Non-pou Domain Containing Octamer-binding Protein Nono/p54nrb Promotes Rhodopsin Expression and Rod Photoreceptor Survival
Sharda Prasad Yadav, Marie-Audrey I. Kautzmann, Jacob Nellissery, and Anand Swaroop
Retinal Development & Genetics Section, Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health
Rhodopsin is the major structural protein in outer segment discs of rod photoreceptors. Daily renewal of membrane discs requires high yet stringently controlled expression of rhodopsin. Quantitative changes in rhodopsin expression are associated with photoreceptor degeneration. The promoter region of Rhodopsin includes two key cis-regulatory elements a proximal promoter region termed RPPR and a distal enhancer element DER. RPPR contains binding sites for NRL and CRX and is sufficient for rod-specific expression in transgenic mice. However, a larger upstream region, including DER, is believed to contain additional regulatory information. The goal of my studies is to identify the proteins that modulate and maintain the quantitatively precise expression of rhodopsin.
We are using a proteomic approach to identify DER-binding proteins from bovine nuclear extract. A biotin tagged oligo derived from DER was incubated with bovine nuclear fraction and bound protein complexes were further separated on SDS-PAGE. Proteins isolated were identified by mass spectra analysis. Our mass data suggest that DER bound proteins mostly involve in splicing, transcription, cell fate determination and signal transduction.
To further investigate the role of these identified proteins in rhodopsin regulation we focused on a candidate protein known as non-pou domain containing octamer-binding protein (Nono/p54nrb) for which we got maximum number of peptides in our analysis. Nono expressed as early as E12.5 in mice retina. Using luciferase reporter assays, we showed that Nono transactivates a 2.2 kb Rhodopsin promoter in the presence of NRL and CRX. Knock down of Nono using shRNA by in vivo electroporation in mice retina at P0, affects the reporter gene expression and leads to death of rod photoreceptors by apoptosis during early stage of its development. Thus, we have identified a novel co-activator that binds to DER of Rhodopsin promoter and regulates its activity and required for rod photoreceptor survival.
Understanding the Brain Learning Circuit Using Antidromic and Orthodromic Stimulation Studies
Simon Hong and Okihide Hikosaka
Neuronal Networks Section, Laboratory of Sensorimotor Research, National Eye Institute, National Institutes of Health
Many of our skillful daily actions are a result of constant positive and negative reinforcements. We investigated brain areas involved in making us act motivated or discouraged. We used the monkey’s eye movement as a probe for this study. More specifically, we gave a small amount of juice (low motivation) when the monkey looked at one position (which results in a slower eye movement) and gave a large amount (high motivation) for another position (which results in a faster eye movement). While the monkey was doing this task, we probed the activities of neurons in several brain regions: the basal ganglia, the lateral habenula, the rostromedial tegmental nucleus, and dopamine neurons. We used techniques called antidromic and orthodromic stimulation. With these techniques, we could see the flow of signals throughout many serially connected regions by simultaneously probing two connected areas at a time. Through this study, we discovered that the motivational signal in the brain is generated in the basal ganglia and traveled to the lateral habenula –> the rostromedial tegmental nucleus –> dopamine neurons –> back to the basal ganglia. Since it is assumed that the level of dopamine represents the level of motivation, we hypothesize that the motivational signal generated by the basal ganglia influences the eye movement controlling neurons situated in the basal ganglia. More studies are needed to answer some unknown questions, such as, why the motivational signal travels through those many areas to go back to its origin.
