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2008 Focus on Fellows Abstracts


Recovery of RPE65 mutations: Studies of low activity mutants

Acitores Alicia, Susan Gentleman, Eugenia Poliakov, Preethi Chander and T. Michael Redmond. Laboratory of Retinal Cell and Molecular Biology. Molecular Mechanisms Section.

RPE65 is the isomerase in the visual cycle, yielding 11-cis-retinol from all-trans retinyl esters. Mutations in the gene for RPE65 are associated with a variety of retinal dystrophies such as Leber congenital amaurosis. Over 30 missense mutations are known to cause dystrophies. Many of these mutations retain residual activity and produce lower amount of protein because they are unstable and/or misfolded.

ER-associated degradation (ERAD) is the machinery involved in elimination of unfolded or misfolded proteins for degradation by the proteasome. VCP (p97, valosin containing protein) and gp78, are two proteins that participate in the ERAD machinery. We are investigating whether blocking VCP protein allows rescue of these missense mutant proteins. We are using two approaches, first VCP-specific interference RNA with ShRNA mir to block VCP expression. Second, we use lactacystin, a specific proteasome inhibitor.

In this study we are using a heterologous expression model of the visual cycle in FreeStyle 293-F cells. Cells transfected with wildtype or mutant RPE65 plasmids are either cotransfected with ShRNA mir vector or control vector, or treated with lactacystin (10 mM). 24 to 72 hours after transfection, all-trans retinol is added to 2.5 M and the cells are grown for a further 6 hours and harvested. Protein expression is monitored by Western blot with specific antibodies. Retinoids were extracted and analysed by HPLC to measure RPE65 enzymatic activity.

Treatment with ShRNA increased RPE65 protein expression measured by Western blot but no increase in enzymatic activity was apparent. Further work is needed to establish conditions for rescue of low activity mutants.

Association of RAGE and HTRA1 gene polymorphisms with diabetic retinopathy in type 2 diabetes

Balasubbu Suganthalakshmi, Sundaresan P, Kim R, Anand R, Namperumalsamy P, Hejtmancik JF, Ophthalmic Genetics and Visual Function Branch

Diabetic retinopathy (DR) is the prevalent cause of visual morbidity worldwide. This microvascular complication of the retina due to hyperglycemia, displays a remarkable complexity in its pathogenesis due to the interplay of genetic, environmental and biochemical factors, resulting in mild non-proliferative DR or severe sight threatening proliferative DR. Genetic factors have been reported to influence the pathogenicity of DR in various ethnic groups worldwide. However, detailed investigations pertaining to Indian population is limited. Hence, this study proposes to investigate the association of various candidate genes with the development of DR in South Indian cohort. Here, we addressed the association of RAGE and HTRA1 gene ploymorphisms with DR. Five SNPs were genotyped in 211 DR and 237 diabetic patients without retinopathy (DNR). The results indicate that SNP rs2070600 in RAGE (P-0.003) and SNP rs11200638 in HTRA1 (P-0.03) showed significant association with DR, when compared with DNR. In order to further confirm the association of these two SNPs, sample size was increased in additional 134 DR (Total n=345) and 122 DNR (Total n=359). The SNP rs2070600 again showed significant association with DR (P-0.008), whereas the SNP rs11200638 did not show association with DR. Therefore, our findings suggest that RAGE gene polymorphism plays a vital role in developing DR.

Genetic screen for a novel autosomal dominant retinitis pigmentosa (ADRP) gene

Friedman, J.S., M. Brooks11, R. Khanna, E.H. Trager, K.E. Branham, V. Ponjavic, L. Gränse, G.R. Abecasis, S. Andréasson, A. Swaroop1, Neurobiology-Neurodegeneration and Repair Laboratory; Department of Ophthalmology, University of Michigan, Ann Arbor MI; Department of Ophthalmology, Lund University Hospital, Lund, Sweden; Center for Statistical Genetics, Department of Biostatistics, University of Michigan, Ann Arbor, MI

Retinitis Pigmentosa (RP) constitutes a group of genetic eye diseases in which the photoreceptor cells in the retina degenerate, leading to irreversible vision loss. To date, 17 genes have been identified to cause autosomal dominant retinitis pigmentosa (RetNet). We are studying a large Scandinavian family with a slower than average progression rate and apparent autosomal dominant pattern of inheritance. We performed linkage analysis using the data obtained by hybridiziation of affected individual DNAs to 250K gene chip SNP arrays from Affymetrix. SNP data was examined using standard methods (Pedstats, GRR etc), and linkage was performed using Merlin. We identified a peak lod score of 5.0 on chromosome 7p15. No mutations were identified in a previously-reported RP9 gene, Pim-1 kinase associated protein, at 7p14. Twelve genes within the critical region are being screened for mutations in the Scandinavian family. Candidate alterations will be tested against ethnically matched and North American controls. The putative disease causing gene will then be screened using DNA from a panel of retinopathy patients from North America. The identification of this ADRP gene will assist in the better understanding of the mechanisms underlying RP and could lead to targeted therapies to alleviate this disease.

