Abstracts
Spatial Attention to Color in the Superior Colliculus
James Herman, Richard Krauzlis
Section on Eye Movements & Visual Selection, Laboratory of Sensorimotor Research, National Eye Institute, National Institutes of Health
Primates rely on covert spatial attention to selectively process portions of the visual field without moving their eyes. One of the most important visual features is color, especially for catarrhine primates (humans, apes, and Old world monkeys), and yet very little work has focused on the allocation of spatial attention to color. The control of attention involves both cortical and subcortical structures, but the processing of color in primates is mostly localized to areas of the visual cortex. Is the ability to attend to color a specialization of the primate cortex, or is it part of a more general brain mechanism?
To address this question, we examined the role of a subcortical brain area, the superior colliculus (SC), in the control of attention to color. We used two techniques to determine the SC’s role: (1) we extracellularly recorded the electrophysiological activity of single neurons in the SC of a macaque monkey during a color change task, and (2) in separate sessions, we chemically inactivated a portion of SC neurons while the monkey was performing the task. The animal’s task was to release his hold on a joystick if one of two peripherally presented stimulus patches (the “cued” patch) changed color, and to ignore changes in the other (the “foil”) by maintaining joystick press. Our single neuron recordings revealed both an expected transient response to the initial stimulus appearance, as well as a notable response to the color change that was, in most cases, larger than the response to the initial appearance. This change-related activity rose more rapidly in response to cued changes than to foil, and its amplitude was predictive of the animal’s behavioral response. Meanwhile, our inactivation data showed strong spatially specific impairment of the animal’s task performance. When the cued patch was presented inside the region affected by the chemical inactivation, the animal’s performance was dramatically (and significantly) impaired, but when it was presented outside the affected region, there was no impairment. Our results show that the SC participates in the detection of near-threshold changes in stimulus color, and that it is causally involved in the allocation of spatial attention to color. These results demonstrate that the SC is a necessary component of the neural circuits for attending to color stimuli, and suggests that the SC is part of a general brain mechanism for processing behaviorally relevant visual events, regardless of the particular visual features involved.
A New Model to Study Mucosal Immunity at the Ocular Surface
Anthony St. Leger1, Hatice Karauzum2, Warren Strober3, Sandip Datta2, Ivan Fuss3, Rachel Caspi1
1Immunoregulation Section, Laboratory of Immunology, National Eye Institute; 2Bacterial Pathogenesis Unit, Laboratory of Clinical Infectious Diseases, 3Mucosal Immunity Section, Laboratory of Host Defenses, National Institute of Allergy & Infectious Diseases, National Institutes of Health.
Dysregulation of immune homeostasis in ocular mucosa is associated with inflammation, ocular discomfort, and potential blindness. Conjunctiva-associated lymphoid tissue (CALT) has been implicated as a major regulator of ocular homeostasis due to the presence of immune follicles within the tissue that increase in size and number concurrently with states of inflammation due to infections, allergies, dry eye, etc. Even though functional roles for CALT have been proposed, no mechanisms governing CALT functionality have been defined. We aimed to develop a mouse model to study the dynamics and functional parameters of CALT in normal and immunologically perturbed hosts. Towards that end, we found that cytokines, IL-17 and IL-22, are absolutely necessary to prevent spontaneous ocular inflammation due to outgrowth of opportunistic bacteria. Furthermore, we defined the sources of these cytokines within ocular tissue, which include γδ T cells, innate-like memory T cells, and group 3 innate lymphoid cells (ILC3s), a population of immune cells that has yet to be defined in ocular tissue. Lastly, we provide mechanistic details that control cytokine production in CALT, which include substantial contributions of ocular commensal bacteria and the ligand-activated transcription factor, aryl hydrocarbon receptor (AhR). Our studies lay the basis for a working mouse model to analyze the functional parameters of CALT, an understudied component of the mucosal immune system. Additionally, our model may permit the identification of targets for potential therapies to alleviate anterior ocular diseases.
Whole Exome Sequencing Reveals Genetic Defects Associated with Cone and Cone-rod Dystrophy in Israel.
Csilla Lazar1,2, Mousumi Mutsuddi1,3, Adva Kimchi4, Lina Zelinger1,4, Liliana Mizrahi-Meissonnier4, Devorah Marks-Ohana4, Alexis Boleda1, Rinki Ratnapriya1, Dror Sharon4, Anand Swaroop1, Eyal Banin1,4
1Retinal Development, Genetics, & Therapy Section, Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health; 2Molecular Biology Center, Interdisciplinary Research Institute on Bio-Nano Sciences, Babes-Bolyai-University, Romania; 3Department of Molecular & Human Genetics, Banaras Hindu University, India; 4Department of Ophthalmology, Hadassah–Hebrew University Medical Center, Israel.