Influence of the Commensal Intestinal Flora on the Development of Spontaneous Autoimmune Uveitis
Carlos R. Zrate-Blads, Reiko Horai, Jun Chen, Phyllis B. Silver, Chi-Chao Chan and Rachel R. Caspi
Immunoregulation Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health
A fundamental function of the immune system is to discriminate between foreign and self-components. Nonetheless, one of the most intriguing questions of contemporary immunology is how commensal microorganisms, present in trillions on the mucosal surface of the gut, apparently do not trigger an immune response. Our group is dedicated to the study of autoimmune uveitis, the cause of 1015% of blindness in which patients exhibit immunological responses to retinal proteins, with no clear etiology. It is well accepted that T lymphocytes must be activated outside the eye to be able to penetrate the blood-retinal barrier to induce uveitis. Recently, we developed a transgenic mouse model (R161H) which expresses T cell receptor (TCR) specific to a retinal protein and that develops autoimmune uveitis spontaneously. Importantly, when R161H mice received broad-spectrum antibiotic treatment (ampicillin, metronidazole, neomycin and vancomycin = AMNV), which depletes the commensal bacterial flora; a significant reduction of retinal inflammation (uveitis) was observed. This suggested that commensal microbiota regulates the immune response in the gut to modulate the development of uveitis. AMNV-treated mice presented both decreased incidence and intensity of inflammation. The analysis of the fecal material confirmed that AMNV-treated mice exhibit a substantial reduction in the total bacterial content as defined by 16S rRNA gene detection. Interestingly, this lower bacterial content in the gut was correlated with an increase in the size of the cecum, but no alteration in the histological structure along the gut was noted. The percentages of IL-17+CD4+ and IL-17+CD4-CD8- lymphocytes were lower in the lamina propria of the small intestine of AMNV treated mice, as compared to untreated mice. In addition, the percentage of IL-17 producing T lymphocytes in the eye-draining (submandibular) lymph nodes was decreased in AMNV-treated mice. These results suggest that (a) the commensal gut microflora influences the systemic T lymphocyte- responses of R161H mice to retina, and (b) that it does so by reducing the IL-17 producing cells, which were shown to be important in the pathogenesis of autoimmune uveitis.
Mutations in FYCO1 Cause Autosomal Recessive Congenital Cataract
Jianjun Chen,1 Zhiwei Ma,1 Xiaodong Jiao,1 Robert Fariss,2 Wanda Lee Kantorow,3 Marc Kantorow,3 Eran Pras,4 Moshe Frydman,5 Elon Pras,5 Sheikh Riazuddin,6,7 S. Amer Riazuddin,6,8 and J. Fielding Hejtmancik1
1Ophthalmic Molecular Genetics Section, Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health; 2Biological Imaging Core, National Eye Institute, National Institutes of Health; 3Dept Biomedical Science, Florida Atlantic University, 4Department of Ophthalmology, Assaf Harofeh Medical Center, Zerifin, Israel; affiliated to the Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; 5The Danek Gertner Institute of Human Genetics, Sheba Medical Center, Ramat Gan, Israel; 6National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan; 7Allama Iqbal Medical College, Lahore, Pakistan; 8The Wilmer Eye Institute, Johns Hopkins University School of Medicine
Congenital cataracts (CCs), responsible for about one-third of blindness in infants, are a major cause of vision loss in children worldwide. Autosomal-recessive congenital cataracts (arCC) form a clinically diverse and genetically heterogeneous group of disorders of the crystalline lens. To identify the genetic cause of arCC in consanguineous Pakistani families, we performed genome-wide linkage analysis and fine mapping and identified linkage to 3p21-p22 with a summed LOD score of 33.42. Mutations in the gene encoding FYVE and coiled-coil domain containing 1 (FYCO1), a PI(3)P-binding protein family member that is associated with the exterior of autophagosomes and mediates microtubule plus-end-directed vesicle transport, were identified in 12 Pakistani families and one Arab Israeli family in which arCC had previously been mapped to the overlapping CATC2 region. Nine different mutations were identified, including c.3755 delC (p.Ala1252AspfsX71), c.3858_3862dupGGAAT (p.Leu1288TrpfsX37), c.1045 C>T (p.Gln349X), c.2206C>T (p.Gln736X), c.2761C>T (p.Arg921X), c.2830C>T (p.Arg944X), c.3150+1 G>T, c.4127T>C (p.Leu1376Pro), and c.1546C>T (p.Gln516X). Fyco1 is expressed in the mouse embryonic and adult lens and peaks at P12d. Expressed mutant proteins p.Leu1288TrpfsX37 and p.Gln736X are truncated on immunoblots. Expressed wild-type and p.L1376P FYCO1, the only missense mutant identified, migrates at the expected molecular mass. Both wild-type and p. Leu1376Pro FYCO1 proteins expressed in human lens epithelial cells partially colocalize to microtubules and are found adjacent to Golgi, but they primarily colocalize to lysosomes, including autophagosomes. Thus, FYCO1 is involved in autophagy in lens development and transparency in humans, and mutations in this gene are one of the most common causes of arCC in the Pakistani population.