7-Ketocholesterol is present in oxidized lipid deposits in the primate retina: potential implication in the induction of VEGF and CNV formation

Larrayoz, Ignacio M. E.F. Moreira, J.W. Lee, I.R. Rodriguez, Laboratory of Retinal Cell and Molecular Biology, Mechanisms of Retinal Diseases Section.

In this study we have identified and localized 7-ketocholesterol (7kCh) in the primate retina and examined the potential consequences of its presence in those specific areas of the retina.

7-Ketocholesterol is a highly toxic oxysterol found in abundance in atherosclerotic plaques and believed to play a critical role in atherosclerosis.

The localization of 7-Ketocholesterol was mainly to deposits in the choriocapillaris and Bruch’s membrane and on the surface of vascular endothelial cells of the neural retina. The RPE/choriocapillaris regions contained approximately 4 times more 7kCh than the neural retina. In ARPE19 cells and HMVECs oxidized LDL and 7kCh induced VEGF 8-10 fold above controls. Vascular endothelial growth factor (VEGF) is best known for its role in promoting angiogenesis, maintaining vascular permeability, promoting hematopoietic cell development and wound healing. VEGF expression is known to be regulated by hypoxia, inflammation-related events and transcription factors such as Sp1.

We found that HIF-1a levels were not increased as a result of 7kCh treatment. On the other hand, cholesterol sulfate, a liver X receptors (LXRs) antagonist had a marked attenuation of the 7-ketocholesterol-mediated VEGF induction. Similar results were obtained when we used LXR-specific siRNAs.

The location of 7-ketocholesterol in the retina and its induction of VEGF in cultured RPE and HMVEC cells suggest it may play a critical role in choroidal neovascularization which is a key event in aging diseases like age-related macular degeneration. The pathway for VEGF induction seems to be independent of HIF-1a but partially regulated by LXRs.

STAT1 inhibits ocular inflammatory disease by inducing dendritic cells (DCs) and retinal microglial cells to produce IL-27

Lee Yun Sang, Cheng-Rong Yu, Ahjoku Amadi-Obi and Charles E. EgwuaguMolecular Immunology Section, Laboratory of Immunology

Previously, we implicated Th17 cells in etiology of human uveitis and showed that IL-2 promotes their expansion. We also showed that IFN-γ inhibits Th17 proliferation by inducing retinal cells to produce IL-27 that in turn inhibits IL-2 production through SOCS3-dependent mechanism. However, recent reports propose that role of IL-27 in suppressing CNS inflammation is indirect; acting by converting effector T-cells into IL-10-producing regulatory T-cells through STAT3-dependent mechanism. Here we have used STAT1-deficient mice to examine whether suppression of ocular inflammation is mediated by IL-10- producing T cells or by IL-27/STAT1-dependent mechanisms. Despite the fact that STAT1 knockout mice develop more severe EAU, they contain similar amount of IL-10 secreting T-cells as WT mice suggesting that level of IL-10-secreting T-cells does not correlate with severity of uveitis. We show that STAT1-deficient DCs secrete substantially reduced level of IL-27 that is associated with reduced production of IL-10 by DCs and enhanced proliferative response of STAT1-deficient uveitogenic T-cells, suggesting that EAU severity may depend on the relative amount of IL-27 and IL-10 produced by innate immune cells. We further show that retinal cells produce IL-27 and demonstrate that retinal cells respond to autocrine IL-27-stimulation by expressing anti-inflammatory proteins IL-10, SOCS1 and SOCS3. Taken together these data suggest a model of immune privilege of the eye based on IFN-g secretion in retina during uveitis by Th1 cells, IFN-g-mediated IL-27 production by retinal microglial cells and the eventual suppression of T-cell expansion in the retina through SOCS3 and IL-10-dependent mechanism

The relationship between spatial selection and object recognition in covert visual search

Monosov I.E., and K.G. Thompson

Laboratory of Sensorimotor Research. Perception and Action Section.

