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2009 Focus on Fellows Abstracts


The Role of Dopamine in Information-Seeking

Ethan Bromberg-Martin, Okihide Hikosaka
Neuronal Networks Section, Laboratory of Sensorimotor Research,

The purpose of the eyes is to see; and the purpose of sight is to gain information about the surrounding world. Our eyes dance in constant motion, a ceaseless pursuit of information that begins the day we are born and continues every waking moment of our lives. Yet it is unknown how this search for information is motivated by neurons in the brain.

Here we will present a candidate for this role in the spiking activity of midbrain dopamine neurons. Dopamine neurons are known to be crucial for motivated behavior in pursuit of basic forms of reward such as food and water. We recorded dopamine neuron activity while rhesus monkeys performed an eye movement task to gain water rewards. We found that animals preferred to gain advance information about the size of upcoming rewards - as if information about the reward size was itself rewarding. Furthermore, the same dopamine neurons that signaled the animal's anticipation of water rewards also signaled the animal's anticipation of information about those rewards. On days when dopamine neurons had stronger information-anticipating signals, animals had stronger information-seeking behavior. This unified neural code for "appetitive" and "informational" rewards suggests that dopamine neurons motivate both reward-seeking and information-seeking, and do so through a common neural mechanism.

Local Mesenchymal Stem Cell Transplantation Confers Neuroprotection in Experimental Glaucoma

Thomas Johnson, Natalie Bull, David Hunt, Keith Martin, Stanislav Tomarev Cambridge Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK, Molecular Mechanisms of Glaucoma Section, Laboratory of Molecular and Developmental Biology, National Eye Institute, NIH

Glaucoma, a group of chronic optic neuropathies characterized by retinal ganglion cell (RGC) death and progressive visual field loss, is a leading cause of irreversible blindness worldwide. Retrograde neurotrophic factor transport blockade has been implicated in glaucomatous pathophysiology. Stem cell transplantation ameliorates some neurodegenerative conditions in the brain and spinal cord, apparently by neurotrophic factor secretion. The current study was conducted to evaluate bone marrow-derived mesenchymal stem cell (MSC) transplantation as a novel neuroprotective glaucoma therapy.

MSCs were isolated from adult transgenic rats that ubiquitously express green fluorescent protein. In vitro, MSCs were co-cultured with organotypic retinal explants for one week and RGC survival was assessed by immunohistochemistry. In vivo, MSCs were transplanted intravitreally one week before, or intravenously on the day of, ocular hypertensive glaucoma induction by trabecular meshwork laser photocoagulation. Four weeks later, ocular MSC localization and integration were determined by immunohistochemistry. Optic nerve damage was assessed by axon quantification within optic nerve cross-sections.

In vitro, MSCs proliferated on the vitreal surface of retinal explant tissue, but minimal engraftment occurred. In this system, MSC transplantation increased RGC survival significantly by 2-3-fold. In vivo, following intravitreal transplantation, MSCs survived for at least five weeks. Cells were found mainly in the vitreous cavity, though a small proportion of cells migrated into the host retina. Intravitreal MSC transplantation resulted in a significant >65% reduction in optic nerve axon loss and a significant >50% reduction in the rate of optic nerve degeneration normalized to cumulative intraocular pressure exposure. Following intravenous delivery, MSCs did not migrate into the glaucomatous eye and had no effect on pathology.

We concluded that MSCs were neuroprotective when cultured with retinal tissue in vitro and when transplanted locally but not systemically in a rat glaucoma model in vivo. Autologous intravitreal MSC transplantation should be considered as a potential neuroprotective therapy for glaucoma.

