Retina Research

Pka Activation Promotes Microvilli Assembly Mediated by Ezrin in Retinal Pigment Epithelium
V.L. Bonilha¹, E.Rodriguez-Boulan². ¹Ophthalmic Research, Cole Eye Institute, Cleveland, OH; ²Dyson Vision Institute, Weill Medical College of Cornell University, New York, NY.
Purpose: Previous work has shown that Ezrin, an adaptor protein that links plasma membrane and the actin cytoskeleton promotes the formation of microvilli and basal infoldings in cultured RPE cells (Bonilha et al., J. Cell Biology, 1999). Independent work has shown that ezrin phosphorylation promotes its association with both plasma membrane and the actin cytoskeleton. Here, we tested the hypothesis that PKA promotes morphogenesis of RPE microvilli by phosphorylating ezrin.
Methods: In vivo studies quantified whole cells lysates from rat RPE collected during the 3 weeks after birth. To further investigate the role of PKA in elongation of RPE microvilli, we treated rat RPE primary cultures (which preserve microvilli and and express high levels of ezrin) with myristoylated-PKI (14-22) amide a specific inhibitor of PKA. On the other hand, D407 cells (human RPE cell line with scattered short microvilli at their apical surface, and low levels of ezrin) were treated with the PKA activators Sp-cAMPS and 8-bromo-cAMP. In addition, these cells were infected with a replication-deficient adenovirus carring the ezrin cDNA. Results were analyzed by immunofluorescence, scanning electron microscopy and western blot.
Results: Quantification of western blots of post-natal rat RPE (P1 to adult) demonstrated that PKA expression increased proportionally to the elongation of the microvilli during postnatal maturation, as previously shown for ezrin. PKA inhibition decreased the length and number of apical microvilli and the triton X-100 resistant immunofluorescent staining of PKAc (PKA catalytic subunit) and ezrin in primary cultures of RPE. In D407 cells, PKAc localized to the Golgi complex but shifted to a punctated plasma membrane pattern (consistent with the appearance of microvilli) after overexpression of ezrin cDNA and PKA activation with Sp-cAMP and 8-bromo cAMP. We are currently investigating whether PKA activation results in direct ezrin phosphorylation.
Conclusions: PKA activation promotes plasma membrane localization of PKAc and ezrin and increased number of microvilli in RPE cells.

Glucose Utilization by Human RPE Cultures
K.G. Shadrach^1A, P.Senanayake^1A, K.Nishiyama^1A, J.W. Lee^1A, J.G. Hu^1A, A.Calabro^1B, D.Bok², J.G. Hollyfield^1A.

 ^ACole Eye Institute, ^BBiomedical Engineering, ¹The Cleveland Clinic Foundation, Cleveland, OH; ²Jules Stein Eye Institute, UCLA, Los Angeles, CA.
Purpose: To evaluate the distribution of glucose transporters (GLUT) and glucose utilization by confluent retinal pigment epithelium (RPE) cultures that have established high resistance junctions
Methods: Human RPE was cultured in Millicell- [PCF] culture plates in medium containing 1 mg/ml glucose. Efficiency of separation of the apical and basal compartments was determined by transepithelial resistance measurements. Glucose utilization was evaluated with glucose either in the apical (1) or basal (2) medium or both (3). Cells with associated matrices (CM), and apical and basal media (Am, Bm) were collected, digested with proteinase K, ethanol precipitated and derivatized with the fluorescent tag 2-aminoacridone (AMAC). After separation by electrophoresis, the AMAC labeled bands were digitized and their fluorescent intensities quantitated. GLUT 1, 3 and 5 expression were evaluated with RT-PCR and GLUT 1, 2 and 3 were localized with immunocytochemistry using confocal imaging.
Results: (1) Glucose decreased by 50% from the Am in 1 h and was depleted in 24 h. Glucose reached the Bm in 1 h and remained detectable for 15 h. (2) Glucose decreased by 50% from the Bm in 2 h and was depleted in 48 h. Glucose reached the Am in 2 h and remained detectable in the for 65 h. (3) Glucose decreased by 50% from the Am and Bm in 16 and 7 h respectively and was depleted from both in 72 h. Expression of GLUT 1, 3 and 5 were detected by RTR-PCR . GLUT 1 was the dominant isoform expressed. GLUT 3 and 5 showed very low levels of expression. Confocal imaging also showed that GLUT 1, GLUT 2 and GLUT 3 were present in the RPE cultures. The transporters were localized preferentially on the apical side of the RPE cells. The density of GLUT 1 was significantly higher than GLUT 2 and 3.
Conclusions: Glucose is utilized rapidly from both Am and Bm. Restricting glucose to either Am or Bm increased the rapidity of its utilization by the RPE cells. These data demonstrate bi-directional transport of glucose by the RPE cell cultures. Preferential apical localization of GLUT transporters may account for the increased, sustained levels of glucose in the Am when glucose is applied only to the basal surface of the RPE

Glycogen in RPE and Choroid of the Diabetic Rat
E.Rungger-Brandle¹, P.S. Senanayake², A.A. Dosso¹, J.G. Hollyfield². ¹Department of Ophthalmology, University Hospital, Geneva, Switzerland; ²The Cleveland Clinic Foundation, Cole Eye Institute, Cleveland, OH.
Purpose: To evaluate steady state levels and storage of glycogen in the RPE-choroid complex after streptozotocin injection.
Methods: The RPE-choroid complex was isolated from pigmented Long Evans rats at one, 4, and 12 weeks after induction of diabetes, digested with proteinase K, and twice ethanol precipitated. Supernatant and precipitate fractions were derivatized with the fluorescent tag 2-aminoacridone (AMAC), either directly to identify endogenous saccharides such as free glucose with free aldehyde or, after digestion with glucoamylase to determine total glycogen. After separation by electrophoresis, the bands were digitized and their intensities quantified. For EM histochemical demonstration of particulate glycogen (glycosomes), thin sections were stained by the periodic acid-thiocarbohydrazide-silver proteinate method.
Results: The levels of glucose, total glycogen and the ratio of glycogen to glucose in the fasting state were similar in the two groups, 4 weeks after the induction of diabetes. In the fed state, glucose and total glycogen were increased in diabetic rats but the ratio of glycogen to glucose was similar to the control. At 12 weeks postinjection, levels of glucose and total glycogen in the fed state were higher in the diabetic rats but the ratio of glycogen to glucose was lower than in the controls. Glycosomes were present in choroidal fibroblasts and smooth muscle cells in the controls and significantly enriched in the diabetic tissue as soon as one week postinjection. By contrast, virtually no glycosomes were detectable in the RPE and the endothelium of the choriocapillaris.
Conclusions: Our data document the progression from normal to altered glucose and total glycogen levels during the development of diabetes. The decreased ratio of glycogen to glucose at later time points of diabetes suggests that the glycogen stock is not replenished. The fact that almost no glycosomes can be detected in the RPE of hyperglycemic rats may be due to rapid glycogen turnover and minimal storage. Moreover, our observations hint to cell type-specific stability of glycogen, storage being stable in choroidal fibroblasts and smooth muscle cells and highly labile in RPE and endothelium.