Frontal eye fields (FEF) is a brain area located in the prefrontal cortex of macaque monkeys and humans. Spatial signals recorded in FEF are thought to be involved in eye-movement planning. Do these signals also play a role in mediating covert spatial attention? And, are these signals required for accurate object perception? We investigated these questions by inactivating the frontal eye fields (FEF) of monkeys performing a cued covert visual search task that required the monkeys to report the identity of a target stimulus amongst distractors. We compared performance accuracy and reaction times before and after unilateral muscimol inactivation of FEF. The effectiveness and spatial extent of the inactivation was assessed by measuring the monkeys’ ability to generate saccades in a memory guided saccade task. In the cued covert visual search task FEF inactivation resulted in decreased performance accuracy and increased reaction times in the visual hemifield contralateral to the muscimol injection. These results provide direct evidence that FEF activity plays an important role in mediating spatial attention that is necessary for accurate object recognition in covert visual search.

Photoreceptors from human embryonic stem cells and role of epigenetics

Nasonkin Igor, K-Y Park, M. William, D. Hambright, L. Dong, R. McKay and A. Swaroop. Neurobiology Neurodegeneration & Repair Laboratory (N-NRL).

MacLauren et al. (Nature 2006) have demonstrated the feasibility of cell-based therapies for retinal degenerative disease; however, the success if such strategies relies on our understanding of molecular mechanisms of retinal development to recapitulate the key induction signaling steps that can produce transplantable cells from pluripotent human embryonic stem cells (HESCs). As part of this effort, we are designing new methods to characterize the process of retinal differentiation of HESCs by using fluorescent reporters and refining subretinal cell transplantation into mice. At this stage, a number of HESC subclones carrying Nrlp-EGFP reporter have been generated from two HESC lines, and a few clones display initial EGFP fluorescence upon application of retinal differentiation protocol. Further efforts are being focused on new generation of reporters based on bacterial artificial chromosomes. A second line of investigation is focused on inducing in vivo regeneration in retina using epigenetic mechanisms, specifically, DNA methylation. To understand the potential of epigenetic signaling in influencing retinal cell fate, we are using a series of floxed alleles of three key methyltransferase enzymes conducting DNA methylation in mice and humans - Dnmt1, Dnmt3a, and Dnmt 3b (Li, Bestor, Jaenisch,1992). Some of the floxed mice show promising retinal/eye phenotypes when crossed to Rx-Cre driver line, indicating a critical role of epigenetic signaling in eye development. Further studies along these lines should provide new insights into retinal differentiation and assist in developing new therapies.

Cdk5 activity regulates stress fiber organization and contraction by controlling rho-activation and rho-dependent myosin phosphorylation

Tripathi Brajendra K. and Peggy S. Zelenka

Laboratory of Molecular and Developmental Biology, National Eye Institute, NIH

Myosin II dependent cytoskeletal contraction, which is essential for cell adhesion and migration, is regulated by phosphorylation of myosin regulatory light chain (MRLC) at Thr18/Ser19. We have previously shown that Cdk5 regulates cell adhesion and migration in epithelial cells, raising the possibility that Cdk5 might affect myosin phosphorylation and contraction. To test this, lens epithelial cells were allowed to spread on fibronectin for 120 min, during which time cells formed stress fibers and appeared contracted, as indicated by a characteristic transition from convex to concave cell boundaries. This was accompanied by an increase in Cdk5 kinase activity and MRLC diphosphorylation. Immunofluorescence microscopy showed that Cdk5 and its activator, p35, co-localized with Thr18/Ser19 diphosphorylated MRLC (dpMRLC) on contracting stress fibers. Inhibiting Cdk5 activity with a pharmacological inhibitor (olomoucine), dominant negative GFP-dnCdk5(D144N), or siCdk5, significantly reduced the level of dpMRLC at 120 min and reduced the number of contracted cells. Moreover, blocking Cdk5 activity significantly reduced dpMRLC immunofluorescence and altered stress fiber organization, with loss of central stress fibers. Since MRLC itself lacks a favorable site for phosphorylation by Cdk5, we tested whether Cdk5 affects the Rho-ROCK upstream signaling cascade, which is primarily responsible for MRLC diphosphorylation under these conditions. Both olomoucine and siCdk5 significantly reduced Rho-GTP formation and consequently ROCK activation, confirming the involvement of Cdk5 in activation of Rho in spreading cells. These findings demonstrate a novel requirement for Cdk5 in Rho-dependent myosin contraction and provide a mechanism for previously observed effects of Cdk5 on epithelial cell adhesion and migration.