Adaptive Mller Cell Responses to Microglial Activation Mediate Neuroprotection and Coordinate Inflammation in the Retina
Minhua Wang1, Wenxin Ma1, Lian Zhao1, Robert N. Fariss2, and Wai T. Wong1
1Unit on Neuron-Glia Interactions in Retinal Disease, & 2Biological Imaging Core, National Eye Institute, National Institutes of Health
Purpose: Microglia and Mller cells are prominent participants in retinal responses to injury and disease that shape eventual tissue adaptation or damage. This investigation examined how microglia and Mller cells interact with each other following initial microglial activation.Methods: Mouse Mller cells were cultured alone, or co-cultured with activated or unactivated retinal microglia, and their morphological, molecular, and functional responses were evaluated. Mller cell feedback signaling to microglia was studied using Mller cell-conditioned media. Corroborative in vivo analyses of retinal microglia-Mller cell interactions in the mouse retina were also performed.
Results: Our results demonstrate that Mller cells exposed to activated microglia, relative to those cultured alone or with unactivated microglia, exhibit marked alterations in cell morphology and gene expression that differed from those seen in chronic gliosis. These Mller cells demonstrated in vitro (1) an upregulation of growth factors, and provide neuroprotection to photoreceptor cells, (2) increased pro-inflammatory factor production, which in turn increased microglial activation in a positive feedback loop, and (3) upregulated chemokine and adhesion protein expression, which allowed Mller cells to attract and adhere to microglia. In vivo activation of microglia by intravitreal injection of lipopolysaccharide (LPS) also induced increased Mller cell-microglia adhesion, indicating that activated microglia may translocate intraretinally in a radial direction using Mller cell processes as an adhesive scaffold.
Conclusion: Our findings demonstrate that activated microglia are able to influence Mller cells directly, and initiate bidirectional microglia-Mller cell signaling that can mediate adaptive responses within the retina following injury. In the acute aftermath following initial microglia activation, Mller cell responses may serve to augment initial inflammatory responses across retinal lamina and to guide the intraretinal mobilization of migratory microglia using chemotactic cues and adhesive cell contacts. Understanding adaptive microglia-Mller interactions in injury responses can help discover therapeutic cellular targets for intervention in retinal disease.
Myocilin-induced Glaucoma: Oxidative Stress Contributes to a Severity of Phenotype
Myung Kuk Joe, Naoki Nakaya, Stanislav I Tomarev
Section of Molecular Mechanisms of Glaucoma, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health
Mutations in the myocilin gene are associated with juvenile and adult-onset primary open-angle glaucoma (POAG). However, the pathogenic mechanisms of myocilin-induced glaucoma are still largely unknown. To investigate these pathogenic mechanisms and develop a genetic mouse model of the disease, we produced stably transfected HEK293 cell lines expressing wild-type or Y437H mutant myocilin under an inducible promoter. This mutation in myocilin leads to severe glaucoma in human. Induction of mutant but not wild-type myocilin expression in HEK293 cells led to endoplasmic reticulum (ER) stress and increased apoptosis after treatment of cells with hydrogen peroxide. Several antioxidant genes were down-regulated in the Y437H mutant myocilin cell line, and also in transgenic mice (Tg-MYOCY437H) expressing the Y437H mutant myocilin in the eye drainage structures. These results suggested that expression of mutant myocilin in the eye drainage structures could make them more sensitive to oxidative stress and lead to a severe glaucoma phenotype under oxidative stress compared with mice expressing mutant myocilin alone. To test this hypothesis, we produced double mutant mice of Tg-MYOCY437H and heterozygous knockout of superoxide dismutase 2, SOD2 (+/-). The Y437H mutant myocilin induced ER stress in the trabecular meshwork (TM) and led to apoptosis of TM cells in the Tg-MYOCY437H / SOD2 (+/-) mice. Compared with the characteristics of littermates carrying only SOD2 (+/-) or Tg-MYOCY437H, the double mutant mice showed 50% higher intraocular pressure [MYOCY437H / SOD2 (+/-) versus SOD2 (+/-),18 mmHg versus 12 mmHg], greatly reduced the number of retinal ganglion cells, and demonstrated severe depletion of the retinal nerve fiber layer. These data suggest that a treatment with antioxidative agents alone or in a combination with small chemical chaperones to reduce ER stress may be considered as a viable therapy for myocilin-induced glaucoma. The double mutant mice represent an excellent animal model to develop novel effective treatments of POAG.
