibers.
Treatment of Laser-Induced Retinal Injury and Visual Loss Using Sustained Release of Intra-vitreal Neurotrophic Growth Factors
H. Kecova1, R. H. Kardon2,3, E. Lavik4, S. A. Park1, S. Jacobson1, K.Hamouche1, E. Alward1, M. M. Harper1,3, S. D. Grozdanic1,3
1Veterinary Clinical Sciences Iowa State University, Ames, IA; 2Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA; 3Veterans Administration Medical Center, Iowa City, IA; 4Case Western University - College of Engineering, Cleveland, OH
Purpose: To develop a novel therapeutic approach to laser retinal damage using intravitreal injection of microspheres with sustained release of glial cell-derived neurotrophic factor (GDNF).
Methods: Laboratory beagles (n=20) with experimentally induced laser retinal damage were divided into 3 groups: group 1 received intravitreal injection of GDNF-PLGA spheres (with degradation and release kinetics of 3 months) 1 hour after damage induction (GDNF1), group 2 received empty spheres (E2) and group 3 received laser damage but no additional treatment (LT3). Functional (pattern and ISCEV ERG) recordings were done at 14, 30, 90 and 180 days post laser exposure.
Results: Ninety days after laser exposure, significant decrease in scotopic a-wave amplitude was present in dogs which received empty microspheres (mean ± SEM: 86.51 ± 3.97µV, p=0.0044, unpaired t-test) and dogs which received laser damage only (56.95 ± 3.77µV, p<0.0001), but not in GDNF treated dogs (113.8 ± 11.03µV, p=0.5041) when compared to healthy controls (123.3 ± 8.49µV). However, 180d post laser injury, a-wave amplitude significantly decreased in GDNF-treated group (48.4 ± 7.27µV, p<0.0001) when compared to control values and was not significantly different when compared to values in dogs, which received empty spheres (45.63±4.39µV, p=0.9455) or dogs with laser damage only (42.75±5.58µV, p=0.671). Almost identical trend of changes has been observed with inner retina function (b-wave amplitudes and oscillatory potentials).
Conclusions: Intravitreal injection of microspheres with sustained release of GDNF protected retinal function in laser-damaged eyes for 3 months. At six months post laser injury, when GDNF was no longer present in the eyes, functional deficits continued to progress, which is suggestive of progressive loss of retinal function even months after initial laser damage in an environment depleted of neurotrophic support.
Voted Outstanding Oral Presentation
Identifying QTL Contributing to Murine Central Corneal Thickness
D. Koehn1, G. D. Lively1, A. Hedberg-Buenz1, K. Wang2, M. G. Anderson1,3
Departments of 1Molecular Physiology and Biophysics, 2Biostatistics, 3Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, Iowa
Purpose: Central corneal thickness (CCT) varies widely in humans and is an independent risk factor for primary open angle glaucoma (POAG). Although there is an established association between CCT and POAG, little is known about the biology of this striking health disparity. Our previous work demonstrated that CCT also varies in inbred mouse strains. QTL analysis of an intercross of mice with thin [C57BLKS/J (KS)] and thick [SJL/J (SJL)] corneas identified a significant locus on chromosome 7 (Cctq1), spanning a 20 Mb region. Here, we further examine Cctq1 using congenic mice. We also show evidence for two additional QTL which contribute to CCT through their interaction.
Methods: For creation of the low-generation congenic KS.Cctq1SJL strain, KS mice were crossed to SJL to produce F1s. Progeny carrying the SJL alleles at Cctq1 were then backcrossed to KS for four generations. N4 mice were intercrossed and F2 offspring were measured for CCT using ultrasound pachymetry (at 100-120 days). To identify interacting QTL with the intercross between KS and SJL mice, we performed the scantwo function of R/qtl using the same SNP genotype data that identified Cctq1. Significant interactions were tested through stratified permutation analysis.
Results: KS.Cctq1SJL mice heterozygous for the congenic alleles (i.e., KS/SJL) had an average CCT value of 100.0 +/- 3.9 microns (mean +/- SD; n = 24 mice). This is significantly higher than inbred KS mice (P<0.05, Student’s t-test). From the two-dimensional QTL analysis, interacting loci were found on chromosomes 11 and 17 (full LOD score = 9.2; P=0.10; suggestive significance).