Nuclear Export of TIP120A in Retinal Pigment Epithelium
J.W. Lee¹, K.Nishiyama¹, K.G. Shadrach¹, T.-A.Tamura², J.G. Hollyfield¹. ¹Cole Eye Institute, The Cleveland Clinic Foundation, Cleveland, OH; ²Chiba University, Chiba, Japan.
Purpose: TIP120A is reported to be a nucleus transcription factor, but little has been investigated about TIP120A in retinal pigment epithelium (RPE). We have previously reported the presence of TIP120A in RPE cells, and more recently we have recognized TIP120A in the interphotoreceptor matrix, the extracellular space facing RPE cells. We have also discovered that TIP120A is localized in the cytoplasm as well as in the nucleus in RPE cells, while it is generally localized only in the nucleus of non-RPE cells. In this study, we investigate the capacity of TIP120A to export from the nucleus to the cytoplasm or to shuttle between these compartments
Methods: Human donor eye tissue and transformed RPE-type cell lines (RPE-J cells and ARPE-19 cells) were analyzed by RT-PCR for TIP120A mRNA. To determine the biochemical localization of TIP120A, subcelluar fractionation of RPE cells was performed, and then the fractionated cell samples were blotted for TIP120A. Immunohistochemistry for TIP120A was also done on donor ocular tissue containing RPE cells and cultured RPE cells.
Results: The mRNA for TIP120A is clearly present in human donor RPE cells as well as transformed RPE cell lines. Western blotting for TIP120A also indicated that TIP120A protein exists in RPE cell lines. Immunohistochemistry data showed that TIP120A is localized not only in the nucleus but also in the cytoplasm in donor RPE cells and RPE culture cell lines. Furthermore, it was shown that the deduced amino acid sequence of TIP120A possesses three potential nuclear export signals.
Conclusions: The present data suggests that TIP120A is a nuclear export-type transcription regulating molecule, and that it is exported from the nucleus to the cytoplasm. Such export or shuttling of TIP120A may be a unique feature of RPE cells. Additional studies are planned to determine the mechanism underlying the nuclear export of TIP120A in RPE cells.

Angiotensin II and its Receptor Subtypes in the Human Eye
P.D. Senanayake^1A, S.-I.Miura^1B, S.Karnik^1B, J.G. Hollyfield^1A. ^ACole Eye Institute, ^BMolecular Cardiology, ¹The Cleveland Clinic Foundation, Cleveland, OH.
Purpose: To evaluate the distribution of Angiotensin II (Ang II) and its receptors in human ocular tissues.
Methods: Donor eyes were obtained from the Cleveland Eye Bank within 12 hours of postmortem and dissected on a chilled tray and the tissues were stored at -80°C. Ang II receptors were characterized and quantified in optic nerve, RPE-choroid complex, retina and ciliary body-iris by competitive membrane binding assays using Ang II, [Sar1 Ile 8] Ang II, and the subtype specific antagonists DUP 753 (AT1 -specific) and PD123319 (AT2-specific). Ang II in optic nerve, RPE-choroid complex, retina, vitreous, and ciliary body-iris was extracted with chilled HCL-Ethanol and concentrated using Water’s C18 Sep-Paks. Ang II was quantified by RIA.
Results: Ang II receptors were present in the four tissues studied: retina,12.1 ± 0.3; RPE-Choroid complex, 6.6 ± 1.1; optic nerve, 3.4 ± 0.1; ciliary body-iris, 2.20 ± 0.4 fmol/mg protein (mean ± se; n=3). In the ciliary body-iris the receptors were exclusively AT1, however in the other tissues, both AT1 and AT2 were present. In the retina AT1 was predominant, in the RPE-choroid complex, the percentage of AT1 was higher than AT2. In the optic nerve, the percentage of AT1 and AT2 was comparable. The highest levels of Ang II were in the optic nerve, ranging from 8 to 819 pg/g (n=12, median 174). Vitreous had the lowest levels, ranging from 3-32 pg/ml (n=27, median 9), The retina (1-367 pg/g, n=19.median =123), RPE-choroid complex (9-271 pg/g, n=12.median = 43), and ciliary body-iris (5-179 pg/g, n=11,median =44), had comparable levels.
Conclusions: High levels of Ang II and Ang receptors are present in the vascularized ocular tissues. The variability in the levels of Ang II within each tissue may be a reflection of the heterogeneity of peptide expression and/or the accompanying therapeutic regimens. Local Ang II may be involved in blood supply and/or pathological processes such as neovascularization in diabetic retinopathy.

Retinal Cone Toxicity in an Ovarian Cancer Patient Treated with Irofulven
M.S. Lee¹, N.Gupta², J.Loewenstein³, M.Wepner², A.M. Milam². ¹Cole Eye Institute/Desk i-32, Cleveland Clinic Foundation, Cleveland, OH; ²F.M. Kirby Center for Molecular Ophthalmology and the Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA; ³Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA.

Purpose: Four of 19 patients at one center with platinum-resistant, advanced epithelial ovarian cancer in a phase II clinical trial using high doses of Irofulven developed signs and symptoms consistent with retinal cone dysfunction. Each complained of photophobia and reduced vision in bright light, while 3 noted various positive visual phenomena. ERG results revealed predominantly cone abnormalities. Improvement in symptoms, visual function testing, and ERG responses occurred with lower doses or cessation of Irofulven. One woman, who received cisplatin prior to entering the trial and carboplatin afterwards, developed AML 10 weeks after her visual symptoms began and died. We describe the clinical, perimetric, electroretinographic, retinal histopathology and immunocytochemistry features of this patient.
Methods: The patient underwent comprehensive neuro-ophthalmic evaluation including Goldmann visual fields and electroretinography. Post mortem eyes of the patient and age matched normal human eyes were processed for histopathology and immunocytochemistry. Mouse monoclonal antibodies specific for cones and rods and a rabbit polyclonal antibody against glial fibrillary acidic protein (GFAP; specific for astrocytes and reactive Müller cells) were used.
Results: The visual acuities were 20/30 and she identified 3/10 Ishihara color plates OU. Goldmann visual fields revealed dense midperipheral and paracentral scotomas. Dilated fundus examination was unremarkable and carcinoma associated retinopathy testing was negative. Two months later her vision off Irofulven was 20/30 OD; 20/25 OS and 10/10 color plates. The ERG was extinguished under bright photopic conditions and 30 Hz flicker testing revealed markedly low b-wave amplitudes and prolonged implicit times. Scotopic conditions showed prolonged implicit times and borderline b-wave amplitudes. Compared to normal retinas the patient’s retina had reduced numbers of macular cones and almost no cones in the peripheral retina. Near normal numbers of rods were present in all regions examined. Müller cells had undergone reactive gliosis and were positive for GFAP, consistent with retinal cone cell death.
Conclusions: High dose Irofulven may cause cone specific retinal toxicity with relative sparing of rods.

Annexins in Retina-choroid Complex: Gene Expression and Protein Distribution
K.Nakata, K.G. Shadrach, H.Sakaguchi, Q.Chen, K.Nishiyama, J.W. Lee, M.E. Rayborn, J.W. Crabb, J.G. Hollyfield. Ophthalmology, Cole Eye Institute, The Cleveland Clinic Foundation, Cleveland, OH.
Purpose: The annexins constitute a family of calcium-dependent phospholipid-binding proteins. Several annexins have been identified in our proteomic studies of drusen. To localize the distribution of annexins I-VI in the retina-choroid complex of human eyes, we pursued Western and immunocytochemical analyses using a series of anti-annexin antibodies and also conducted RT-PCR.
Methods: The ocular tissues analyzed were retina, retinal pigment epithelium, and Bruch’s membrane / choroid. RT-PCR and western blots were performed for the demonstration of annexins. Tissues from human eyes embedded in paraffin were used for immunohistochemistry. Commercially available annexin antibodies were used for light microscopy employing the ABC method.
Results: All annexins studied except annexin-III, were localized using immunocytochemistry throughout the retina, in the RPE, Bruch’s membrane / choroid. Annexins I and II were expressed more strongly in Bruch’s membrane and choroid than in the retina and RPE. Annexins IV, V and VI were expressed more strongly in retina than in the RPE, Bruch’s membrane and choroid.
Conclusions: All the annexins we studied except annexin III are present in RPE, choroid and retina. Although annexin function is still not clearly defined, detected annexins exhibit a characteristic expression pattern in the retina-choroid complex.
Supported by NIH-NEI and FFB.