Cone and cone-rod dystrophies are clinically and genetically heterogeneous retinal disorders. Mutation in several genes have been identified, however these explain less than 25% of cases and finding the remaining fraction of underlying genetic determinants is essential for developing personalized treatment strategies. The genetic make-up of the Israeli population is unique as it results from high prevalence of consanguineous marriages and autosomal recessive diseases. In rod-dominated phenotypes, disease-causing genes and mutations often differ from those seen in other populations.
The goal of the present study was to identify genetic defects in Israeli families with cone-dominated retinal phenotypes using whole exome sequencing (WES). Families were ascertained after detailed clinical analysis. WES was performed on 27 individuals from 8 cone-dominated retinopathy families for whom prior mutation analysis had not revealed the causative gene. Targeted genetic screening was performed on 106 additional families with cone-dominated phenotypes.
WES analysis revealed mutations in known retinopathy genes in seven out of eight families: two pathogenic variants in the GUCY2D gene in three families; two known CDHR1, C8orf37 and two possible novel CACNA1F and ALMS1 gene variants. Segregation analysis confirmed their presence in the studied families. Targeted screening of additional cone-dominated families led to identification of GUCY2D mutations, including two highly probable novel disease-causing variants, in four other families.
In conclusion, WES allows a comprehensive and rapid analysis that can be followed by targeted screens of larger samples to gain insight in to the genetic structure of retinal disease in unique population cohorts. Our study suggests that GUCY2D is a major cause of autosomal dominant cone and cone-rod dystrophy in Israel, similarly to other Caucasian populations and in contrast to the genetic architecture of primary rod disease observed in the Israeli population.
Rates of Vision Loss in Patients with Geographic Atrophy in AREDS and AREDS 2
David Valent1, Emily Chew1, Elvira Agrón1, Frederick Ferris1, Philip Rosenfeld2, Traci Clemons3, Wai Wong4 Age-Related Eye Disease Study Research Group, Age-Related Eye Disease Study 2 Research Group.
1Clinical Trials Branch, Division of Epidemiology & Clinical Applications, National Eye Institute, National Institutes of Health, 2Bascom Palmer Eye Institute, University of Miami Leonard M. Miller School of Medicine; 3EMMES Corporation; 4Unit on Neuron-Glia Interactions in Retinal Disease, National Eye Institute, National Institutes of Health.
Purpose: To evaluate the rates of vision loss compared with baseline in participants with geographic atrophy (GA) at baseline and those that developed GA in two major clinical trials.
Methods: The Age Related Eye Disease Study (AREDS) was a multi-center, double masked randomized clinical trial that enrolled 4757 participants with varying degrees of macular degeneration. The AREDS 2 was a multi-center, double masked phase 3 study which enrolled 4203 participants who were at risk for progression to advanced age related macular degeneration. In both studies, best-corrected visual acuity was obtained at baseline and at yearly intervals using the standard protocol from the Early Treatment of Diabetic Retinopathy Study (ETDRS).
Results: In AREDS, 259 eyes of 217 participants were identified as having GA at baseline. Of these, 79 had central GA. By the 5-year follow-up, 1267 eyes of 908 participants developed GA, 259 eyes were defined as having central GA. For those with GA at baseline, 48.7% of participants lost more than 10 letters and 36.8% lost more than 15 letters at 5 years compared with baseline. For those that developed GA during follow up, 47.1% of eyes lost more than 10 letters of vision, while 36.8% lost more than 15 letters at 5 years. Participants that developed GA during the study that had better initial visual acuity (20/80 or better), on average lost more letters than those with worse visual acuity when diagnosed with GA at 5 years (20 letters vs. 5.7 letters). In AREDS 2, 524 eyes of 418 participants were identified as having GA at baseline, of which 170 eyes had central GA. By 5 years of follow-up, an additional 868 eyes of 791 participants developed GA, 291 of which had central GA. For those with GA at baseline, 44.2% of participants lost more than 10 letters, and 33.5% lost more than 15 letters at 5 years follow up. For those that developed GA during follow up, 50.0% of participants lost more than 10 letters, and 35.0% lost more than 15 letters at 5 years.