PDGF-CC rescues neurons by regulating GSK3ß phosphorylation

Zhongshu Tang, Pachiappan Arjunan, Chunsik Lee, Yang Li, Anil Kumar, Xu Hou, Bin Wang, Piotr Wardega, Fan Zhang, Lijin Dong, Yongqing Zhang, Shi-Zhuang Zhang, Hao Ding, Kevin Becker, Johan Lennartsson, Yihai Cao, Nobuo Nagai, Xuri Li
Unit on Retinal Vascular Neurobiology, NEI/NIH, Weifang Medical University, Shandong, P. R. China; Ludwig Institute for Cancer Research, Uppsala University, Sweden; NIA/NIH, Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada; Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden; Department of Physiology, Kinki University School of Medicine, Osaka, Japan

Platelet-derived growth factor-CC (PDGF-CC) is the third member of the PDGF family discovered after more than two decades’ studies on PDGF-AA and PDGF-BB. The biological function of PDGF-CC remains largely to be explored. We report here a novel finding that PDGF-CC is a potent neuroprotective factor by modulating glycogen synthase kinase (GSK)3β activity. In several neuro-injury animal models, PDGF-CC protein or gene delivery protected neurons from apoptosis in both retina and brain, while PDGF-CC inhibition or deficiency exacerbated neuronal death. Mechanistically, we revealed that the neuroprotective effect of PDGF-CC was achieved by regulating GSK3β phosphorylation and expression. Our data demonstrated that (1) PDGF-CC is critically required for neuronal survival and may potentially be used to treat neurodegenerative diseases. (2) Inhibition of the PDGF/receptor pathway for different clinical purposes should be conducted with caution to preserve normal neuronal functions.

Molecular and functional changes associated with normal aging in the retina

Jessica Chang, Sunil Parapuram, Radu Cojocaru, Matthew Brooks, Tiziana Cogliati, Ritu Khanna, Naheed Khan, Jerome Roger, Bo Chang, Anand Swaroop
Neurobiology-Neurodegeneration and Repair Laboratory

Age is a major risk factor for age-related macular degeneration and other retinopathies, and it has been suggested that rod photoreceptors are particularly vulnerable to the effects of aging. This study aims to elucidate changes in gene expression associated with normal aging of rod photoreceptors. Flow cytometry was used to enrich for Green Fluorescent Protein (GFP)-tagged rod photoreceptors from dissociated retinas taken from mice at 1.5, 5, and 12 months of age. RNA was extracted from purified rod photoreceptors and gene expression profiles were generated using Affymetrix microarray chips. Quantitative polymerase chain reaction and immunohistochemistry were used to confirm expression level changes in select genes. Changes associated with age were found in the expression levels of several genes associated with mitochondrial function, lipid metabolism, and cellular proliferation, among others. To complement these studies, electroretinograms (ERGs) were also performed to measure retina functional change with age. Consistent with previous findings in mice and humans, ERGs showed decreases in scotopic a and b waves, and in photopic b waves with increasing age.

Aging is a complex process that varies between cell types within the same individual. Evaluating gene profile changes associated with aging in a single postmitotic cell type should allow resolution of subtle changes with age that might otherwise be obscured in whole-tissue gene profiling. The candidate genes found here provide a foundation for future experiments to explore potential pathways of aging and related functional adaptation.

Generation and unique features of antigen-specific “Th9” cell lines

Cuiyan Tan, Barbara Vistica, Guangpu Shi, Jenna Lovaas, Mehak Aziz, Igal Gery Experimental Immunology Section, Laboratory of Immunology