The Human Interphotoreceptor Matrix Proteome-Initial Results
J.G. Hollyfield, K.G. Shadrach, K.A. West, J.Sun, J.W. Crabb. Cole Eye Institute, The Cleveland Clinic Foundation, Cleveland, OH.
Purpose: The interphotoreceptor matrix (IPM) serves important roles in retinal physiology yet less than a dozen IPM proteins have been documented to date. Additional proteins must function in this critical retinal extracellular matrix and we have initiated efforts to define the IPM proteome.
Methods: Human IPM was isolated from dissected retina and posterior eye cup containing RPE by sequential rinses with PBS pH 7 and extractions with TBS at pH 8. Four IPM fractions were obtained: 1) the retina rinse and 2) the eye cup/RPE rinse containing readily soluble IPM components; 3) the retina extraction and 4) eye cup/RPE extraction containing less soluble IPM components. Extracted IPM samples were digested with Chondroitinase ACII to remove chondroitin sulfate-type GAGs from proteoglycans. Proteins were separated by SDS-PAGE, excised from the gel, digested in situ with trypsin and identified by LC MS/MS sequence analysis using Swiss Pro and NCBI databases.
Results: Over 100 different proteins have been identified from initial analyses of the IPM, including interphotoreceptor retinoid-binding protein, SPACR and SPACRCAN. Particularly striking is the number of hypothetical and unknown proteins, accounting for about 15% of these initial protein identifications.
Conclusions: Proteomic methods are revealing the identity of proteins in the IPM. Hypothetical and unknown proteins may be possible candidate genes for inherited retinal disease. Complete definition of the IPM proteome will lead to a better understanding of the molecular interactions taking place in this important compartment supporting photoreceptor and RPE functions.

CRALBP Interacts with a PDZ-domain Protein in Extracts of RPE
J.C. Saari¹, M.Nawrot¹, K.A. West², J.W. Crabb². ¹Ophthalmology, University of Washington, Seattle, WA; ²Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH.
Purpose: Analysis of the phenotype of cellular retinaldehyde-binding protein (CRALBP) knockout mice led us to conclude that the visual cycle was delayed at the isomerase step because of the lack of an efficient acceptor for 11-cis-retinol. Earlier studies in vitro demonstrated that 11-cis-retinol bound to CRALBP was a good substrate for a cis-specific dehydrogenase of RPE. Thus, it is likely that apo-CRALBP accepts 11-cis-retinol from an isomerase and facilitates its oxidation to 11-cis-retinal. In order to understand the mechanism of release of 11-cis-retinal from CRALBP and from RPE, we sought proteins that interact with CRALBP in RPE.
Methods: Interacting proteins were detected with an overlay assay. RPE microsomes were subjected to 1D or 2D SDS PAGE and blotted to a PVDF membrane, which was blocked, incubated with CRALBP, washed and probed with anti-CRALBP. Proteins were excised from the gel, digested in situ with trypsin and identified by LC MS/MS sequence analysis.
Results: CRALBP bound to a protein with an apparent molecular weight of 54 kDa in 1D gels. Other proteins substituted for CRALBP in the assay did not bind, suggesting a specific interaction. The protein was resolved into several components on 2D gels and each was identified by sequence analysis as ERM-binding phosphoprotein 50 (EBP50), also known as sodium/hydrogen exchanger-3 regulatory factor (NHERF-1).
Conclusions: ERM (ezrin, radixin, moesin) proteins link plasma membrane proteins with the actin cytoskeleton. EBP50 is a phosphoprotein that binds to ERM proteins through its C-terminal domain. It is known to colocalize with ezrin in apical RPE. It also interacts through its two PDZ-domains with a number of other proteins, including sodium/hydrogen exchanger-3. The functional significance of the affinity of EBP50 for CRALBP is not known, but it may involve recruitment of the binding protein to an RPE apical protein complex involved in releasing or processing 11-cis-retinal.

Five-Year Results of Verteporfin Therapy for Subfoveal CNV Due to AMD: Third Year of an Open-label Extension of the TAP Investigation
P.K. Kaiser, TAP Study Group. Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH.
Purpose: To report 5-year results from the Treatment of AMD with Photodynamic therapy (TAP) Investigation that include 3-year results from an open-label extension evaluating verteporfin therapy (Visudyne^®, Novartis AG) in AMD patients with classic-containing subfoveal CNV for vision outcomes of patients with predominantly classic lesions and safety outcomes for all participants.
Methods: Patients who completed 24 months of the TAP Investigation and who it was judged might benefit further from verteporfin therapy, were enrolled into the TAP Extension. Methods were similar to those in the TAP Investigation, except extension patients with fluorescein leakage from CNV received open-label verteporfin therapy irrespective of their treatment assignment (verteporfin or placebo) at baseline.
Results: The enrolled participants included 124 (78%) of the original 159 verteporfin-treated patients with lesions composed of predominantly classic CNV at baseline, of whom 93 (58%) of the 159 completed the month 48 examination. Visual acuity measurements were done for 92, 90, and 93 patients at month 24, 36, and 48, respectively. A loss of ≥15 letters of visual acuity from baseline occurred in 33 (36%) at month 36, and 40 (43%) at the month 48 examination, with a mean letter score loss of 8.7, 9.9, and 10.4, respectively. During the extension no additional safety concerns were noted in any patient receiving verteporfin. Two patients originally assigned to placebo had acute severe vision decrease (loss of ≥20 letters of visual acuity within 7 days of treatment) in the study eye between months 24 and 36. No additional case of acute severe vision decrease was reported between months 36 and 48. Vision and safety outcomes through the month 60 examination will be presented.
Conclusions: Vision outcomes remained relatively stable for verteporfin-treated patients with predominantly classic lesions from the month 24 to the month 48 examination. Caution in the interpretation of these results appears warranted in the absence of a comparison with an untreated group during the extension and because not all patients in the TAP Investigation participated in the TAP Extension.

Potential Binding Partners of Tissue Inhibitor of Metalloproteinase-3
P.A. Klenotic, J.Qi, L.Y. Marmorstein, B.Anand-Apte. Ophthamology, Cole Eye Institute, Cleveland, OH.
Purpose: Tissue Inhibitor of Matrix Metalloproteinases-3 (TIMP-3) is a 24kDa protein synthesized by the retinal pigment epithelium and present in Bruch’s membrane. TIMP-3 can inhibit matrix metalloproteinases (MMPs) as well as ADAMs (a-disintegrin and a metalloproteinases domain) and ADAMTSs (ADAM with thrombospondin-like repeats) within the adamalysin family. Mutations of certain cysteine residues to serine result in deposition of excessive amounts of TIMP-3 in Bruch’s membrane, producing early blindness in patients with Sorsby fundus dystrophy. In this study, we proposed to identify potential binding partners of TIMP-3 in the retina.
Methods: cDNA inserts of a primary human fetal RPE library were transformed into the yeast reporter strain, AH109, along with a second plasmid for in vivo recombination to generate cDNA-GAL4 activation domain fusion constructs targeted to the nucleus. The complete coding sequence, or the N- or C-terminal domain of human wild type TIMP-3 was used to transform AH109 to generate TIMP-3-GAL4 binding domain fusion constructs. Growth on nutritionally deficient plates denoted an interaction. Sequencing of the clones followed by BLAST searches allowed identification of potential binding partners. Co-transformation and mating experiments confirmed the interactions and ruled out false positives.
Results: We have identified 16 clones that appear to be good candidates for putative binding partners of TIMP-3. Binding region has also been determined by using truncated TIMP-3 protein as bait. Some interactions have been confirmed by co-immunoprecipitation experiments in eukaryotic cells systems.
Conclusion: Detailed analysis of the interactions of TIMP-3 and potential binding partners will contribute significantly to the understanding of the molecular function of TIMP-3.