Conclusions: Participants in the AREDS and AREDS2 study with geographic atrophy trended toward progressive visual loss. As expected, those participants with better initial visual acuity tended to lose more letters than those with worse initial visual acuity. This data may guide other investigators in planning for future studies of GA associated with AMD.
The Link Between RPE65 Retinol Isomerase and the Lipid Metabolism
Abdulkerim Eroglu, Susan Gentleman, Eugenia Poliakov, T Michael Redmond
Molecular Mechanisms Section, Laboratory of Retinal Cell & Molecular Biology, National Eye institute, National Institutes of Health
RPE65 is the key isomerase in the RPE visual cycle that catalyzes the conversion of all-trans retinyl ester (ATRE) into 11-cis retinol. We are interested in potential parallels between the RPE65 catalytic mechanism and that of lipid metabolism enzymes. We tested inhibitors of lipid metabolism to determine their effect on RPE65 isomerase activity. HEK 293-F cells were transiently transfected with expression vectors for visual cycle proteins (RPE65, LRAT, CRALBP, and RDH5) and isomerase activity was measured in cellulo in the presence of added substrate and inhibitors. Membrane preparations of transfected cells were used to test the effect of additives on isomerase activity in vitro. To determine if these fatty acid analogs compete with the ATRE substrate specifically, we incubated membranes prepared from transfected cells with liposomes containing a range of ATRE concentrations. We have identified fatty acid analogs used in studies of lipid metabolism as potent inhibitors of RPE65 and that compete with the ATRE substrate of RPE65 for binding, thereby inhibiting its isomerase activity. The effects of these inhibitors provide further insight into the catalytic mechanism of RPE65 retinol isomerase.
Digoxin Inhibits Induction of Experimental Autoimmune Uveitis in Mice, but Causes Severe Retinal Degeneration
Samuel H. Hinshaw1, Osato Ogbeifun1, Wambul Wandu1, Haohua Qian2, Yichao Li2, Igal Gery1
1Experimental Immunology Section, Laboratory of Immunology, 2Visual Function Core, National Eye Institute, National Institutes of Health
Purpose: Digoxin, a major medication for heart disease, was recently reported to inhibit the development of experimental autoimmune disease. Here, we determined the immunosuppressive property of digoxin on the development of experimental autoimmune uveitis (EAU).
Methods: B10.A mice immunized with an emulsion of interphotoreceptor retinoid-binding protein (IRBP) in complete Freund’s adjuvant were treated daily with digoxin or vehicle control. On day 14 post immunization (p.i.) the mouse eyes were examined histologically, while spleen cells were tested for proliferation and cytokine production in response to IRBP. To investigate the degenerative effect of digoxin on the retina, wild-type (FVB/N x B10.BR) F1 mice were similarly treated with digoxin and electroretinography (ERG) was performed on days 7 and 14.
Results: Histological analysis of the eyes showed that the treatment of mice with digoxin inhibited the development of EAU, as well as the cellular response to IRBP, i.e., proliferation and production of IFN-γ and IL-17. Importantly, digoxin treatment also induced severe retinal degeneration, determined by histological analysis as early as day 4, with thinning across all layers of the retina. Vision loss was also monitored by ERG, showing dose responsiveness, reaching its peak by day 7, without change on day 14.
Conclusions: Treatment of mice with digoxin inhibited the development of EAU and cellular immune response to IRBP. However, the treatment induced severe damage to the retina. Thus, the use of digoxin in humans cannot be recommended due to its toxicity to the retina.
Microglial Phagocytosis of Living Rod Photoreceptors Contribute to Inherited Retinal Degenerations
Matthew Zabel1, Lian Zhao1, Xu Wang1, Parth Shah1, Robert Fariss2, Haohua Qian3, Christopher Parkhurst4, WenBiao Gan4, Wai Wong1
1Unit on Neuron-Glia Interactions in Retinal Disease, 2Biological Imaging Core, 3Visual Function Core, National Eye institute, National Institutes of Health; 4Skirball Institute, Department of Neuroscience & Physiology, New York University School of Medicine
Retinitis pigmentosa (RP) is a set of inherited retinal degenerations lacking current comprehensive treatment. We report the discovery that microglia in the retina contribute directly to rod photoreceptor degeneration by the primary phagocytosis of living rods. Using the rd10 RP mouse model, we found that rod degeneration is accompanied by the early infiltration of microglia into the photoreceptor layer and the upregulation of phagocytic molecules in microglia and “eat-me” signals on mutated rods. Using live-cell imaging, we found that infiltrating microglia interact dynamically with photoreceptors via motile processes and engage in the rapid engulfment of non-apoptotic rods. The contribution of primary phagocytosis to rod demise is evidenced by the amelioration of photoreceptor degeneration by genetic ablation of retinal microglia, and by the inhibition of microglial phagocytosis. Our findings highlight primary microglial phagocytosis as a key mechanism underlying cell death in RP and implicate it as a target for therapeutic approaches.