T helper (Th) lymphocytes play an important role in the immune system. Research in recent years has shown that naïve lymphocytes can differentiate into lineages (Th1, Th2, Th17, and Treg cells) with distinct effector functions and signature cytokines. Two recent publications (Nat. Immunol., 2008, 9:1341; 1347) reported the generation of Th cell lines that specifically express high levels of IL-9. These lines were generated by activation of naïve lymphocytes with antibodies against surface antigens (CD3/CD28), in the presence of the polarizing cytokines IL-4 and TGF-. “Th9” line cells also produced considerable levels of IL-10. The mode of activation used in these studies generated non-antigen specific lymphocyte lines. Here, we generated lines of antigen-specific “Th9” cells, by activating T-cells transgenically expressing T-cell receptor (TCR) specific to hen egg lysozyme (HEL), with this antigen in the presence of the polarizing cytokines IL-4 and TGF-. Production of cytokines by the line cells was determined at different time points by measuring their mRNA transcript and the proportions of cells positive for intracellular staining. Profound differences were seen between the kinetics of IL-9 and IL-10 production. IL-9 was expressed at the early phase of culture, with the highest levels of its mRNA transcript being found on days 2 and 3 and its peak of intracellular staining (~45% positive cells) on day 3, followed by sharp decline of these two parameters thereafter. In contrast, IL-10 transcript expression was negligible on day 2, increased by day 3 and remained at the elevated level till day 6. Strikingly, unlike IL-9, intracellular IL-10 was marginal on day 2 and gradually increased, to ~40%, on day 6. To determine the plasticity of the “Th9” lines, we switched their medium to cytokine “cocktails” used to polarize Th1 or Th17 lines. The great majority of “Th9” cells retained their phenotype, thus indicating the relative stability of the “Th9” cell lines.

ASCC3L1 Gene Mutation in a Chinese Family with Autosomal Dominant Retinitis Pigmentosa

Ningdong Li, Han Mei, XiaoDong Jiao, Ian MacDonald, Kanxing Zhao and J. Fielding Hejtmancik
Tianjin Eye Hospital, Tianjin, China, Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health Department of Ophthalmology, University of Alberta, Edmonton, Canada

The purpose of this study was to localize and identify the pathogenic gene in a Chinese Family with autosomal dominant retinitis pigmentosa. A Chinese family with adRP was ascertained and patients underwent complete ophthalmological examinations. Blood samples were collected and DNA was extracted after informed consent. A linkage scan of genomic regions containing known candidate genes was performed using 54 polymorphic microsatellite markers on genomic DNA from affected and unaffected family members and lod scores were calculated. Candidate genes were sequenced and mutations analyzed.

A genome wide scan a lod score of 3.5 at θ = 0 with D2S2333 and 3.46 at θ = 0 with D2S2216. This region harbors the ASCC3L1 gene. Sequencing of ASCC3L1 shows a heterozygous single base pair change; c.3269G > T, single base pair change predicted to result in a R1090L amino acid change. These results provide strong evidence that mutations in ASCC3L1 result in autosomal dominant retinitis pigmentosa in this Chinese family.

RPE65: Adaptation of a Carotenoid Oxygenase to a Visual Cycle Isomerase Function

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

The retinal pigment epithelial layer of the eye harbors a crucial visual cycle enzyme called RPE65. This 61kDa protein plays an indispensible role in the regeneration of 11-cis retinal in the visual cycle, the compound that confers sensitivity to light on rhodopsin to make vision happen. Without RPE65 activity, patients suffer Leber Congenital Amaurosis (LCA) and retinal dystrophies with symptoms such as night blindness and severe loss of rod and cone photoreceptor sensitivity. The spectrum of conditions ranges from rapidly progressing severe early-onset forms to milder late-onset slowly progressing forms, depending on the type of mutation. Understanding the mechanism of action of this visual isomerase has been a focus of the Molecular Mechanisms section, LRCMB. RPE65, a unique member of the carotenoid oxygenase family of enzymes, is the only mammalian/vertebrate protein known to date that is evolved to perform a retinoid isomerase function. Comparative analyses with related enzymes of this family, BCMO1 and ACO, have aided in understanding the structural details of the adaptation of the RPE65 to perform its isomerase activity. Using a model-based approach, residues crucial for RPE65 activity have been identified and tested for functionality. Site-directed mutagenesis of conserved groups of residues such as catalytic iron coordinating residues have established fundamental mechanisms in the function of carotenoid oxygenases as well as illuminating previously unknown concepts in the visual system. We now show that conservation of aromatic amino acid residues in the substrate-binding cleft of RPE65 helps modulate isomerase specificity. This in turn defines the chemistry underlying vitamin A isomerization in the vertebrate eye and provides a common mechanism for all the carotenoid oxygenases. The molecular model is expected to help further understand the structure-function relationship of the isomerase activity of RPE65.