Thirty Year Clinical Follow-Up of a Patient with Novel RPE65 Mutations and Leber Congenital Amaurosis
K.Al-khayer¹, S.Hagstrom¹, H.Zegarra², G.Pauer¹, E.I. Traboulsi¹. ¹Ophthalmic Research, Cleveland Clinic Foundation, Cleveland, OH; ²Ophthalmology, Retinal Associate of Cleveland, Beachwood, OH.
Purpose: To report a North American family with heterozygous compound mutations in the RPE65 gene associated with Leber Congenital Amaurosis, and to present a long-term follow-up of the ocular findings in the proband
Methods: RPE65 mutation screening was performed on 30 patients with Leber Congenital Amaurosis (LCA) using PCR amplification of the 14 exons of RPE65, and search for sequence changes using SSCP and direct sequencing of abnormal bands. Ophthalmic examinations included visual acuity testing, ophthalmoscopy, color vision testing, dark-adapted threshold perimetry, and electroretinography.
Results: The proband, a 35 year-old female carried two RPE65 mutations in a compound heterozygous fashion: a maternal K303X (A961T) nonsense mutation and a paternal Y431C (A1346G) missense mutation. She had severe visual deficits and an absence of rod and cone electroretinographic responses. Visual acuity of 20/60 and color recognition during early childhood declined over time to 20/100 OD and 20/50 OS with total absence of color recognition during the teenage years, and only 2/200 OD and 1/200 OS at the age of 30. She graduated from high school in regular classroom setting. Both parents had normal visual function and a sister carried one of the mutations.
Conclusions: The RPE65 mutations K303X and Y431C in compound heterozygous form cause progressive visual compromise that starts in childhood and advances to almost total visual loss by the fourth decade of life. The identification and characterization of the clinical course of patients with RPE65 mutations is important in preparation for future trials of gene therapy for retinal degeneration.

Clinical Correlation for Two Novel Mutations in the RPGR Gene for X-linked Retinitis Pigmentosa
D.C. Chung¹, Y.K. Demirci², H.Zegarra³, A.J. Locastro⁴, M.B. Gorin², E.I. Traboulsi¹. ¹Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH; ²Ophthalmology and Genetics, University of Pittsburgh, Pittsburgh, PA; ³Retina Associates of Cleveland, Cleveland, OH; ⁴Pediatric Eye and Oculoplastic Surgeons, Children's Hospital Medical Center of Akron, Akron, OH.
Purpose: X-linked Retinitis Pigmentosa (XLRP) is a hereditary retinal degeneration that leads to the early onset of night blindness with progressive loss of peripheral vision and eventual legal blindness in most patients in later decades of life. We describe the clinical manifestations of two novel mutations in the RPGR gene in 3 patients. Mutations in RPGR have been associated with XLRP (RP3) and X-linked cone-rod dystrophy (COD1).
Methods: Snellen visual acuity, ocular alignment, color vision assessment, intraocular pressure measurements and cycloplegic or manifest refractions were preformed, followed by complete slit lamp bimicroscopy and dilated fundus examination, electroretinograms, and Goldmann visual fields. Family ophthalmic histories were noted. Appropriate informed consent was obtained and DNA was extracted from whole blood samples. Coding regions plus ORF15 were polymerase chain reaction (PCR) amplified with intronic primers specific for the 19 exons and ORF15 of the RPGR gene. The amplified exon fragments underwent mutation analysis by standard direct sequencing techniques.
Results: Patient #1, age 12 years, had a best corrected visual acuity (BCVA) of 20/25-, with onset of visual symptoms in the first decade; he had a mild to moderate myopic error of refraction, and carried a novel mutation in RPGR Exon 4 (333_336dup4). Patient #2, age 19 years, had BCVA of 20/40-, with onset of symptoms at age 13 years and a high myopic refractive error. Patient #3 was 27 years old; his BCVA was 20/200 with onset of night blindness at age 4 years; he was moderately to highly myopic. Both patient #2 and #3 had a novel mutation in RPGR ORF15 (ORF15+483_484delGA). Fundus appearance was typical of classic retinitis pigmentosa in all patients. Electroretinographic tracings were severely reduced or non-recordable in all patients.
Conclusion: There appears to be no significant difference in the clinical presentation between these two particular mutations in the RPGR gene that cause classic XLRP with myopia.

Identification of Lipid Oxidation Products and Proteins in Bruch’s Membrane from Normal and AMD Donor Eyes
X.Gu^1A, K.Shadrach^1A, M.Sun², K.A. West^1A, L.Shan^1B, S.Hazan^1B, R.G. Salomon², J.G. Hollyfield^1A, J.W. Crabb^1A. ^AOphthalmic Research, ^BCell Biology, ¹Cleveland Clinic Found, Cleveland, OH; ²Chemistry, Case Western Reserve University, Cleveland, OH.
Purpose: To identify lipid oxidation products and proteins in Bruch’s membrane possibly associated with the pathogenesis of age-related macular degeneration (AMD).
Methods: Bruch’s membrane was isolated free of adjacent tissues and in the presence of antioxidants from 5 normal and 4 AMD donor eyes. Lipids were extracted with chloroform and methanol and analyzed by LC MS. Proteins were subjected to SDS-PAGE, blotted to PVDF membranes and/or gel bands were excised and proteins identified by LC MS/MS. Western analyses were used to screen for oxidative protein modifications.
Results: Lipid oxidation products from docosahexaenoyl phosphatidylcholine (DHA-PC), arachidonoyl (AA)-PC, and linoleyl (LA)-PC were detected in relatively greater amounts from AMD than age matched healthy donor eyes. Common drusen proteins were also detected in Bruch’s membrane, including tissue inhibitor metalloproteinase-3, clusterin, vitronectin and serum albumin. Carboxyethylpyrrole protein adducts generated from the oxidation of DHA containing lipids were more abundant in Bruch’s membrane from AMD donors.
Conclusions: These observations are consistent with recent drusen analyses (2002 Proc. Natl. Acad. Sci. USA 99: 14682) and support a role for lipid oxidation products in Bruch’s membrane changes associated with AMD.

Endogenous Oxidoreductase Expression is Induced by Aminoglycosides
J.E. Sears, Y.Chai, G.Hoppe. Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH.
Purpose: To determine the effect of aminoglycosides on protein glutaredoxin-1 (grx-1) levels in cultured human retinal pigment epithelial cells.
Methods: Varied concentrations of gentamycin, kanamycin, and hygromycin were used to treat ARPE-19 cells. Western blot was used to detect grx-1 content. Reactive oxygen species (ROS) formation was measured by a dichlorofluorescein diacetate (DCFDA) fluorescent assay after aminoglycoside exposure. The effect of phorbol 12-myristate 13-acetate (PMA), N-acetyl cysteine (NAC), and t-butyl hydroperoxide (tBHP) were tested in order to correlate oxidoreductase expression to ROS production.
Results: All aminoglycosides increased grx-1 expression in a time and dose dependent fashion. Hygromycin was most effective, creating a 10-fold increase in grx-1 expression at a concentration of 400 μg/ml at 48 hours. PMA similarly increased grx-1 expression. NAC decreased grx-1 expression. ROS formation was correlated directly to aminoglycoside effect.
Conclusions: In summary, aminoglycosides increase grx-1 protein levels. This effect is blunted by NAC, enhanced by PMA, and may be directly correlated to reactive oxygen species induced by tBHP. The effect of aminoglycoside selection on strategies for grx-1 investigation should take this observation into account. Further studies are warranted to understand the mechanism of reactive oxygen species induction of grx-1 protein levels.