Conclusions: These results confirm the presence of a QTL on chromosome 7 that contributes to CCT and identify two new interactive QTL. Future efforts will identify the specific CCT gene in the Cctq1 region and the specific interactive genes on chromosomes 11 and 17. We will also extend the QTL approach to identify additional CCT-regulating genes and test roles of these genes in glaucoma.
A New Mouse Model of Early Onset Glaucoma Caused by Sh3pxd2b
M. Mao1, M.G. Anderson1,2
1Department of Molecular Physiology and Biophysics; 2Department of Ophthalmology and Visual Sciences; The University of Iowa, Iowa City, Iowa
Purpose: A spontaneously derived mouse mutant, nee, which contains a 1 bp deletion in Sh3pxd2b, was recently shown to exhibit ocular defects suggestive of glaucoma. Here, we present a detailed phenotypic characterization of nee mutants and expression analysis of SH3PXD2B to closely examine the potential contributions of Sh3pxd2b to glaucoma pathology.
Methods: neemutants at different ages and their age matched wild-type littermates were compared using slit-lamp examination, intraocular pressure (IOP) measurements, and histology. Ocular expression of Sh3pxd2b was analyzed with western blot and immunofluorescent labeling.
Results: In the anterior segment, slit-lamp examination showed that nee homozygotes exhibit peripheral anterior synechia at P17. Increased anterior chamber depth and elevated IOP was observed in mice over one month of age. In the posterior segment, P17 neehomozygotes initially exhibit a normal retina and optic nerve. By 3 months of age, neehomozygotes had a severe degeneration of the ganglion cell layer and optic nerve. In addition, nee mutants rapidly developed optic nerve head excavation. To further determine the mechanisms through which SH3PXD2B might contribute to glaucoma, we examined its expression in human tissue, finding that SH3PXD2B is wildly expressed in the eye, including the trabecular meshwork and retina.
Conclusions: We have identified a new mouse model which exhibits phenotypes resembling a form of early onset glaucoma. neemutant mice represent a valuable resource for mechanistic studies of glaucoma that does not require expensive aging of mice. SH3PXD2B is also suggested to be a candidate potentially contributing to human glaucoma.
Functional Retinal Ganglion Cell PERG Deficits in an Acute and Chronic Mouse Model of Parkinson’s Disease
K. Mohan1,2, M.M. Harper1,2, K. Schipull1, A. Gosh2, 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.
Using Information Theory to Quantify Functional Capacity in Retinal Disease
E. L. Nylen1, S. F. Stasheff1,2
Departments of 1Biomedical Engineering, 2Pediatrics (Neurology), University of Iowa, Iowa City, IA
Purpose: Information theoretical methods have provided a useful means of quantifying characteristics of communication in neural systems. We use such methods to describe how inherited retinal disease influences the message the brain receives from the eye.
Methods: Extracellular action potentials were recorded simultaneously from 30-90 retinal ganglion cells in the in vitro retina of 14 day old wild type (wt) and rd1 mice, using a multi-electrode array. Spontaneous activity was monitored and full field light flashes were presented over a range of illuminance values. The mutual information between stimuli and responses was calculated for each recorded cell, and background spontaneous activity was quantified in terms of spike count, rate, and Shannon entropy.
Results: The entropy of all spike trains in rd1 cells (N=99) was 15.6 bits, and in wt cells (N=79) 16.5 bits. This measure of entropy estimates the overall cellular signaling capacity for a large group of cells. The input-output fidelity of these cells can be characterized by isolating each cell's unique responses to stimuli, described as the mutual information between stimulus and response. The wt cells showed a mean mutual information of 5.4 bits per cell, while the rd1 displayed a significantly less mean mutual information of 2.1 bits per cell (Mann-Whitney U Test, p <0.001).
Conclusions: In our study of the rd1 mouse, an animal model of retinitis pigmentosa, we quantitatively show that an increase in spontaneous firing activity of retinal ganglion cells may affect these cells' capacity to send distinguishable messages. At postnatal day 14 though, the spontaneous activity observed in rd1 does not completely mask the retina's response to light stimulation. Our findings suggest that, despite aggressive photoreceptor degeneration and an increase in noise, retinal ganglion cells' signal capacity may be sufficient for administration of successful visual restoration therapies in early stages of disease.