The Protective Effect of Quercetin Against Oxidative Stress in Cultured Human RPE and Retinal Lesions of Ccl2/Cx3cr1 Double Deficient Mice

Xiaoguang Cao, Defen Shen, Jingsheng Tuo, Youngeun Cho, Chi-Chao Chan Immunopathology Section, Laboratory of Immunology

Oxidative stress in retinal pigment epithelium (RPE) plays a role in age-related macular degeneration (AMD) development. The flavonoid quercetin has been extensively studied for its antioxidant effect. This study investigates the mechanisms of the protective effect of quercetin on cultured human RPE cells and in Ccl2/Cx3cr1 double deficient (DKO) mice, which spontaneously develop retinal lesions mimicking AMD.

Cultured ARPE-19 cells were exposed to 1mM H2O2 with or without 50µM quercetin for 2 hours. Cellular viability was measured by MTT assay, and apoptosis was detected by Comet assay. Expression of Bcl-2, Bax, p53, Caspase-3 and -9 mRNA were measured by RQ-PCR. DKO and wild type (WT) mice were randomly divided into quercetin-treated and vehicle control groups. Quercetin in 40% v/v DMSO/PBS was injected i.p. (25mg/kg/day) for two months. Funduscopy was performed monthly. After treatment for two months, the mice were sacrificed. Serum from each mouse was collected for COX, PGE-2 measurement, and nitrite. The eyes were harvested for histology. Expression of bcl-2, bax, p53, cox-2, and iNOS mRNA in DKO and WT retinas were also measured by RQ-PCR.

H2O2-treated RPE cells showed a significant decrease in cell viability, whereas quercetin diminished this decrease. H2O2-induced apoptosis was significantly attenuated by quercetin by Comet Assay. The ratio of Bcl-2/Bax mRNA expression decreased after H2O2 treatment (0.77-fold), which was reversed by quercetin treatment (3.08-fold). H2O2 also increased caspase-3 and -9 levels. This effect was abolished by quercetin, from 2.09- and 1.83- to 1.26- and 1.32-fold, respectively. Furthermore, quercetin stabilized p53 mRNA expression in H2O2-treated cells. Funduscopy of DKO receiving quercetin showed less retinal lesions than that of controls. Quercetin lowered serum COX from 0.53+/-0.23 to 0.27+/-0.21nmol/min/ml, PGE-2 from 95.41+/-5.34 to 85.98+/-8.44pg/ml, and nitrite from 24.49+/-13.94 to 11.32+/-3.04µM. Retinal expression of bax in DKO was significantly increased as compared with WT, and quercetin suppressed the increase.

Quercetin is able to protect RPE cells against oxidative damage. Quercetin slows the progression of retinal lesions in the DKO, which may act via suppression of the COX/PGE-2 pathway and the intrinsic apoptotic signal pathway. Therefore, we recommend taking antioxidative dietary flavonoids such as quercetin to prevent the development of AMD.