Genetic Epidemiology of Autosomal Recessive Retinitis Pigmentosa and Congenital Cataracts in Pakistan
Yabin Chen1, Lin Li1, Zhiwei Ma1, Xiaodong Jiao1, Dan Jiang1, Li Huang1, Qiwei Wang1, Xiaoyin Ma1, Chongfei Jin1, Mukesh Tanwar2, Wenmin Sun3, Jianjun Chen4, Sheikh Riazuddin5,6, S. Amer Riazuddin5,7, J. Fielding Hejtmancik1
1Ophthalmic Molecular Genetics Section, Ophthalmic Genetics & Visual Function Branch, National Eye Institute, National Institutes of Health; 2Laboratory for Molecular Reproduction & Genetics, Department of Anatomy, All India Institute of Medical Sciences, India; 3State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, China; 4Department of Ophthalmology, Shanghai Tenth People’s Hospital & Tongji Eye Institute, Tongji University School of Medicine, China; 5National Centre of Excellence in Molecular Biology, University of the Punjab, Pakistan; 6Allama Iqbal Medical College, Pakistan; 7The Wilmer Eye Institute, Johns Hopkins University School of Medicine
Because of its high consanguinity rate and large family size Pakistan provides an ideal population for identifying the genetic causes of autosomal recessive cataracts (arCC) and retinal degenerations (arRD). Since 2004, we have ascertained 198 arRD and 114 arCC families in a study of inherited ophthalmic diseases in this population. Between 2004 and 2012, we identified genes or loci in 67 arRD families and 27 arCC families using linkage analysis followed by sequencing of candidate genes in the linked region. Some of the remaining families are too small to provide significant linkage scores. In order to identify the causative genes and mutations in the unmapped families, we carried out homozygosity exclusion mapping in which we screened for homozygosity at highly informative microsatellite markers within 1 cM of known candidate genes for arCC and arRD. After identifying homozygous markers at candidate loci, standard linkage and haplotype analysis were carried out. Mutations were detected in 25 additional arRD families and 10 additional arCC families by this approach. Since selection of both families and candidate loci was unbiased, this approach allows an accurate estimate of the contributions of various genes to arCC and arRD in Pakistan. Overall, in the Pakistani population, FYCO1 contributes about 15% and Crystallins about 7% of the arCC burden, with no other gene contributing more than 3% of cases of arCC. No currently identified gene or locus appears to contribute more than 3% of cases of arRD. The types and frequencies of mutations seen in arRD and arCC in Pakistan are similar to those reported in Western populations. Finally, approximately 58% of arRP and 65% of arCC families were excluded from known genes and loci, suggesting that this population remains a largely untapped resource for identification of novel genes contributing to arRD and arCC.
NRL Downstream Target Syntrophin α1 is Essential for Rod Photoreceptor Presynaptic Development.
Soo-Young Kim1, D. Thad Whitaker1, Christine Park1, Juthaporn Assawachananont1, Matthew Brooks1, Andreas Klingenhoff5, Yichao Li2, Haohua Qian2, Tiziana Cogliati1, Robert N. Fariss3, Wei Li4, Anand Swaroop1
1Retinal Development, Genetics, & Therapy Section, Neurobiology-Neurodegeneration & Repair Laboratory, 2Visual Function Core, 3Biological Imaging Core, 4Retinal Neurophysiology Section, National Eye Institute, National Institutes of Health; 5Genomatix Software GmbH, Germany
The mammalian brain displays a wide variety of neuronal types with characteristic morphologies. Questions about the development of neuronal diversity and the formation of specific circuits remain for the most part unanswered. Retinal photoreceptors are highly specialized neurons that detect photons and transduce visual signals to second-order neurons, ultimately transmitting visual information to the brain. Presynapses of rod and cone photoreceptors, called spherules and pedicles respectively, have specialized ribbon structures, which are different from conventional synapses in the brain. Furthermore, spherules and pedicles exhibit distinct morphologies and connect with defined sets of bipolar neurons in separate sublaminae of the retinal outer plexiform layer (OPL). However, the differential molecular mechanisms that determine spherule and pedicle ribbon synapses remain unknown. Here, we investigate the molecular mechanisms underlying presynaptic morphogenesis and OPL sublamination of rod photoreceptors. We report that the transcription factor NRL, a rod photoreceptor-fate determinant, and its downstream effectors are critical for rod photoreceptor presynaptic morphogenesis. We found that Nrl-/- photoreceptors display pedicle-like synaptic forms that terminate in the lower portion of the retinal OPL, below rod bipolar dendritic tips, like wild type pedicles. To detect candidate genes that regulate presynaptic morphogenesis of rod photoreceptors, we performed gene ontology analysis of NRL downstream targets (containing NRL ChIP-Seq peaks) that are enriched in developing rod photoreceptors (by differential expression in Nrl-GFP vs. Nrl-/-/Nrl-GFP sorted cells). shRNA screening by in vivo electroporation was used to identify genes affecting synaptic morphogenesis in rod photoreceptors. We found that neurofascin (Nfasc), alpha2, 8-sialytransferase1 (St8sia1), phospholipase 2 group7 (Pla2g7), alpha1-syntrophin (Snta1) and beta2-syntrophin (Sntb2) are involved in spherule morphogenesis. Finally, we have examined the retinas of Snta1-/- and Sntb2-/- mice. Our data indicate that the rod-fate determinant transcription factor NRL and its targets control synaptic morphogenesis and connections to specific neuron types.