Voted Outstanding Poster Presentation
Early Functional and Structural Optic Nerve Deficits in a Canine Model of Compressive Optic Neuropathy
R. N. Nzokwe1,3, H. Kecova1,3, R. H. Kardon2,3, S. A. Park1,3, K. Hamouche1,3, S. Jacobson1,3, E. Alward1,3, M. M. Harper1,3, S.D. Grozdanic1,3
1Veterinary Clinical Sciences, Iowa State University, Ames, IA; 2Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA; 3Center for Prevention and Treatment of Vision Loss-Veterans Administration Medical Center, Iowa City, IA.
Purpose: To evaluate functional and structural optic nerve deficits in a canine model of compressive optic neuropathy (CON).
Methods: CON was induced in healthy laboratory beagles (n=10) by implanting an expandable silicone implant into the orbital space. Controlled optic nerve compression was achieved by inflation of the implant with saline injection. Fluorescein angiography was performed to demonstrate perfusion of the optic nerve and retinal vasculature. Thickness of the retinal nerve fiber layer (RNFL) was evaluated using optical coherence tomography (Spectralis SD-OCT system), while pattern ERG (pERG) was recorded to evaluate retinal ganglion cell (RGC) function.
Results: Despite relatively normal optic nerve perfusion observed by fluorescein angiography, orbital implant inflation resulted in significant pERG deficits only 2 minutes after induction of optic nerve compression (0.36+0.13 µV; mean+SEM; p<0.0001, Student’s t-test), when compared to control values (6.2+0.35 µV). OCT linear scan analysis showed significant increase in the RNFL thickness of area centralis (macula) region in CON dogs when compared to control dogs (p<0.0001). Peripapillary circular scan analysis showed significantly decreased RNFL thickness in CON dogs when compared to controls (p=0.009). Linear correlation analysis showed statistically significant negative correlation between macular NFL thickness and pERG amplitudes (r2= 0.43, p=0.03), while there was no statistically significant relationship between peripapillary RNFL thickness and pERG amplitudes.
Conclusions: Experimental optic nerve compression resulted in macular RNFL thickening and correlated with pERG deficits. Inclusion of blood vessel profile in the peripapillary RNFL analysis seems to decrease detection sensitivity and specificity for RNFL changes associated with compressive optic nerve injury in the dog model.
Support: Veterans Administration Rehabilitation Division Merit Grant and Iowa City VA Center for the Prevention and Treatment of Vision Loss
The BALB/cByJ Tyrosinase Allele is Linked to Resistance to Vaso-Obliteration in the Oxygen-Induced Retinopathy Model
B.E.H. O’Bryhim1,2, H. Niu2, J. Radel2, R.C.A. Symons1,3
Departments of 1Molecular and Integrative Physiology, 2Occupational Therapy Education, and 3Ophthalmology, University of Kansas Medical Center, Kansas City, KS
Purpose: Retinopathy of prematurity (ROP) is a common cause of blindness in children. The development of ROP is known to be influenced by gestational age at delivery, birth weight, and over-use of oxygen in the nursery. It is unknown whether genetic factors influence ROP severity. This study aims to use mouse genetics to identify genes which play a role in the development of oxygen-induced retinopathy (OIR) model of ROP.
Methods: A mapping cross was established between BALB/cByJ and C57BL/6ByJ, which are, respectively, relatively resistant and susceptible to central retinal vaso-obliteration in the OIR model. 217 neonatal F2 mice were subjected to 120 hours of 75% inspired O2, beginning on day 7 of neonatal life. 96 hours after return to room air, mice were anesthetized, perfused with fluorescein-isothiocyanate (FITC)-labeled dextran and enucleated. Retinas were excised and flat-mounted, and the avascular area was measured using ImageJ software.
Results: Susceptibility to the development of ROP was not shown to be associated with sex (p>0.05), but was shown to correlate with weight at P16 (p<0.0001) and with non-albino versus albino coat color (p<0.0001).
Conclusions: In this cross, mouse weight and allelotype at the tyrosinase locus were associated with susceptibility to vaso-obliteration. However, these factors explain only a small proportion of the total variation, and it is expected that whole genome analysis will lead to the discovery of further loci.