Glutathione as a Second Messenger of Oxidation: Hsc70 Is a Substrate of Glutaredoxin-1
G.Hoppe, Y.-C.Chai, K.West, J.E. Sears. Cole Eye Institute, Cleveland Clinic, Cleveland, OH.
Purpose: To determine the protein S-glutathiolated substrates of glutaredoxin-1 (grx-1) in cultured human retinal pigment epithelial cells.
Methods: Protein-S-glutathiolation was induced in ARPE-19 cells by treatment with 250 μM diamide following pre-loading of cells with 14 μM reduced recombinant grx-1. Reversible glutathiolation by grx-1 was detected using monoclonal antibody specific for glutathione adduct and heat shock cognate protein 70 (Hsc70) identified by Q-TOF mass spectrometry. A luciferase aggregation assay was used to test Hsc70 activity in various redox states as well as in the presence of grx-1.
Results: A 70 kD band that demonstrated reversible glutathiolation by grx-1 was identified as Hsc70. Recombinant Hsc70 was glutathiolated in vitro by oxidized glutathione, and Hsc70 protein-S-glutathiolation (Hsc70-SSG) reversed by reduced grx-1. Hsc70-SSG was twice as effective in preventing luciferase aggregation at 42 °C than reduced Hsc70. Pre-incubation with grx-1 enhanced the activity of Hsc70-SSG.
Conclusions: Glutathione may become adducted to Hsc70 in order to enhance the activity of this heat shock protein under oxidative stress. The synergistic effect of grx-1 and Hsc70-SSG suggests that glutathiolation acts as a signal for cooperative binding between these two proteins to enhance chaperone activity.

TIMP-3 Distribution and Content in Bruch’s Membrane and the Choroid Is Different in Caucasian and African American Donor Eyes
H.Sakaguchi, K.G. Shadrach, M.E. Rayborn, J.G. Hollyfield. Cole Eye Institute, The Cleveland Clinic Foundation, Cleveland, OH.
Purpose: Age-related macular degeneration (AMD) occurs more frequently in lightly pigmented individuals of Northern European extraction than in more heavily pigmented individuals of African extraction. To determine whether specific molecular differences are present in the connective tissue below the RPE of the macula, we defined the distribution and content of TIMP-3 in Bruch’s membrane between age-matched Caucasian and African American donor tissues.
Methods: Donor eyes used were between 50 and 85 years of age, 11 from African American donors and 12 from Caucasian donors. Bruch’s membrane and choroid from the macula from each donor eye were prepared for immunocytochemistry and Western blotting and differences in immunoreactivity were quantitated.
Results: TIMP-3 immunoreactivity was present in broader areas in Bruch’s membrane and connective tissue surrounding the choriocapillaris in the Caucasian samples than was observed in the African American samples. Additionally, quantitation of Western blots indicated that Caucasian tissues show a progressive increase in TIMP-3 content with age, whereas African American tissues show near steady state levels over the same age ranges.
Conclusions: TIMP-3 has been proposed to be one of the candidate proteins involved in age-related macular degeneration. Our study suggests that the susceptibility of Caucasians to AMD may be related to the progressive accumulation of proteins in Bruch’s membrane and surrounding tissues that could alter the exchange of metabolites between the RPE and choriocapillaris.

Immunohistochemical Analysis of the Outer Plexiform Layer in the Nob Mouse
S.L. Ball^1A, M.T. Pardue², M.A. McCall^3A, R.G. Gregg^3B, N.S. Peachey^1B. ^APsychology, ^BOphthalmic Research, ¹Cleveland VA Hospital; Case Western Reserve University; Cole Eye Institute, CCF, Cleveland, OH; ²Ophthalmology, Atlanta VA Hospital, Emory University, Decatur, GA; ^ADepartments of Psychological & Brain Sciences and Ophthalmology & Visual Sciences, ^BOphthalmology & Visual Sciences Biochemistry & Molecular Biology, ³University of Louisville, Louisville, KY.
Purpose: In nob (no-b-wave) mice, a mutation in the gene that encodes for the nyctalopin protein results in a defect in communication between photoreceptors and depolarizing bipolar cells (DBCs). As the role of the nyctalopin protein is unknown, we investigated possible effects of this mutation on the distribution of proteins that are necessary for DBC activation or the generation of a b-wave.
Methods: Adult normal and nob mice were enucleated, and the eyes were immersion fixed in 4% paraformaldehyde for 10 min, cryoprotected, embedded and sectioned on a cryostat at 10 μm. Immunohistochemistry reactions were performed using standard protocols with antibodies to the following proteins: PKC, PSD-95, the α_1F subunit of voltage gated calcium channels, mGluR6, G_0α, bassoon, trkB, and dystrophin.
Results: In control retinas, each antibody showed a labeling pattern in the OPL that was comparable to those previously described for mouse retina. In nob mice, the labeling pattern was comparable to controls.
Conclusions: The normal distribution of these OPL synaptic proteins in nob mice leads us to two possible explanations. First, although these proteins are correctly localized, in the absence of nyctalopin one or more may not be functional. Alternatively, the defect associated with nob may involve a novel role for nyctalopin in synaptic transmission or the DBC signal transduction cascade.

Stimulus-Response Characteristics of the Mouse DC-ERG
J.Wu^1A, A.D. Marmorstein^1B, N.S. Peachey^1A. ^ACole Eye Institute, ^BCole Eye Institute, Cell Biology, ¹Cleveland Clinic Fndn, Cleveland, OH.
Purpose: To characterize ERG components generated by non-neuronal tissues of the mouse eye.
Methods: After overnight dark adaptation, adult mice (C57BL/6J and BALBc/ByJ) were sedated with ketamine/xylazine and placed on a heating pad. Two Ag/AgCl electrodes were fashioned with capillary tubes filled with Hanks BSS; one was placed in contact with the test eye and the other contacted the fellow eye, which was shielded from light stimulation delivered to the test eye. The dc-ERG signal was amplified (dc-100 Hz), digitized (20 Hz) and stored off-line. Full-field stimuli, 7-min in duration, were presented using a Uniblitz shutter. Flash intensity was controlled with neutral density filters.
Results: In response to a 7-min flash, the mouse dc-ERG included an initial b-wave which was followed by a c-wave, fast oscillation (FO), light peak (LP), and an off-response at flash offset. As flash intensity increased, the c-wave, FO, and LP first increased and then decreased in amplitude. The polarity of the off-response was negative for low intensity stimuli and positive at the high end of the stimulus range; polarity reversal occurred at a lower intensity for BALBc/ByJ than C57BL/6J mice.
Conclusions: The major components of the dc-ERG are readily measured in the mouse. Therefore, this recording technique may be used to examine the effects of genetic manipulation on the electrical activity of the RPE, and how this is altered in retinal degenerative disorders.