Light Guiding by Cone Photoreceptor Mitochondria
John Ball, Wei Li
Retinal Neurophysiology Section, National Eye Institute, National Institutes of Health
The vertebrate retina possesses an inverted structure in which light must pass through each retinal layer before finally reaching light-sensitive photoreceptors in the deepest layer. This feature might be expected to cause scattering of photons entering the eye, thus compromising visual acuity. Recently, it has been discovered that the retina might possess an adaptation to overcome this difficulty; In the guinea pig retina, Müller cells, support cells that span all layers of the retina, funnel incident light toward cone photoreceptors, increasing the flux of photons into cones by as much as 5- to 10-fold. However, light that enters a cone must still pass through its cell body and inner segment of the cone itself before being absorbed by photosensitive pigment in its outer segment. We recently obtained high-resolution images of ground squirrel cones using serial block-face electron microscopy (SBEM). The ground squirrel is a valuable animal model because its retina, like the human fovea, is dominated by cones (approximately 90%). The obtained SBEM images revealed cone inner segments that are densely packed with large mitochondria—analysis suggests a packing density of the inner segment by 70-80%. These mitochondria, many of which span the entire length of the inner segment, exhibit cylindrical shapes and are largely well aligned with the main axis of the inner segment. This orientation evokes the notion of a bundle of biological light guides that aligns photons for absorption in the cone outer segment. Despite this hypothesis, the question remains: Do these dense mitochondria enhance light transmission to cone outer segments, or do they instead scatter light, opposing any light funneling by Müller cells? To address this question, we directly utilized 3D reconstructions of cone mitochondria in electromagnetic simulations of light propagation. These simulations were performed in MEEP, a free finite-difference time-domain (FDTD) package typically used in optics device engineering applications. These simulations indicated that light entering a cone inner segment is focused by its bundle of mitochondria into a volume corresponding to the approximate location of the photosensitive cone outer segment. This focusing, which was largely wavelength-independent, increased the flux of light through the outer segment by as much as 6-fold. Taken in the context of the previous findings concerning Müller cell light focusing, these simulations suggest that cumulatively, instead of scattering light and hindering visual acuity, the inverted structure of the retina might focus light onto cone outer segments by 30- to 60-fold more than what would be expected if light were distributed uniformly across the photoreceptor layer.
Increased Mature CD1c+ mDC1 Reflect Disease Activity in Non-infectious Uveitis
Ping Chen1, Alastair Denniston2,3, Susan Hannes1, William Tucker1, Lai Wei1, Baoying Liu1, Tiaojiang Xiao4, Sima Hirani1, Zhiyu Li1, Shayma Jawad1, Richard Lee5, H. Nida Sen1, Robert Nussenblatt1
1Clinical Immunology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health; 2Ophthalmology Department, Queen Elizabeth Hospital Birmingham, University Hospitals Birmingham NHSFT, UK; 3Centre for Translational Inflammation Research, University of Birmingham, UK; 4Physical Chemistry Section, Laboratory of Molecular Biology, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health; 5Department of Clinical Sciences, University of Bristol, Bristol, UK
In this study we investigated the role of blood CD1c+ myeloid dendritic cells 1 (mDC1), a key mDC subtype, in patients with non-infectious uveitis. We observed a significant increase of blood CD1c+ mDC1 in uveitis patients. The increased CD1c+ mDC1 exhibited high HLADR expression and less antigen uptake. CD1chi mDC1 subpopulation showed less antigen uptake and higher HLADR expression compared to CD1clo mDC1 subpopulation. Importantly, the CD1chi mDC1 subpopulation was increased in uveitis patients. We found that mature monocyte-derived dendritic cells (MoDCs), characterized by lower levels of antigen uptake, induced more CD62L-CD4+ T helper cell proliferation. We also provide evidence that the mature phenotype and function of CD1c+ mDC1 may be regulated by TNFα via a p38 MAPK-dependent pathway. These data show that alterations in the systemic immune response are involved in the pathogenesis of human uveitis and invite the therapeutic possibility of attenuating uveitis by manipulating blood CD1c+ mDC1.