Molecular Characterization of a Feline Model of Congenital Glaucoma
A. Pfleging1, G. J. McLellan2, N. .M. Ellinwood3, M. Menotti-Raymond4, M. H. Kuehn1
1Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, IA; 2Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health; 3Department of Animal Science, Iowa State University, Ames, IA; 4 Laboratory of Genomic Diversity, National Cancer Institute, Frederick, MD
Purpose: We recently identified a novel, spontaneously occurring animal model of congenital glaucoma in a pedigree of Siamese cats. These cats were found to have bilateral moderate buphthalmos and elevated intraocular pressure (IOP). Mean IOP is 30.6 mmHg, significantly above the normal feline IOP of 19 mmHg. Elevated pressure and bupthalmos is obvious in affected kittens by 3 weeks of age. Here we examine the ocular morphology of affected cats and conduct a screen to detect the genetic defect leading to the observed phenotype.
Methods: Histological examinations of the retina, optic nerve head, and anterior segment of affected and unaffected animals were conducted using frozen tissue sections. The sequence of several candidate genes were determined by direct DNA sequencing and candidate genetic loci were evaluated for linkage using nucleotide repeat markers.
Results: Post-natal development of the structures of the ciliary cleft appears to be arrested in these animals. The uveal trabecular meshwork and angular aqueous plexus remains rudimentary in affected animals and many outflow channels appear collapsed. The retina and the optic nerve head appear unremarkable in young affected animals. However, older affected cats frequently present with pronounced optic nerve head cupping and retinal degeneration. In a genetic screen STR markers on chromosome B3 yielded a LOD score of 18.08 with zero recombination. Sequence analysis of the LTBP2 gene, which is located in this locus, demonstrated that affected cats contain a 4-bp insertion disrupting the open reading frame and leading to premature truncation of the gene product.
Conclusions: Our data suggest that mutations in LTBP2 lead to the development of congenital glaucoma in cats. Data from several other laboratories have suggested that mutations in LTBP2 are also responsible for congenital glaucoma in several Middle Eastern populations. Thus these animals represent a model for human disease and may aid in the development of treatment modalities or the elucidation of biochemical pathways involved in the morphogenesis of the anterior segment of the eye.
Functional analysis of the BBS3 and BBS3L A89V mutation in vision
P.R. Pretorius1,2, M.A. Aldahmesh6, F.S. Alkuraya 6,7,8, E. M. Stone2,4, V.C. Sheffield33,4, D.C. Slusarski5
The University of Iowa 1Interdisciplinary Program in Genetics, 2Ophthalmology and Visual Sciences, 3Pediatrics, 5Biology, 4 Howard Hughes Medical Institute; 6Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia; 7Department of Pediatrics, King Khalid University Hospital and College of Medicine, King Saud University, Riyadh, Saudi Arabia; 8Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia
Purpose: We are studying the mechanism underlying a heterogeneous syndromic form of retinal degeneration, Bardet-Biedl Syndrome (BBS). We have previously identified a second longer transcript of BBS3, BBS3L, and have demonstrated that BBS3L is required for retinal organization and function in both the zebrafish and mouse. Homozygosity mapping of a consanguineous family from Saudi Arabia identified a missense mutation (A89V) in BBS3 that results in non-syndromic retinitis pigmentosa (RP). The functional relationship of this mutation between non-syndromig RP and BBS remains to be elucidated.
Methods: To evaluate the functional role of the BBS3 A89V in non-syndromic RP and BBS, rescue experiments were performed in the zebrafish by co-injecting bbs3 antisense oligonucleotides (morpholinos) with either human BBS3 A89V or BBS3L A89V RNA. Two previously described phenotypes of bbs3 knockdown in the zebrafish were assessed: the time of epinephrine induced melanosome transport and visual function.
Results: We have demonstrated that BBS3L RNA is sufficient to rescue the vision defect observed with loss of bbs3 in zebrafish. In contrast, the BBS3L A89V RNA is not sufficient to rescue the vision defect; however, BBS3 A89V RNA is able to suppress the cardinal BBS phenotype of melanosome transport, similar to wild-type BBS3 RNA.
Conclusions: These data provide strong functional evidence that the BBS3 A89V mutation identified in patients with non-syndromic RP is critical and specific for the vision defect as the mutant transcript is able to suppress the cardinal BBS phenotype of melanosome transport in zebrafish.