VEGF-B Prevents Excessive Angiogenesis
Chunsik Lee1, Anil Kumar1, Lasse Jensen2, Masashi Narazaki3, Harald Langer4, Fan Zhang1,Yang Li1, Zhongshu Tang1, Pachiappan Arjunan1, Ling Li5, Bin Wang6,7, Shizhuang Zhang6, Tom Doetschman8, David Ornitz5, Triantafyllos Chavakis9, Yihai Cao2, Xuri Li1
1Unit on Retinal Vascular Neurobiology, National Eye Institute, National Institutes of Health; 2Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Sweden; 3Osaka University Graduate School of Medicine, Japan; 4University of Tbingen, Germany; 5Washington University School of Medicine; 6Weifang Medical University, P. R. China; 7Binzhou Medical University, P. R. China; 8University of Arizona; 9University Dresden, Germany
Little is known about how excessive blood vessels are prevented despite the universal and abundant expression of certain potent angiogenic factors, such as the FGFs and their receptors, which play important roles in angiogenesis. For example, loss of even one Fgf2 allele reduced cardiac blood vessel density by 25% in mice. Notwithstanding, how the potent angiogenic activities of the FGFs/FGFRs are counteracted to avoid excessive angiogenesis is unclear. VEGF-B is a VEGF-A homologue discovered a long time ago. However, the biological function of VEGF-B remains controversial. Here, we report our novel finding that VEGF-B prevents excessive angiogenesis by acting as a natural inhibitor of the angiogenic activity of the FGF/FGFRs pathways. VEGF-B protein treatment or gene delivery inhibited FGF2-induced angiogenesis, whereas VEGF-B deficiency by gene deletion in mice or morpholino knockdown in zebrafish increased blood vessel density. Mechanistically, we revealed that VEGF-B antagonistically binds to FGFR1/2 and inhibits FGF2-induced Erk activation by upregulation of Sprouty4, a major inhibitor of the FGF/FGFR signaling pathway. Thus, our data uncovered an unexpected novel function of VEGF-B as a “vascular surveillance”, rather than a “vascular growth” factor. This unique function of VEGF-B may have therapeutic implications in modulating angiogenesis.
Zebrafish Modeling of a Novel 5q Deletion Syndrome in a Patient with Coloboma
Holly E. Babcock1, Sunit Dutta1, Ramakrishna P. Alur1, Igor B. Dawid2, Brian P. Brooks1
1Pediatric, Developmental and Genetic Ophthalmology Section, Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health; 2Section on Developmental Biology, National Institute of Child Health & Human Development, National Institutes of Health
Ocular development begins with an outpocketing of forebrain neuroepithelium followed by an asymmetrical invagination to form the optic cup. In order for the eye to form a closed sphere, the two ventral edges of the optic cup (the optic fissure) must approximate and fuse; failure to do so results uveal coloboma-a potentially blinding congenital ocular malformation.
Despite a low incidence rate, uveal coloboma may be responsible for approximately 10% of childhood blindness and may be accompanied by other congenital defects. Though the embryology has long been understood, the genetic and developmental mechanisms behind optic fissure closure defects are still uncertain. We previously identified two genes, nlz1 and nlz2, that are necessary for optic fissure closure.
The current research stemmed from a maternally-inherited microdeletion of a patient with bilateral coloboma, dysmorphic facial features, and shortened metacarpals. We analyzed the expression pattern of each of the deleted genes in both the developing eye and fin of zebrafish and identified aldehyde dehydrogenase 7 family, member A1 (aldh7a1) as a gene necessary for proper eye development. Mutations in human ALDH7A1 have been linked to pyridoxine-dependent epilepsy. In these clinical cases, the eye has been largely ignored, although some patients are reported to have optic nerve hypoplasia. We find that morpholino knockdown of aldh7a1 in zebrafish results in a similar syndromic phenotype including coloboma, craniofacial malformations, and shortened pectoral fins. In the morphants, cell proliferation in the eye is reduced. Morphant rescue experiments suggest that aldh7a1 is mechanistically upstream of nlz1 and folic acid. We are also moving forward with this project clinically by sequencing the ALDH7A1 gene in many of our coloboma patients as well as performing whole-exome sequencing on the proband. In brief, we hope to pinpoint genetic players in eye development, identify the cause of the patient’s syndrome, and complement our zebrafish findings with human data.
Single Cell Analysis of Early Retinal Ganglion Cell Development
Szilrd Sajg1,2, Miruna Ghinia1,2, Tudor C. Badea1
1Retinal Circuit Development and Genetics Unit, Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health; 2Interdisciplinary Research Institute in Bio-Nano-Sciences, Babes-Bolyai University, Cluj-Napoca, Romania
BRN3 is a group of related POU family transcription factors. Brn3a, Brn3b, and Brn3c are expressed in distinct, but largely overlapping subsets of ganglion cells in the retina, and in many other sensory neurons. Brn3a mutant mice die at birth, as a result of neuronal loss in the Dorsal Root Ganglia (DRG) and Trigeminal Ganglia (TGG). Mutation of Brn3b causes a significant loss of Retinal Ganglion Cells (RGC), and surviving RGCs exhibit axonal misguidance. In Brn3b mutants, RGCs projecting to the accessory optic tract are completely missing. Brn3c null mice are characterized by inner ear hair cell loss.