The N-terminal Extension of βB1-crystallin: Identification of a Critical Region Which Modulates Protein Interactions with βA3-crystallin

Monika Dolinska, Yuri Sergeev, May Chan, Ira Palmer, Paul Wingfield
National Eye Institute and 2National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892

The human lens proteins β-crystallins are subdivided into acidic (βA1-βA4) and basic (βB1-βB3) subunit groups. These structural proteins exist at extremely high concentrations and associate into oligomers under physiological conditions. Crystallin acidic-basic pairs tend to form strong heteromolecular associations. The long N-terminal extensions of ß-crystallins may influence both homo- and heteromolecular interactions. However, identification of the critical regions of the extensions mediating protein associations has not been previously addressed. This was studied by comparing the self-association and heteromolecular associations of wild-type recombinant βA3- and βB1-crystallins and their N-terminally truncated counterparts

(βA3ΔN30 and βB1ΔN56) using several biophysical techniques, including analytical ultracentrifugation and fluorescence spectroscopy. Removal of the N-terminal extension of βA3 had no effect on dimerization or heteromolecular tetramer formation with ßB1. In contrast, the level of self-association of βB1ΔN56 increased, resulting in homotetramer formation, and heteromolecular association with βA3 was blocked. Limited proteolysis of βB1-produced βB1ΔN47, which is similar to intact protein formed dimers but in contrast showed enhanced heteromolecular tetramer formation with βA3. The tryptic digestion was physiologically significant, corresponding to protease processing sites observed in vivo. Molecular modeling of the N-terminal βB1 extension indicates structural features that position a mobile loop in the vicinity of these processing sites. The loop is derived from residues 48-56 which appear to be critical for mediating protein interactions with βA3-crystallin.

ADRP-associated human NRL mutations in codons 50 and 51 affect photoreceptor survival under bright light exposure in mouse retina.

Jerome Roger, Keerthi Ranganath, Jessica Chang, Avinash Hiriyanna, Anand Swaroop Neurobiology-Neurodegeneration and Repair Laboratory

NRL (Neural Retina Leucine zipper) is the key transcription factor, essential and sufficient, for rod differentiation in mouse retina. Indeed, rods are transformed to functional S-cones in Nrl-/- mouse retina. NRL is a member of the Maf transcription factor family and its activity is regulated by post-translational modifications. We have demonstrated that multiple phosphorylated isoforms of NRL are expressed in rod photoreceptors. Previous studies showed that two mutations in codons 50 (S50T) and 51 (P51S) are associated with ADRP in human. In cultured cells, these 2 mutations alter NRL phosphorylation and lead to increased NRL transcriptional activity. This study was undertaken to create mouse models of human autosomal dominant Retinitis Pigmentosa (adRP) resulting from S50T and P51S mutations in NRL and elucidate mechanism(s) of photoreceptor cell death.

We generated 2 transgenic lines expressing mouse p.S50T or p.P51S under the control of 2.5kb Nrl promoter, named Nrlp::S50T and Nrlp::P51S, respectively. Mice were mated to Nrl knockout mice. ERGs were performed to assess retinal function. For light exposure experiments, mice were exposed under bright light (10,000 Lux) for one hour, and eyeballs were harvested 2 weeks later. Retinal degeneration was assessed by immunohistochemistry.

We identified by western blot analysis, founders for each lines expressing different amounts of NRL protein. In each case, expression of NRL mutant protein in Nrl knockout mice resulted in the conversion of cones back to rods. The retinas of these mice were indistinguishable from wild type retina demonstrating that these two mutations do not affect essential NRL function during rod differentiation. In contrast with the human disease, these two mutations didn’t lead on their own to retinal degeneration even in more than one-year-old mice. However, retinal degeneration was observed after bright light exposure. Very strong Muller glial cells activation was also observed.

We show that S50T or P51S mutations don’t affect rod differentiation and photoreceptor survival under normal conditions. Nonetheless, the mutant NRL proteins somehow affect rod survival under bright light exposure suggesting the importance of phosphorylation in controlling NRL function under stress conditions.

Photoreceptor/RPE Interactions Mediated by Metabolic Waste

Jeffrey Adijanto, Qin Wan, Nancy Philp, Rong Li, Tina Banzon, Sheldon Miller
Section on Epithelial and Retinal Physiology and Disease

The detection and conversion of light quanta into information by the visual systems is energy expensive. Therefore, a high retinal metabolism is needed to maintain the Na dark-current and the flow of retinal information (i.e., phototransduction). Most of this energy is provided by mitochondria that are densely packed in the photoreceptor inner segments. More than 80% of all glucose metabolized by the retina is converted to lactic acid while the remainder is converted to CO2 and water, and these waste products are released to the extracellular space or subretinal space (SRS).