Using Genomic Tools to Identify Causative Genes for Congenital Eye Anomalies
G. Raca1,2, C. Jackson1, B. Warman3, L. A. Schimmenti3
1UW Cytogenetic Services, State Laboratory of Hygiene, Madison, Wisconsin; 2Department of Pathology, University of Wisconsin-Madison; 3Department of Pediatrics and Ophthalmology, Institute of Human Genetics and Developmental Biology Center, University of Minnesota
Purpose: To discover additional genetic causes of three related ocular birth defects - anophthalmia, microphthalmia and coloboma - by using advanced genomic technologies of high resolution array comparative genomic hybridization (aCGH) and massively parallel next generation sequencing (NGS).
Methods: Patients with anophthalmia, microphthalmia and coloboma are tested for deletions and duplications affecting genes involved in eye development, using 2 million probe (HD2) arrays from NimbleGen. To detect point mutations in candidate genes for eye anomalies, we designed a custom NimbleGen sequence capture array with 112 selected genes. The array was validated in combination with the GS FLX sequencing by testing whether these methods could detect two known mutations in the renal-coloboma (Papillorenal) syndrome associated gene PAX2.
Results: aCGH testing of 31 patients with congenital eye malformations detected one case with a deletion of the PAX2 gene. Non-polymorphic copy number changes were also observed at several candidate chromosomal regions, including 13q31.3, 8q23.1q23.2, 7q34 and 20q13.13. Our preliminary studies using sequence capture and GS FLX sequencing successfully detected two known mutations in the PAX2 gene.
Conclusions: Our study showed that aCGH could detect deletions and duplications associated with ocular birth defects, but that copy number abnormalities were not a common cause of isolated anophthalmia, microphthalmia and coloboma.
Glia Maturation Factor Gamma Expression in Glaucoma
T. Risma1, A. Cook1, Q. Ding1, G. Zode2, S. D. Grozdanic3, M. H. Kuehn1
Departments of 1Ophthalmology and Visual Sciences and 2Pediatrics, The University of Iowa; 3Iowa State University Department of Veterinary Clinical Sciences, Ames, IA
Purpose: : Glaucoma is one of the leading causes of glaucoma worldwide. This study aims to evaluate the retinal expression of glia maturation factor gamma (GMFG) and its possible link to glaucoma pathogenesis. Recent studies have suggested that GMFG may play a role in a range of possible functions including hematopoiesis, heat shock response, and phagocytosis in antigen presenting cells. Importantly, preliminary studies have suggested that GMFG is expressed at elevated levels in the retina of glaucoma patients.
Methods: Expression levels for GMFG were evaluated in the retina of dogs with and without glaucoma by gene array expression analysis and RT-PCR. The distribution of GMFG was further evaluated in human donor retinas with primary open angle glaucoma (n=6), and controls with no history of ocular pathology (n=6), aged 72-89. Tissue was sectioned to 7µm thickness, blocked with BSA to prevent nonspecific binding of antibodies and incubated with rabbit GMFG antibody (Abgent). Secondary controls received no primary antibody to assure the absence of nonspecific antibody labeling. Images were obtained using Olympus BX41 fluorescent microscope. Synthesis of GMFG in the human retina was further evaluated by Western blot analysis.
Results: RT-PCR analysis demonstrated that expression levels of GMFG are significantly elevated in the glaucomatous retina. While expression levels in control retinas were uniformly low, considerable differences in expression level were apparent within the group of glaucoma samples. Similarly, immunohistochemical analysis of glaucomatous human donor tissue showed variable GMFG fluorescent signal intensity. GMFG antibody signal was localized to the nerve fiber layer of the retina in all samples which displayed fluorescence. All healthy and control retinas showed little to no staining with GMFG antibody.
Conclusions: Our data demonstrate that elevated GMFG expression occurs both in human and canine glaucomatous retinas and localizes within the nerve fiber layer and basal lamina of the retina. The strong variability of GMFG expression levels between glaucoma samples is intriguing and may be related to the disease state of the donor or perhaps to donor’s genetic background. The molecular function of GMFG remains largely unknown, but recent studies have suggested possible immunological functions for GMFG. Immunological factors have also become increasingly implicated in the pathogenesis of glaucoma. Our results suggest that GMFG may play an important role in some glaucoma patients.