Our present work is focusing on the study of axon guidance mechanisms at single cell resolution in the developing mouse visual system. Our approach consists in sparse Cre mediated recombination of conditional alleles expressing AP (alkaline phosphatase) under the Brn3 promoter of interest. By using a modified Tet-ON system with dual pharmacological control of Cre, we are able to efficiently control the amount of recombination. This allows us to visualize individual neurons and axonal fibers.
Using this methodology, we are able to reconstruct the early steps in RGC birth and axon formation. In addition, we are describing the stages of progression of single RGC axons through the optic nerve and tract. Surprisingly, we also define a novel cell population transiently expressing Brn3b at Embryonic day 12.5, in a unique region of the developing floor plate, potentially located at the future optic chiasm.
Enhanced Apoptosis in Retinal Pigment Epithelium Under Inflammatory Stimuli and Oxidative Stress
Yujuan Wang1,2, Defen Shen1, Vinson M. Wang1, Cheng-Rong Yu3, Ren-Xi Wang3, Jingsheng Tuo1, C.C. Chan1
1Immunopathology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health; 2Zhongshan Ophthalmic Center, Sun Yat-sen University, P.R. China; 3Molecular Immunology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health
Age-related macular degeneration (AMD) is a neurodegenerative disease that causes irreversible central vision loss in the elderly. Retinal pigment epithelium (RPE) has been found to be a key element in AMD pathogenesis. The Ccl2-/-/Cx3cr1-/- (DKO) mouse is created as an AMD model, developing AMD-like retinal lesions. Our study aimed to examine RPE apoptosis in DKO mice and human ARPE-19 cell line. In vivo, DKO RPE has higher FasL and Fas expression when compared with age-matched WT RPE in physiological conditions. Apoptosis of primary mouse RPE was evaluated under stimulation of lipopolysaccharide (LPS) for inflammatory stress and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or H2O2 for oxidative stress in vitro. Compared with WT RPE, DKO RPE was more susceptible to apoptosis under both inflammatory and oxidative stress, with less cell viability and higher expression of FasL and Fas transcripts and cleaved caspase-3 and caspase-9 proteins. Less cell viability was also observed in ARPE-19 cells under each stimulus. We also explored the anti-apoptotic effects of decoy receptor 3 (DcR3), a decoy receptor for FasL, on ARPE-19 cells under inflammatory and oxidative stress. DcR3 pre-incubated ARPE-19 cells showed decreased apoptosis, with more cell viability and less expression of FasL and Fas transcripts and cleaved caspase-3 and caspase-9 proteins under the stimuli. On the contrary, knockdown of DcR3 in ARPE-19 cells showed totally opposite results. Our study demonstrates that Ccl2-/-/Cx3cr1-/- DKO RPE is prone to FasL-mediated apoptosis under inflammatory and oxidative stress. DcR3 has anti-apoptotic effects in ARPE-19 cells under similar stress. Inflammatory and oxidative stress on RPE cells may play a pivotal role in AMD pathogenesis.
From Isolated Experiments to the Big Picture: a Mathematical Model of Eye-Head Coordination
Pierre M. Daye and Lance Optican
Neural Modeling Section, Laboratory of Sensorimotor Research, National Eye Institute, National Institutes of Health
During everyday life, we continuously reorient our gaze (gaze = eye-in-head + head-in-space) to new objects of interest. Gaze movements have been characterized for more than a century (Dodge, 1903) and a gazillion studies have analyzed neural areas involved in controlling and coordinating eye and head movements during the reorientation of the visual axis. Most of those studies reported important findings regarding the effect of neural discharges in specific areas on behavior, but they did not account for the interactions between different areas. Building a mathematical model is a key step in understanding global brain function because it tests hypotheses about the interactions between areas, which are difficult to test experimentally. Almost always, the exact behavior of some part of the model is not known. Therefore, the model must generate predictions about its performance. These predictions can guide experimentalists in the design of new protocols either to confirm or to refute the model structure.
This presentation will focus on how we built a model of the control and coordination of eye and head movements during a head-unrestrained saccade (quick reorientation of the visual axis from one center of interest to another). To explain the model structure, parts of the model will be presented in parallel with experimental data. Special care will be taken to emphasize the model’s behavior without mathematical details.