Dicer and microRNAs are essential for development of lens and cornea in mice

Li Yan, and Joram Piatigorsky

Laboratory of Molecular and Developmental Biology, Sections of Molecular Genetics

Although it is known that microRNAs (miRNAs) play important roles in regulating gene expression in many tissues, little is known of miRNA functions in eye development. Processing of miRNAs requires the ribonuclease Dicer. We found abundant expression of Dicer mRNA and mature miRNAs in 6-week-old mouse cornea and lens. Particularly, expression of mir-184 was highly cornea- and lens- specific. In addition, 13.7% of all the verified mouse miRNAs showed differential expressions in the mouse cornea before and after eye opening, consistent with a role of miRNAs in eye development. We conditionally deleted Dicer in cornea and lens and demonstrated specific reduction in Dicer and miRNA expressions in the developing lens by E12.5. Lens morphogenesis in the mutant mice progressed normally at E12.5 but started to degenerate at E14.5. Increased cell death and reduced cell proliferation were observed as early as E12.5 in lens epithelia. Expressions of key transcription factors for lens development, such as Pax-6 and Prox1, were not affected in the E12.5 lenses but later started to decrease due to lens degeneration. Except for much smaller size, the corneal epithelia in the mutant mice maintained normal morphology and Pax-6 expression even after birth. However, the corneal epithelial cells did not stratify. Adult 8-week-old mice lacking Dicer in lens and cornea were severely microphthalmic. These results reveal critical roles for Dicer and miRNAs in the control of cell growth and differentiation during the development of lens and cornea.

Role of the lateral habenula in motivational control of animal behaviors

Matsumoto Masayuki, and Okihide, Hikosaka

Neuronal networks Section, Laboratory of Sensorimotor Research

An approaching behavior to reward is more frequently and quickly elicited when a big reward is expected than when a small reward is expected. Our brain achieves such reward-based control of behavior by the contribution of dopamine neurons. These neurons provide excitatory signals when a big reward is expected and inhibitory signals when a small reward is expected. However, it was unknown how dopamine neurons acquire such reward-related signals. Here we show that the lateral habenula, a small brain structure, provides dopamine neurons with the signals. We used an experimental procedure in which monkeys were required to make an eye movement toward a visual target presented on the left or right side of a screen. Eye movements toward left target were followed by reward but eye movements to the right target were not (or vice versa). Therefore, the monkeys could predict reward or no-reward based on the target position. Indeed, eye movements were initiated more quickly toward the reward-predicting target than the no-reward-predicting target. We found that many lateral habenula neurons of the monkeys were inhibited by the reward-predicting target and excited by the no-reward-predicting target. In contrast, dopamine neurons were excited by the reward-predicting target and inhibited by the no-reward-predicting target. These reciprocal responses between lateral habenula and dopamine neurons suggest the causality of their responses. Consistent with this view, artificial activation of the lateral habenula elicited an inhibition of dopamine neurons. This suggests that the lateral habenula can cause the inhibitory signals of dopamine neurons. Thereby the lateral habenula could contribute to the reward-based control of behavior especially when a small or no reward is expected.

Inflammatory processes induced by TH1 or TH17cells remarkably differ in heir kinetics, apparently due to differences in their susceptibility to reactivation induced cell death (RICD)

Shi Guangpu, Barbara P. Vistica1, Madhu Ramaswamy, Cuiyan Tan1, Eric F. Wawrousek, Richard M. Siegel and Igal Gery

Laboratory of immunology, Laboratory of Molecular and Developmental Biology, NEI/NIH; Immunoregulation Unit, Autoimmunity Branch, NIAMS/NIH

Both Th1 and Th17 cells are involved in immune-mediated inflammation. To learn about specific features of immunopathogenic activities of the two Th lineages, we used an experimental system in which TCR-transgenic Th1 or Th17, specific against hen egg lysozyme (HEL), induce ocular inflammation in recipient mice expressing HEL in their eyes. Polarized Th1 divided in recipient mice considerably faster than Th17 cells, as measured by CFSE dilution. Th1 also invaded the recipient eyes earlier than Th17 cells: onset of the inflammatory process was day 3 for Th1 and day 4 for Th17 cells. After reaching the severity peak, however, the inflammatory process in Th1 recipient eyes receded rapidly, but was sustained in Th17 recipient eyes. Importantly, Th1 cell number and their proportion among infiltrating cells rapidly declined after day 5, whereas Th17 remained the majority among eye infiltrating cells and their actual number even increased slightly during the observation period of 15 days. The rapid decline of Th1 in inflamed eyes, as compared to Th17, was due at least in part to their high susceptibility to RICD, a major pathway whereby stimulated Th are eliminated. The difference in susceptibility RICD between Th1 and Th17 could be attributed to the profoundly higher expression levels of Fas ligand (FasL) by Th1 cells. On the other hand, the two cell lineages expressed similar levels of Fas. Thus, inflammatory processes induced by Th1 and Th17 differ remarkably in their kinetics, due in part to the high susceptibility of Th1 to RIDC.