USP2, a De-ubiquitinating Enzyme, is a new component of the molecular clock
H. Scoma1, M. Humby1, S. Thompson2, Q. Zhang1, J. Fogerty1, A. Lewis1, J. C. Besharse1
1Department of Cell Biology, Neurobiology and Anatomy Medical College of Wisconsin, Milwaukee, Wisconsin; 2Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, IA
Abstract: Daily entrainment to environmental light dark cycles involves appropriate expression of several clock components. We have identified a highly circadian de-ubiquitinating enzyme, USP2, involved in entrainment following day lengthening. Early evening dim light exposure produces enhanced phase delays in mice lacking USP2 (USP2-/-). USP2 is found in complexes containing several clock components. BMAL1 abundance, stability, and ubiquitination-state are altered by USP2 expression. This identifies USP2 as a new component of the molecular clock machinery directly regulating BMAL1 stability. However, the simple regulation of BMAL1 stability does not readily explain the entrainment phenotype of USP2-/- mice. One possibility is that loss of USP2 could enhance BMAL1 transcriptional activity; BMAL1 transcriptional activation is closely coupled to its ubiquitination. We have found that PER1 stability is not directly regulated by USP2 and that loss of Per1 does not enhance sensitivity to early evening light. Nonetheless, altered expression of CLOCK/BMAL1 controlled genes, like PER1, during early evening dim light exposure may account for the increased sensitivity observed in USP2-/- mice. On the other hand, USP2 may function upstream of the SCN during light resetting by targeting other circadian clock components or other proteins within the retinohypothalamic tract (RHT). We are currently investigating a potential role for USP2 in retina or RHT following light exposure.
Distinct Patterns of Hyperactivity Characterize Varied Forms of Retinal Degeneration
S. F. Stasheff1
1Department of Pediatrics (Neurology), University of Iowa, Iowa City, IA.
Purpose: Increased levels of spontaneous activity have been demonstrated among ganglion cells in several animal models of hereditary retinal degeneration with primary deficits in the phototransduction cascade. We now report that such hyperactivity occurs in genetic mouse models of widely varied human retinal and more generalized neurologic degenerative diseases. Furthermore, distinctive spatiotemporal patterns of this activity characterize underlying cellular dysfunction as diverse as defects in retinoid recycling (RPE65), phototransduction (Pde6-beta & rhodopsin), ciliary transport (CEP290), lysosomal function (cln3), and regulation of cell growth Tsc-2.
Methods: Using a multielectrode array, extracellular action potentials were recorded simultaneously from 30-90 retinal ganglion cells in the in vitro retina of young adult mice with mutations in Rpe65, rod phosphodiesterase (Pde-6b), Cep290, cln3, or Tsc-2. Various spatiotemporal parameters of this spontaneous activity were compared quantitatively among the strains, including average firing rate, periodicity, power spectral density, and correlation index.
Results: Among this variety of neurodegenerative disease models, elevated spontaneous activity was a common feature. However, distinctive patterns of this activity included: constant but supranormal firing rates in Rpe-65, rd1 and rd10, modestly elevated rates in cln3, periodic sudden elevations in firing rate in Tsc-2, and extremely slowly migrating waves of correlated activity in Cep-290 mice.
Conclusions: Spontaneous hyperactivity among retinal ganglion cells is a hallmark feature of a variety of fundamental causes of retinal and neurologic dysfunction; distinctive patterns of this activity are associated with particular underlying etiologies. We speculate that such patterns not only identify specific disorders, but represent unique distortions of the neural code of vision that is used by the retina to communicate with the brain. We predict that these specific firing patterns will cause distinctive patterns of reorganization in retinal projections to downstream visual system targets in the central nervous system, and consequently distinguishable disruptions of normal vision.
Voted Outstanding Oral Presentation
Light-aversion in mice originates with irradiance detection mechanisms that are independent of spatial-vision
S. Thompson1, A. Recober2, T. W. Vogel3, A. Kuburas4, V. C. Sheffield5,6, A. F. Russo4, E. M. Stone1,6
Departments of 1Ophthalmology and Visual Sciences, 2Neurology, 3Neurosurgery, 4Physiology, 5Pediatrics, The University of Iowa; 6Howard Hughes Medical Institute
Purpose: Detection of light in the eye takes two distinct forms: spatially mapped detection of light for vision, and detection of irradiance (gross brightness) to regulate adaptive responses in behavior and physiology. The goal of this study was to identify the photo-sensory basis for light-aversion behavior in mice.
Methods: Comparison of wild-type, Rpe65-/- and rd1 mice allowed distinction between effects of vision and irradiance detection on behavior. Rpe65-/- have functional spatial-vision but severely attenuated irradiance detection, and rd1 mice have no spatial-vision and an intermediate reduction in irradiance detection. A suppression of running wheel activity known to originate with irradiance detection is termed negative masking.