Mutation Screening of RPGR in Male Patients With X-Linked or Isolated Forms of Retinitis Pigmentosa or Cone-Rod Dystrophy
F.Y. Demirci¹, A.L. Radak², B.W. Rigatti¹, E.I. Traboulsi³, T.Alitalo⁴, T.S. Mah¹, M.B. Gorin⁵. ¹Ophthalmology, Univ. of Pittsburgh SOM, Pittsburgh, PA; ²Human Genetics, Univ. of Pittsburgh GSPH, Pittsburgh, PA; ³Ophthalmology, Cleveland Clinic Foundation, Cleveland, OH; ⁴Ob/gyn, Helsinki Univ. Hospital, Helsinki, Finland; ⁵Ophthalmology and Human Genetics, Univ. of Pittsburgh SOM and GSPH, Pittsburgh, PA.
Purpose: Mutations in the RPGR (retinitis pigmentosa GTPase regulator) gene isolated from RP3 region (Xp21.1) have been reported to be responsible for up to 70% of X-linked (XL) retinitis pigmentosa (RP) families. RPGR exon ORF15 mutations have also been shown to cause XL-atrophic macular degeneration and COD1 type XL-cone-rod dystrophy (CRD). This study is intended to determine the spectrum and frequency of RPGR mutations in our male patients with XL or isolated forms of RP or CRD.

Methods: RPGR exons 1-14 and exon ORF15 were screened for mutations by direct PCR sequencing of samples from 5 XLRP and 11 XL-CRD families and a total of 24 male patients with isolated forms of either RP or CRD. Unlike previous reports that primarily screened isolated RP cases with severe phenotypes, we evaluated all available isolated RP males to avoid an ascertainment bias.

Results: Mutations were found in 2 XLRP samples (IVS1+1G>A and ORF15+483_484delGA) and in 2 isolated RP samples (213G>A and 1404C>T). Analyses of additional samples are underway. The 213G>A (G52R) is a novel missense mutation, which would also be predicted to disrupt normal splicing and was not detected in 100 control chromosomes. The 1404C>T (R449X) mutation was found in an affected male whose mother had both normal alleles, thus representing a de novo mutation. Despite a sampling bias created by our previous efforts to recruit COD1 families, more than half of our XL-CRD families have not been mapped to COD1 region and/or lack RPGR mutations, supporting the importance of genetic heterogeneity. No RPGR mutations were detected in any isolated CRD cases.

Conclusions: Our data supports the involvement of RPGR in isolated RP. The determination of the mode of inheritance by molecular testing has major implications for genetic counseling. The comparison of phenotypes of RPGR mutation-positive patients versus negatives may provide indicators for prediction of the isolated cases at high-risk for RPGR mutations. A larger cohort will be necessary to clearly establish any correlation of phenotype with RPGR mutations. However, the phenotypes of our isolated cases with RPGR mutation are consistent with the early onset, severe RP previously reported in RP3 families.

Mutation Screen in the Membrane-Type Frizzled-Related Protein (MFRP) Gene in 113 Patients With Inherited Retinal Degenerations
G.J. Pauer, Q.Xi, E.I. Traboulsi, S.A. Hagstrom. Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH.
Purpose: MFRP is a member of the frizzled-related protein family and contains a cysteine-rich domain essential for Wnt binding and signaling. MFRP is highly expressed in the retinal pigment epithelial cells of the eye. A splice donor mutation in the mouse homolog of Mfrp is responsible for photoreceptor degeneration in the rd6 mouse, whose fundus is characterized by discrete dots distributed across the retina. We investigated MFRP as a candidate gene for a variety of retinal degenerations. Methods: To date, a partial screen (11 of 13 exons) for mutations in 47 unrelated patients with Stargardt’s macular dystrophy, 44 unrelated patients with Retinitis Pigmentosa (RP), and 22 unrelated patients with Leber Congentital Amaurosis (LCA) has been performed using exon-by-exon SSCP. Variant bands detected by SSCP were further analyzed by direct genomic sequencing.

Results: Three missense sequence changes (Arg54Gly, Ile119Val, and Val136Met) were identified in MFRP. Arg54Gly was identified in one autosomal dominant Stargardt’s patient and Ile119Val was identified in one simplex Stargardt’s patient. The Val136Met missense change and three isocoding changes (Asp238Asp, Leu318Leu and Ala545Ala) were found in patients with all three retinal degenerations. An intronic change (IVS11+3G→A) was also identified in one Stargardt’s patient. None of the isocoding changes or the intron change alters a splice site. Cosegregation is pending to determine whether the observed missense sequence anomalies are pathogenic in Stargardt’s macular dystrophy.

Conclusions: We report 7 sequence changes in MFRP in patients with inherited retinal degenerations. The possible pathogenic role of these changes is under further investigation. We are proceeding with an evaluation of the remaining exons in these patients and an evaluation of all exons in additional patients with allied diseases.

Exclusion of GNGT1 Gene as a Positional Candidate for Canine rcd2 Disease
A.V. Kukekova¹, W.Wang², J.K. Lowe³, E.A. Ostrander³, G.D. Aguirre¹, G.M. Acland¹. ¹J.A.Baker Institute for Animal Health, Cornell University, Ithaca, NY; ²Department of Ophthalmology, Cole Eye Institute, Cleveland, OH; ³Fred Hutchinson Cancer Research Center, Seattle, WA.
Introduction: Rod cone dysplasia type 2 (rcd2) is an early onset form of canine progressive retinal atrophy (PRA) phenotypically similar to rcd1 of Irish setters. In difference from rcd1, which is caused by a mutation in PDE6B subunit, a number of phototransduction cascade genes have been excluded as candidates underlying the rcd2 disease.
Purpose: To identify the genetic linkage between rcd2 and chromosomal loci and to evaluate GNGT1 gene as a positional candidate for rcd2 disease.
Methods: A set of rcd2 informative pedigrees has been generated and genotyped with microsatellite markers from standard canine genome wide scan set. Based on identified genetic linkage the positional candidate gene approach has been applied.
Results: 13 rcd2 informative three-generation pedigrees have been screened with 97 canine microsatellites. Genotyping data was analysed by best two-point option of MultiMap software and genetic linkage between rcd2 disease and CFA14 markers (FH3725, lod 2.25; FH2547 lod 1.98; C14.390 lod 1.976) has been found. Comparison of CFA14 with human ortholog (HSA7) has revealed GNGT1 gene as a positional candidate for rcd2. Based on canine RH map 5000 cR the GNGT1 gene is located on CFA14 in the region of interest, its chromosomal position on CFA14 between markers FH3725 and FH2060 was also confirmed on canine RH panel 3000 cR. The cDNAs of GNGT1 from rcd2 affected and normal dogs have been cloned and sequenced. The polymorphism involves an A to G change in the nucleotide 298 in the 3’ UTR of the canine GNGT1 cDNA has been found. A fragment of GNGT1 cDNA was used as a probe for canine BAC library screening and 3 polymorphic microsatellites were found in selected canine BAC clones. Identified microsatellites and SNP were used for genotyping of rcd2 families. Recombinants between GNGT1 and rcd2 have been observed by “Identity by Descent” approach as well as by linkage analyses of rcd2 informative pedigrees.
Conclusions: GNGT1 gene has been excluded as a positional candidate for rcd2 disease.

Supported by EY06855 and EY13132, the Foundation Fighting Blidness, American Border Collie Association , Morris Animal Foundation/ The Seeing Eye, Inc., and the Van Sloun Fund for Canine Genetics Research

Hyaluronan in the Mouse Interphotoreceptor Matrix (IPM) Revisited
M.E. Rayborn, K.G. Shadrach, P.Senanayake, J.G. Hollyfield. Cole Eye Institute, The Cleveland Clinic Foundation, Cleveland, OH.
Purpose: We previously reported that the mouse IPM is free of hyaluronan (HA), a conclusion based on the absence of IPM staining in this species with a specific probe for HA (Exp. Eye Res. 65: 603-608,1997). To determine whether HA could be detected with biochemical methods and with a specific HA probe (bHABC) following attempts to remove HA binding partners, the following studies were performed.