Acid accumulation within the SRS causes acidosis that is detrimental to all of the retinal neurons and retinal pigment epithelium (RPE). Fortunately, the RPE can protect the photoreceptors from this acid load by removing lactic acid from the subretinal space. To facilitate this process, the RPE expresses proton-coupled lactate transporters at the apical (MCT1) and basolateral (MCT3) membranes. Interestingly, lactic acid also affects other ion channels and transporters in the RPE—these are part of a process that helps the RPE adapt to changes in photoreceptor activity following light/dark transitions. By using intracellular pH and microelectrode recordings, we show that apical lactate entry stimulates various potassium and chloride channels at the RPE membrane surfaces. In addition, the apical lactic acid load also activates proton-extrusion mechanisms such as the Na/H exchanger. Without these ion-transport mechanisms, the ionic homeostasis within the subretinal space would be disrupted, and compromise visual function. In this talk, I present experiments from our recent study that illustrates the interplay between lactic acid transport and various ion transport mechanisms in the RPE. The role of these mechanisms in maintaining normal photoreceptor function will also be discussed.

Push-pull organization of binocular receptive fields robustly encodes disparity in monkey V1

Seiji Tanabe, Bruce G. Cumming
Vision Section, Laboratory of Sensorimotor Research

Primates, including humans, have a highly developed visual system for stereo vision. The brain is capable of representing the 3D scene from images projected onto the retina, by using those in both the left and right eyes. The retinal images are slightly disparate between the eyes, because the eyes have different vantage points. The disparity poses a problem to the visual system. It needs to detect corresponding elements in the two images, while rejecting others. A quantitative model, called the disparity-energy model, describes how signals from the two eyes are combined by neurons in the primary visual cortex (area V1). This model predicts that neurons respond not only to the correct disparity, but also to false disparity. In order for animals to correctly perceive the scene in 3D, the visual system needs a mechanism for avoiding the false responses.

We show that the receptive fields of V1 neurons are specially organized to counter the false responses. We trained rhesus monkeys to fixate on a small point on a computer screen. As the monkey maintained fixation, we presented white-noise images on the receptive fields of the recorded neuron. After the recording, we analyzed what properties were common to those noise images that preceded an action potential. This revealed that, for many neurons, some noise patterns had a suppressive effect, in addition to the excitatory inputs of the traditional model. There was a systematic relationship between suppressive and excitatory elements: A disparity that maximally excites the excitatory elements, actually maximally inhibits the suppressive elements, and vice versa. Thus there is a push-pull mechanism for encoding the stimulus disparity.

The suppressive elements counter the excitatory ones when they are activated by false disparity, whereas the excitatory signal is let through when the disparity is correct. Thus V1 is functionally specialized to avoid the representation of disparity from being disrupted by false signals.

Cdk5 Regulates Src Activity by Targeting cSrc for Ubiquitin-dependent Degradation

Qing Pan, Fengyu Qiao, Chun Gao, Peggy Zelenka
Cellular Differentiation Section, Laboratory of Molecular and Developmental Biology

Recent studies from this laboratory have shown that inhibition of Cdk5 in spreading lens epithelial cells reduces myosin-dependent stress fiber contraction, abolishes central stress fibers, and increases the rate of cell migration. These effects were traced to a Cdk5-dependent increase in Src activity. The present study was undertaken to explore the mechanism underlying regulation of Src activity by Cdk5, with particular emphasis on the possible role of a consensus Cdk5 phosphorylation site at S75 in the cSrc N-terminus.