Results: Negative masking sensitivity was increased in rd1 as previously described, but was reduced Rpe65-/- mice - the first example of a loss of sensitivity phenotype for this response. By contrast, light-aversion (preference for the dark) was present in wild-type but abolished in both Rpe65-/- and rd1.
Conclusions: This shows that light-aversion is dependent on irradiance detection but is independent of spatial vision. It is also distinct from the negative masking response. Therefore, multiple irradiance detection mechanisms acutely modify behavior.
Exploring Ganglion Cell Development at Single Cell Resolution
1Department of Genetics, Development and Cell Biology, Iowa State University
Purpose: Previous attempts to uncover the gene expression networks responsible for the generation of retinal ganglion cells (RGCs) relied on whole tissue strategies that obscure the genetic programs present in a small number of cells. This is especially true for RGCs that represent a small proportion of the total cells in the retina. Using a single-cell profiling approach, I have begun to characterize the transcriptional programs of individual RGCs at various developmental stages.
Methods: Single retinal cells were isolated from dissociated retinas at multiple early developmental stages (E12.5 through P0). The cDNAs resulting from individual retinal cells were hybridized to mouse Affymetrix arrays. Almost 200 single retinal cells were profiled in this manner. Different in situ hybridization techniques have been used to validate the array data.
Results: Taking advantage of the Math5-LacZ knock-in mouse, individual cells transitioning from a cycling progenitor cell to an RGC were isolated and profiled. Gene clusters were identified that correlate specifically with these transitioning cells, including several transcription factors whose function in the developing retina is unknown. In addition to the identification of marker genes for newborn RGCs, these single-cell profiles have yielded insight into the gene networks expressed during the initial stages of RGC development.
Conclusions: Single-cell gene expression profiling has revealed considerable heterogeneity among cycling retinal progenitor cells and developing RGCs. Many genes have been found in subsets of retinal cells including several transcription factors. These microarray experiments have yielded both important new markers of developing RGCs and new insights into retinal progenitor cell biology.
Mouse Model of MYOC Glaucoma
G. S. Zode1, K. Bugge1, C. Searby1, V. C. Sheffield1,3, E. M. Stone2,3, M. H. Kuehn2
Departments of 1Pediatrics and 2Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, Iowa City, IA; 3Howard Hughes Medical Institute
Purpose: Myocilin mutations have been associated with congenital glaucoma and 2–4% of primary open angle glaucoma (POAG) cases, but the pathogenic mechanisms remain largely unknown. The objective of this study was to examine the glaucoma phenotype of the Tyr437His mutant of human myocilin in transgenic (Tg) mice.
Methods: Tg mice were generated by expressing the Tyr437His mutant form of human myocilin under the control of the CMV promoter. Myocilin expression in the anterior segment of Tg mice was examined by RT-PCR, immunohistochemistry and western blot analysis. Intraocular pressure (IOP) was measured using a rebound tonometer. Whole mount retina and PPD staining of optic nerve was used to examine retinal ganglion cells (RGC) loss and axonal damage respectively. Western blot analysis and immunostaining of anterior segment was performed to examine the unfolded protein response (UPR) in the trabecular meshwork (TM) of Tg mice.
Results: Tg mice expressed higher levels of myocilin in the irodocorneal angle than wild type (wt) littermates. Starting at 3-months age, Tg mice exhibited significantly higher intraocular pressure and demonstrated progressive loss of RGCs loss and axonal damage. Interestingly, expression of mutant myocilin in the trabecular meshwork of Tg mice induced ER stress and activated UPR, detected by induction of GRP78, GRP94, IRE α, and spliced XBP-1. ER stress in the Tg mice switched from UPR to pro-apoptotic response as demonstrated by upregulation of chop and processing of pro-caspase-12 in Tg mice. In addition, UPR activation by anterior chamber injection of tunicamycin significantly elevated IOP in control mice.
Conclusions: Our observations indicate that Tg mice exhibit pathological changes similar to those in glaucoma patients. In addition, expression of Tyr437His mutant of human myocilin in the TM leads to ER stress and activation of UPR. The sustained UPR may compromise TM function leading to outflow deficiency in glaucoma patients with myocilin mutation.
For questions or comments please contact our Webmaster