Methods: BALB/c and C57Bl/6J mouse eyes were used. For HA biochemistry, IPM was extracted in pH 8.0 tris buffered saline, the complex carbohydrate precipitated with ethanol, digested with Streptococcal hyaluronidase and chondroitinase ABC. Samples were analyzed with FACE and disaccharide bands compared to authentic standards. For bHABC analysis, retinas were isolated and rinsed with PBS and then fixed in 2.5% glutaraldehyde in phosphate buffer. Paraffin sections were stained with bHABC.

Results: HA disaccharides were present in the IPM extract from the mouse retina, along with disaccharides of unsulfated chondroitin and 6-sulfated chondroitin. bHABC decorated the IPM in the rinsed retinas, with the cones showing heavier labeling than the rods.

Conclusions: We conclude that HA is present in the mouse IPM, as evidenced from the presence of HA disaccharides in the IPM extract, and the binding of bHABC to the IPM in the rinsed retinas. The failure of bHABC to decorate HA in the IPM in our previous analysis was probably due to the complete coverage of HA by matrix molecules that saturate the linear HA molecule, preventing attachment of the HA probe.

Induction of Angiogenesis by Active Matrix Metalloproteinses-2 and 9: Role of VEGF
Q.Ebrahem, B.Anand-Apte. Ophthalmic Research, Cole Eye Institute/Cleveland Clinic Foundation, Cleveland, OH.
Purpose: Matrix metalloproteinases (MMPs) are a specialized group of enzymes that participate in extracellular matrix (ECM) degradation and have been postulated to play an important role in angiogenesis. The purpose of this study was to determine if active MMPs could induce angiogenesis in vivo.

Methods: The chick chorio-allantoic membrane (CAM) assay was used as an in vivo angiogenesis model in this study. Methylcellulose discs containing pro or active MMPs were placed on 6 day CAMs and analyzed for their ability to induce a neovascularization response surrounding the disc after 48 hours. The ability of a neutralizing VEGF antibody to inhibit this response was examined.

Results: Active MMPs can initiate an angiogenicresponse in the CAM assay in the form of increasing tortuosity of vessels surrounding the disc. This effect was not observed with pro MMPs. Neutralizing antibodies to VEGF can inhibit this response. In addition, synthetic inhibitor of MMPs can inhibit the angiogenic response of VEGF.

Conclusions: Active MMPs can initiate an angiogenic response by increasing the tortuosity of capillaries mediated by release of low levels of VEGF. Our data suggests that MMPs may act upstream as well as downstream of VEGF during in vivo angiogenesis.

Evaluation of Inner Retinal Structure in the Aged RCS Rat
M.Blum¹, M.T. Pardue², B.Hanzlicek¹, N.S. Peachey³, S.L. Ball⁴. ¹Cleveland VAMC, Cleveland, OH; ²Atlanta VAMC, Department of Ophthalmology, Emory University, Atlanta, GA; ³Cleveland VAMC, Cole Eye Institute, CCF, Cleveland, OH; ⁴Cleveland VAMC, Case Western Reserve University, Department of Psychology, Cleveland, OH.
Purpose: In retinitis pigmentosa (RP), loss of visual function is predominantly due to the loss of rod photoreceptors. A number of labs are currently evaluating whether RP may be treated by replacing the diseased photoreceptors with either healthy cells or a photosensitive prosthetic device. As these efforts are critically dependent upon the maintenance of an intact inner retinal circuitry, we evaluated inner retinal structure and function in a widely used model of photoreceptor degeneration, the RCS rat.
Methods: Eyes were collected from deeply anesthetized pigmented dystrophic RCS rats and controls from 3 weeks to 12 months of age, immersion fixed in 4% paraformaldehyde for 30 min, cryoprotected, embedded and sectioned at 10 μm. Immunohistochemistry was completed using standard protocols with the following antibodies: PKC or recoverin, to label rod and cone bipolar cells, respectively, and calretinin, parvalbumin, choline acetyltransferase, or tyrosine hydroxylase to label subclasses of amacrine cells.
Results: In addition to observing a retraction of rod bipolar cell projections similar to that previously described (Hanitzsch et al., 1998), we observed an increase in the intensity of recoverin label in cone bipolar cell somas. We found a normal laminar labeling pattern for all amacrine cell markers in the RCS inner nuclear and plexiform layers.
Conclusion: This study identified changes in the cone visual pathway which may be important in considering treatment strategies for RP. In comparison, amacrine cell organization appears to be well maintained in the RCS rat during photoreceptor degeneration.

CRALBP Topological Analyses by Mass Spectrometry
J.W. Crabb, A.Hasan, Z.Wu. Cole Eye Institute, The Cleveland Clinic Foundation i31, Cleveland, OH.
Purpose: Cellular retinaldehyde binding protein (CRALBP) interacts with other proteins as well as with ligand in its role as an acceptor of 11-cis-retinol and substrate carrier in the rod visual cycle. CRALBP topological analyses have been pursued as an approach to identifying functional domains and better understanding visual cycle mechanisms.
Methods: Human recombinant CRALBP with a 23 residue His-tag N-terminal fusion sequence was produced in E. coli and purified to apparent homogeneity. Amide hydrogen-deuterium exchange (H-D exchange) and mass spectrometric analyses of pepsin digests of apo- and holo-rCRALBP were used to identifying solvent exposed regions that may interact with other proteins and buried regions that may bind 11-cis-retinoid.
Results: Significant localized differences in deuterium incorporation were found between apo- and holo-rCRALBP. Ligand dependent conformational changes were observed in rCRALBP residues 4-22, 80-94 and 282-316 which are more solvent exposed in the holo-protein and in residues 198-212 and 224-243 which are less solvent exposed in the holo-protein. Other regions exhibited less than 5% difference in deuterium incorporation between the apo- and holo-proteins.
Conclusions: Binding of 11-cis-retinal induces confromational changes in rCRALBP detectable by H/D exchange mass spectrometry. Separate analyses have confirmed retinoid binding pocket components within buried holo-rCRALBP residues 198-212 and 224-243. The present results provide useful hints regarding structural domains in CRALBP available for functional interactions.

Identification of the CRALBP Ligand Binding Pocket by Photoaffinity Labeling
S.K. Bhattacharya¹, Z.Wu², K.Nakanishi³, J.W. Crabb². ¹Ophthalmic Research, Cole Eye Institute Cleveland Clinic Foundation, Cleveland, OH; ²Ophthalmic Research & Department of Chemistry, Cole Eye Institute Cleveland Clinic Foundation & Cleveland State University, Cleveland, OH; ³Department of Chemistry, Columbia University, New York, NY.
Purpose: Noncovalent ligand interactions determine the function of cellular retinaldehyde-binding protein (CRALBP) in the rod visual cycle as an 11-cis-retinol acceptor and substrate carrier. To further characterize the ligand binding cavity of CRALBP, we covalently labeled rCRALBP with a photoaffinity retinoid analogue and identified the modified amino acids.
Methods: Purified human apo-rCRALBP was labeled with 3-diazo-4-keto-11-cis-retinal, excess retinoid removed and covalent incorporation achieved by photolysis with UV-light (254 nm) at –196⁰C for 5s to 20 min. Protein bound retinal was reduced to retinol with NaB³H₄, radiolabeling the ligand incorporation sites. Labeled rCRALBP was alkylated, digested with trypsin and fractionated by RP-HPLC. Radioactive peptide fractions were identified and peptides were analyzed by MALDI-TOF MS and LC MS/MS.
Results: Short irradiation times (5-40s) yielded relatively constant incorporation levels (~1%) therefore 5s photolysis times were used to minimize nonspecific protein modifications. Peptides accounting for 100% of the rCRALBP sequence were identified by mass spectrometric analyses of photolabeled rCRALBP. Eight photoaffinity modified residues were identified in radioactive fractions, each with variable mass additions.
Conclusions: Four of the photoaffinity modified sites have been previously identified as retinoid binding pocket residues. The other four modified residues are also in the retinoid binding domain and may represent newly identified CRALBP ligand binding pocket components. The cause of the variable mass additions is not clear but may be due in part to free radical migration throughout the conjugated double bonds of the retinoid analogue.