It Catches the Eye: Influence of Object Experience on Gaze Orientation
Ali Ghazizadeh, Okihide Hikosaka
Neuronal Networks Section, Laboratory of Sensorimotor Research, National Eye Institute, National Institutes of Health
Let us consider the problem facing a hungry monkey in a fruit tree. He wants to find the ripe fruits before the competition and avoid unripe sour ones. How does he process the crowded visual information to find the ripe fruits among all the leaves, twigs and other distracters? To answer this question, we trained macaque monkeys in an object-reward association task in which half of visual fractals were associated with a large reward (good fractals) and the other half associated with a small reward (bad fractals). On each trial, one fractal was presented and the monkey made a saccade to it to obtain the reward. After extensive training with over 100 fractals, performance was tested in a search task. Multiple fractals (n=3,5,7,9) were simultaneously presented to the monkey after central fixation. A single good object was present in half of the trials. Surprisingly, monkeys made a saccade quickly and accurately to the good fractal (if present) regardless of the number of bad fractals. This independence from object number suggests that the values of peripheral objects are processed simultaneously (parallel search) rather than by sequential scanning (serial search). Interestingly, the saccades to good fractals occurred even when there was no reward. When good and bad fractals appeared, but with no reward outcome, monkeys preferentially looked at good fractals. This gaze preference remained intact months after the last training. This suggests that good fractals have become salient in and of themselves. To investigate the neural mechanism underlying object salience, we performed single unit recording as well as fMRI while monkeys viewed good and bad fractals. Our results suggest that both the cerebral cortex (prefrontal and temporal) as well as the basal ganglia (caudate and substantia nigra) contribute to the automatic detection of good objects and orientation toward them.
Study of Mitochondrial Function in Aging and Degenerating Mouse Retina by a Novel Ex Vivo Assay
Keshav Kooragayala1, Norimoto Gotoh1, Jacob Nellissery1, Tiziana Cogliati1, Wei Li2, Raul Covian Garcia3, Robert Balaban3, Anand Swaroop1
1Retinal Development, Genetics, & Therapy Section, Neurobiology-Neurodegeneration, & Repair Laboratory, 2Retinal Neurophysiology Section, National Eye Institute; 3Laboratory of Cardiac Energetics, National Heart, Lung, & Blood Institute, National Institutes of Health
Purpose
Mitochondrial dysfunction and oxidative stress are believed to play an important role in the manifestation of retinal degeneration and loss of vision. We have optimized an ex vivo assay that uses freshly isolated retinal punches to measure oxygen consumption by microplate-based respirometry. This assay allows the quantification of changes in mitochondrial respiration associated with retinal distress.
Methods
Oxygen consumption rate (OCR), an indicator of oxidative phosphorylation, was measured by the Seahorse XF24 Bioanalyzer. “Punches” of retinal tissue (1 mm diameter) were placed in XF24 Islet FluxPak microplates containing Ames medium (with glucose and pyruvate) or a basic salt solution supplemented with different combinations of substrates (i.e., glucose, pyruvate, lactate, insulin, β-hydroxybutyrate). Two mitochondrial uncouplers, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine (Bam15), were injected during the assay. A complex I inhibitor, rotenone, was administered to terminate the assay. Values were background-subtracted (determined by rotenone administration), to account for non-mitochondrial respiration, and were normalized to cytochrome a content.
Results
OCR was measured in the presence of various combinations of substrates; Ames medium optimally maintained a steady baseline OCR throughout the experiment. To determine the maximal uncoupled OCR, FCCP or Bam15 was injected into the medium after five consecutive basal recordings. At maximal uncoupling concentration, both drugs induced a 15-20% increase of OCR. Retina isolated from 18 and 24 month-old mice showed a similar basal OCR to young mice but exhibited a higher rate of maximally uncoupled OCR.