Human lens epithelial cells (FHL124) were transfected with cDNA for the indicated fusion proteins. V5-tagged proteins were isolated by immunoprecipitation and immunoblotted for Src(pY419), V5, and ubiquitin. Src activity was measured as the ratio of Src(pY419)/total Src. Cdk5 activity was blocked by 15M olomoucine, Cdk5(D144N) (dominant negative), or siRNA oligonucleotides. Proteosomal degradation was inhibited with 50M Z-L3VS.

Expression of dominant negative Cdk5 or suppression of endogenous Cdk5 significantly increased Src activity in FHL124, as previously found using a pharmacological inhibitor. Expression of the mutated Src(S75A) showed that this form was approximately 2.5-fold more active than wild type Src, but its activity was not further increased by inhibiting Cdk5, indicating that this site is required for Cdk5-dependent regulation. Moreover, expression of Src(S75A) significantly reduced stress fiber contraction and formation of central stress fibers, as compared to Src wild type. Since active Src can be de-activated by phosphorylation at Y529 or by ubiquitin-dependent proteolytic degradation, we next tested whether Cdk5 affects the ubiquitylation of Src by inhibiting the ubiquitin ligase, Cullin5. Suppressing Cullin5 expression with siRNA increased Src activity 2 +/- 0.2 fold but this activity was not further increased by inhibiting Cdk5. Thus, Cdk5 increases Src activity by affecting ubiquitin-dependent degradation. We next compared the rate of ubiquitylation of wild type Src and Src(S75A) by inhibiting proteosomal degradation and measuring the level of ubiquitylated Src. Incorporation of ubiquitin into Src(S75A) was approximately 77% that of wild type Src. Since ubiquitylation rate is proportional to the concentration of active Src(pY419), and Src(S75A) was 2.5-fold more active than wild type Src, the rate of Src(S75A) ubiquitylation is approximately 34.3% that of wild type Src. Cdk5 limits Src activity by increasing the rate of ubiquitylation by Cullin5. This effect requires an intact phosphorylation site at Src(S75), a site known to be phosphorylated by Cdk5 in vitro.

Mechanistic insight into the interaction of pigment epithelium-derived factor and its receptor PEDF-R

Preeti Subramanian, Matthew Rapp, Silvia Locatelli-Hoops, S. Patricia Becerra
Protein Structure and Function Section, Laboratory of Retinal Cell and Molecular Biology

Pigment epithelium-derived factor (PEDF) is a protein secreted by the retinal pigment epithelium (RPE) into the interphotoreceptor matrix where it acts on retinal cell survival. Recently, PEDF-R a novel membrane-linked lipase protein has been identified as a potential cell-surface receptor for PEDF. This newly identified protein is present in the surface of cells and has transmembrane topology. It specifically binds with high affinity to PEDF. It exhibits phospholipase A2 (PLA2) activity, which is stimulated by PEDF binding. In this study, we show that PEDF exhibited a concentration-dependent survival effect on R28 retinal cells upon induction of cell death by serum starvation. We demonstrate the presence of PEDF-R protein in the plasma membrane of R28 cells by western blotting. Furthermore, we found that R28 plasma membrane fractions exhibited in vitro PLA2 activity, which was stimulated by PEDF. Peptides from the extracellular loop region of PEDF-R were designed and two peptides, E4b and P1, specifically bound to PEDF by ligand blot. Most importantly, pre-incubation of PEDF with these peptides attenuated the survival effects of PEDF on R28 cells. Lastly, C-terminal truncated PEDF-R (M1- L232), generated using the cell-free system demonstrate PLA activity that increased with PEDF additions as seen with full length PEDF-R (M1-L504). In conclusion, we show that PEDF-R is present as an active 80-kDa protein in the plasma membrane of R28 cells and the interaction of PEDF with the extracellular loop region of PEDF-R plays a key role in mediating cell survival in R28 cells.

Department of Health and Human Services NIH, the National Institutes of Health