Intravitreal administration of AAV8 RS1 vector to the RS1-KO mouse results in cell type-specific retinoschisin expression and morphological improvement

Wu Zhijian, Tae Kwann Park, Sten Kjellstrom, Yong Zeng, Ronald A. Bush, Paul A. Sieving, Peter Colosi1

National Eye Institute and National Institute on Deafness and Other Communication Disorders, National Institute of Health, Bethesda, MD

X-linked juvenile retinoschisis (XLRS) is an early-onset and slowly progressive retinal and macular degeneration in males caused by mutations in the retinoschisin gene. Gene complementation of recessive genetic retinopathies by subretinal injection of adeno-assoicated virus (AAV) vectors is a promising treatment modality for such diseases. This approach has been validated by promising results from 3 clinical trials evaluating the safely and efficacy of AAV vectors for the treatment of Leber congenital amaurosis. However, the use of subretinal injection as a delivery method for an XLRS clinical trial may be problematic due to the unique pathology of this disease. We describe a method of obtaining efficient gene transfer to XLRS retinas without subretinal injection. Simple vitreous injection of AAV8 vectors results in efficient transduction of all retinal layers in the retinoschisin knockout mouse (RS1-KO). Expression can be restricted to the cell types that express retinoschisin in wild type animals by the use of a vector with tissue-specific expression. This vector consists of a human retinoschisin promoter, a human retinoschisin cDNA containing a truncated retinoschisin first intron, and the human beta-globin polyadenylation site. Vitreal administration of this vector to RS1-KO mice results in robust retinoschisin expression with a retinal distribution that is similar to that seen in wild type retina. Little or no off-target expression is observed. RS1-KO mice treated with this vector demonstrate a decrease in size and number of schisis cavities. These results form part of a preclinical data set that will allow evaluation of this therapy in the clinic.

Phagocytosis defects associated with the choroideremia CHM gene

Zein W, Strunnikova N, Gordiyenko N, Sui H, Macdonald I. Ophthalmic Genetics and Visual Function Branch, National Eye Institute.

Choroideremia is an X-linked eye disease, characterized by progressive degeneration of the photoreceptors, retinal pigment epithelium, and choriocapillaris. The diffuse, progressive atrophy begins peripherally and spreads centrally. Affected patients complain of night blindness in the first or second decade of life, followed by progressivevisual field restriction leading to tunnel vision and often blindness. Expression and progression are variable among patients, even within the same family. Carriers show specific fundus changes and may have associated functional loss. Histopathology shows atrophy of the retinal pigment epithelium and photoreceptor layer in an apparently independent manner. Genetic analysis of choroideremia patients shows that most mutations result in loss of function of Rab-escort protein 1 (REP1). REP1 is a 653 aminoacid protein involved in intracellular trafficking. We present a family of three affected hemizygote males and their carrier mother and discuss their phenotypic variability as well as a noteworthy defect in phagocytosis.

VEGF-B is an apoptosis inhibitor by suppression of BH3-only protein gene expression via VEGFR-1

Zhang Fan, Yang Li1, Nobuo Nagai, Zhongshu Tang1, Johan Lennartsson, Kevin G. Becker, Andrew Nash, Carl-Henrik Heldin, Xuri Li1

National Eye Institute, Unit on Retinal Vascular Neurobiology Department of Physiology, Kinki University School of Medicine, Osakasayama, Osaka, Japan. TRIAD Technology Center, National Institute on Aging, NIH, Baltimore, Maryland, USA. CSL Limited, Parkville, Victoria, Australia. Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden.

To present days, a lot of growth factors were found to be involved in the process of angiogenesis and neurogenesis in developing, normal or pathological retina. How to use these growth factors to explore better therapies for treating human neural vascular diseases always attracts our interest. Despite its early discovery and high sequence homology to the other VEGF family members, the biological functions of VEGF-B remain poorly understood. We revealed here a novel function for VEGF-B as a potent inhibitor of apoptosis. VEGF-B inhibited the expression of genes encoding the (pro) apoptosis- and cell deathrelated proteins via activation of VEGFR-1. Consistent with this, VEGF-B treatment rescued neurons from apoptosis in the retina and brain in mouse models of ocular neurodegenerative disorders and stroke, respectively. Simultaneously, VEGF-B treatment at the dose effective for neuronal survival did not cause retinal neovascularization. These findings indicate that VEGF-B may be the first member of the VEGF family that has a potent anti-apoptotic effect while lacking a general angiogenic activity and may potentially offer a new therapeutic option for the treatment of ocular neurodegenerative diseases.