Conclusions
We detected a relatively low induction of maximum OCR upon uncoupling, suggesting that the retina operates near its peak metabolic capacity. Furthermore, maximum retinal respiration seems to be unaffected or even improved with age. We propose OCR measurement in retinal punches as a convenient and reliable method to study mitochondria function in retinal aging and disease models.
Posters
Identification of a Specific Set of Membrane Biomarkers for the Purification of Retinal Pigment Epithelium from Differentiated Induced Pluripotent Stem Cells
Alejandro Morales Martinez1, Vladimir Khristov2, Andrea Li1, Ruchi Sharma1, Fang Hua2, Omar Memon2, Arvydas Maminishkis2, Daniel Riordon3, Rebekah Gundry4, Kenneth Boheler5, Sheldon Miller2, Kapil Bharti1
1Unit on Ocular & Stem Cell Translational Research, Ophthalmic Genetics & Visual Function Branch, 2Section on Epithelial & Retinal Physiology & Disease, National Eye Institute, 3Cardiac Function Section, Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health; 4Department of Biochemistry, Medical College of Wisconsin; 5Department of Physiology, University of Hong Kong.
Gut Microbial Signals Associated with Triggering Spontaneous Autoimmune Disease in the Retina
Carlos Zárate-Bladés1, Reiko Horai1, Patricia Dillenburg-Pilla2, Nadim Ajani3, Mathew Wong3, Phyllis Silver1, Jun Chen1, Yingyos Jittayasothorn1, Chi-Chao Chan4, Hidehiro Yamane5, Joseph Petrosino3, Kenya Honda6, Rachel Caspi1
1Immunoregulation Section, Laboratory of Immunology, National Eye Institute, 2Oral & Pharyngeal Cancer Branch, National Institutes of Dental & Craniofacial Research, National Institutes of Health; 3Alkek Center for Metagenomics & Microbiome Research (CMMR), Department of Molecular Virology & Microbiology, Baylor College of Medicine; 4Immunopathology Section, Laboratory of Immunology, National Eye Institute, 5General Immunology Section, Laboratory of Immunology, National Institute of Allergy & Infectious Disease, National Institutes of Health; 6RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.
Colobomatous Microphthalmia, Macrocephaly, Albinism & Deafness (COMMAD syndrome), a New Syndrome Caused by Biallelic Mutation of MITF: Clinical Characterization and Molecular Analysis
Aman George1, Mariana Rius1, Ramakrishna Alur1, Dina Zand2, Yuri Sergeev1, Kapil Bharti3, Brian Brooks1
1Section on Pediatric, Developmental, & Genetic Ophthalmology, Ophthalmic Genetics & Visual Function Branch, National Eye Institute, National Institutes of Health, 2Department of Genetics, Children’s National Medical Center, Washington, D.C., 3Unit on Ocular & Stem Cell Translational Research, Ophthalmic Genetics & Visual Function Branch, National Eye Institute, National Institutes of Health
Thirteen-lined Ground Squirrel Induced Pluripotent Stem Cells – To Study Hibernation in a Culture Dish
Jingxing Ou1, Barbara Mallon2, Kiyoharu Miyagishima1, Dana Merriman3, Wei Li1
1Unit of Retinal Neurophysiology, National Eye Institute, 2Stem Cell Unit, National Institutes of Health, 3Department of Biology, University of Wisconsin at Oshkosh
Modeling Late-Onset Retinal Degeneration with Human Induced Pluripotent Stem Cells (iPSCs): Insights into the Shared Pathogenesis of Retinal Degenerative Diseases
Katharina Clore-Gronenborn1, Kiyoharu Miyagishima2, Congxiao Zhang2, Vaiskh Rajan2, Jason Silver2, Qin Wan2, Ruchi Sharma1,Catherine Cukras3, Paul Sieving4, Sheldon Miller2, Kapil Bharti1
1Unit on Ocular & Stem Cell Translational Research, Ophthalmic Genetics & Visual Function Branch, 2Section on Epithelial & Retinal Physiology & Disease, 3Clinical Trials Branch, Division of Epidemiology & Clinical Applications, National Eye Institute, 4Section for Translational Research on Retinal & Macular Degeneration, National Institute on Deafness & Other Communication Disorders, National Institutes of Health
Characterization of Retbindin, a Novel Component of Mammalian Photoreceptors
Shruti Shankar1, Sanghamitra Mishra1, Mercedes Campos2, Todd Duncan3, Jianguo Fan1, Katherine Peterson1, Graeme Wistow1
1Molecular Structure & Functional Genomics Section, 2Biological Imaging Core, 3Molecular Mechanisms Section, Laboratory of Retinal Cell & Molecular Biology, National Eye Institute, National Institutes of Health
Optimizing Biodegradable Scaffolds for Developing Induced Pluripotent Stem Cells (iPSC)-Derived Retinal Pigment Epithelium (RPE)Tissue
Vladimir Khristov1, Juliet Hartford2, Qin Wan1, Mostafa Lotfi1, Kiyoharu Miyagishima1, Arvydas Maminishkis1, Juan Amaral3, Sheldon Miller1, Janine Davis2, Kapil Bharti2
1Section on Epithelial & Retinal Physiology & Disease, 2Unit on Ocular & Stem Cell Translational Research, Ophthalmic Genetics & Visual Function Branch, 3Mechanisms of Retinal Diseases Section, Laboratory of Retinal Cell & Molecular Biology, National Eye Institute, National Institutes of Health
Mutated Myocilin and Oxidative Stress Act Synergistically in a Mouse Model of Open-angle Glaucoma
Myung Kuk Joe1, Naoki Nakaya1, Mones Abu-Asab2, Stanislav Tomarev1
1Section on Retinal Ganglion Cell Biology, Laboratory of Retinal Cell & Molecular Biology, 2Histology Core, National Eye Institute, National Institutes of Health
Neuronal Correlates of Change Detection in Basal Ganglia during Covert Spatial Attention
Fabrice Arcizet, Richard Krauzlis
Section on Eye Movements & Visual Selection, Laboratory of Sensorimotor Research, National Eye Institute, National Institutes of Health
Induction of Primary Cilium Helps in Development of Fully Polarized and Mature Retinal Pigment Epithelium (RPE)
Ruchi Sharma1, Juliet Hartford1, Qin Wan2, Helen May-Simera3, Jason Silver2, Janine Davis1, Kiyoharu Miyagishima2, Vladimir Khristov2, Andrea Li1, Kapil Bharti1
1Unit on Ocular & Stem Cell Translational Research, Ophthalmic Genetics & Visual Function Branch, 2Section on Epithelial & Retinal Physiology & Disease, 3Retinal Cell Biology & Degeneration Section, Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health
Development of Induced Pluripotent Stem Cells (iPSCs)-based Therapies for Blinding Retinal Neurodegenerative Diseases
Hiroko Shimada-Ishii1, Jacklyn Mahgerefteh1, Guokai Chen2, Rossukon Kaewkhaw1, Jeanette Beers2, Kohei Homma1, Mahendra Rao3, Tiziana Cogliati1, Brian Brooks4, Samuel Jacobson5, Anand Swaroop1
1Retinal Development, Genetics, & Therapy Section, Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, 2iPSC & Genome Engineering Core Facility, National Heart, Lung, & Blood Institute, 3National Center for Regenerative Medicine, National Institute of Arthritis & Musculoskeletal & Skin Diseases, 4Section on Pediatric, Developmental, & Genetic Ophthalmology, Ophthalmic Genetics & Visual Function Branch, National Eye Institute, National Institutes of Health; 5Center for Hereditary Retinal Degenerations & Retinal Function Department, Scheie Eye Institute, University of Pennsylvania
Leucine-rich Repeat Kinase 2 (LRRK2) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU)
Sylvia Wandu1, Osato Ogbeifun1, Guangpu Shi1, Samuel J.H. Hinshaw1, Cuiyan Tan1, Huaibin Cai2, Igal Gery1
1Experimental Immunology Section, Laboratory of Immunology, National Eye Institute, 2Transgenisis Section, Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health
Separate Groups of Dopamine Neurons Project to Caudate Head and Tail
Hyoung Kim, Ali Ghazizadeh, Okihide Hikosaka
Neuronal Networks Section, Laboratory of Sensorimotor Research, National Eye Institute, National Institutes of Health
Using Stem Cells from Mouse Models to Elucidate Pathogenesis of Leber Congenital Amaurosis (LCA) Caused by CEP290 Mutations
Holly Y Chen1, Pinghu Liu2, Milton English1, Lijin Dong2, Anand Swaroop1
1Retinal Development, Genetics, & Therapy Section, Neurobiology-Neurodegeneration & Repair Laboratory, 2Genetic Engineering Core, National Eye Institute, National Institutes of Health