SPACRCAN Binding to Hyaluronan: Molecular and Biochemical Studies
Q.Chen, K.G. Shadrach, J.G. Hollyfield. Cole Eye Institute, The Cleveland Clinic Foundation, Cleveland, OH.
Purpose: Hyaluronan (HA) is a glycosaminoglycan (GAG) in the interphotoreceptor matrix (IPM). It has been implicated as providing a primary scaffold in the IPM. Some proteins in IPM, such as SPACR and SPACRCAN, have been shown to possess HA binding motifs. Both of them have been demonstrated to bind to HA. However, there is no direct evidence that the binding is through interaction with the HA binding motif. The purpose here is to initiate a study to demonstrate the function of HA motifs in the HA binding identified in mouse SPACRCAN.
Methods: A short polypeptide fragment of mouse SPACRCAN containing each HA binding motif was subcloned into the pGEX-2TK vector and expressed in E. coli BL21 cells. Proteins purified from the cell extracts were subjected to CPC precipitation analysis in which binding of a cationic detergent, cetylpyridinium chloride, to anionic GAGs like HA leads to co-precipitation of proteins interacting with the GAGs. To demonstrate the binding was HA specific, digestion with a HA-specific hyaluronidase, Streptomyces hyaluronidase, was included in the co-precipitation analysis.
Results: Polypeptides containing four of the HA binding motifs in mouse SPACRCAN have been expressed in E. coli BL21 cells. CPC precipitation analysis showed that the polypeptides were precipitated in the pellets. However, pre-incubation of the polypeptides with Streptomyces hyaluronidase dramatically decreased the amount of peptides precipitated in the pellets.
Conclusions: The HA binding motif containing polypeptides expressed in E. coli binds to HA. Further mutagenesis studies are in progress to determine the HA binding is through the HA binding motifs.

Transcription Factors of the Sp Family Synergize With Both Nrl and Crx, and Regulate the Expression of Multiple Retina-Specific Genes
L.E. Lerner¹, Y.Gribanova², D.B. Farber². ¹Cole Eye Institute, Cleveland Clinic Foundation and Jules Stein Eye Institute, UCLA, Cleveland, OH; ²Jules Stein Eye Institute, Los Angeles, CA.
Purpose: The Sp1 and Sp4 transcription factors have been implicated in transcriptional control of a number of genes in various tissues. Although Sp1 is ubiquitously expressed, Sp4 is predominantly expressed in CNS, including the retina. Transcription factors Nrl and Crx are essential for the expression of a number of photoreceptor-specific genes during development and in the adult retina. We reported previously that Sp4 and Nrl, but not Crx had major roles in transcriptional activation of the rod cGMP-phophodiesterase ß-subunit (ß-PDE) promoter. The purpose of this study was to test whether these transcription factors are able to functionally interact with each other on different promoters providing a novel mechanism for combinatorial regulation of multiple retina-specific genes.

Methods: Transient transfection and co-transfection assays were performed in 293 human embryonic kidney (293HEK) cells. Luciferase reporter constructs containing promoters from Rhodopsin, Arrestin, IRBP, RPE65, or α’-PDE genes were employed. In order to test the mutual functional effects of the transcription factors of interest, expression plasmids encoding Sp1, Sp4, Nrl or Crx were added to transfection mixtures individually or in combinations.

Results: Both Sp1 and Sp4 were able to activate promoter activity of multiple retina-specific genes. The Sp4-mediated activation was generally more pronounced than that by Sp1. Both Sp1 and Sp4 showed synergistic effects with Nrl or Crx suggesting the possibility of functional interactions between these transcription factors in vivo.

Conclusions: These results suggest that Sp1 and Sp4 could regulate the expression of multiple retinal genes in combinatorial fashion by interacting with other transcription factors including Nrl and Crx.

Interaction between the Transcription Factors Crx and Sp4 Regulates the Expression of Photoreceptor Genes
G.Peng¹, L.E. Lerner², Y.E. Gribanova³, S.Chen¹, D.B. Farber³. ¹Ophthalmology, Washington Univ Sch of Med, Saint Louis, MO; ²Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH; ³Ophthalmology, Jules Stein Eye Institute, Los Angeles, CA.
Purpose: Crx is a photoreceptor transcription factor essential for the expression of many rod and cone genes. The Sp family of transcription factors has been implicated in controlling the transcription of a wide-range of genes in various tissues, including the retina. The purpose of this study was to test if Crx was able to physically interact with Sp proteins.

Methods: Co-immunoprecipitation assays were used to detect the physical interaction between Crx and Sp4, or Sp1, as well as their truncated mutants, Crx1-107, Crx111-299, Sp4-dZnf and Sp4-Znf.

Results: A positive interaction between Crx and Sp4 was detected by co-immunoprecipitation with in vitrotranslated proteins and an antibody against either Crx or Sp4. Further analysis with deleted forms of Crx and Sp4 showed that the DNA binding domain of Crx (homeodomain) and Sp4 (zinc fingers) are necessary and sufficient for mediating the Sp4-Crx interaction. A positive interaction between Crx and Sp1 was also detected using co-immunoprecipitation with the Crx antibody.

Conclusions: Both Crx and the Sp family members have been recently implicated in the transcriptional regulation of photoreceptor-specific genes. Crx is able to bind both Sp1 and Sp4 providing the physical basis of their potential functional interactions. Furthermore, it appears that the homeodomain of Crx and the zinc finger domains of Sp1 and Sp4 are critical for this binding. Therefore, interactions between Crx and Sp1 or Sp4 (or both) may be implicated in transcriptional regulation of photoreceptor-specific genes.

Effects of Fat-Soluble Vitamins and Cholesterol Supplementation on Retinal Structure and Function in an Animal Model of Smith-Lemli-Opitz Syndrome
D.K. Vaughan¹, D.J. Slotten¹, M.J. Richards^2A, B.A. Nagel^2A, N.S. Peachey³, S.J. Fliesler^2B. ¹Biology, Univ. Wisc. Oshkosh, Oshkosh, WI; ^AOphthalmology, ^BOphthalmology & Pharm. Physiol. Sci., ²Saint Louis Univ. Sch. Med., St. Louis, MO; ³Cleveland VAMC & Cole Eye Inst./Cleveland Clinic Foundn., Cleveland, OH.
Purpose: Patients afflicted with Smith-Lemli-Opitz syndrome (SLOS, an autosomal recessive metabolic disease caused by defective cholesterol biosynthesis) have abnormally low levels of cholesterol (Chol) and excessive levels of 7-dehydrocholesterol (7DHC) in all bodily tissues. The sterol metabolic defect is accompanied by inefficient absorption of dietary fat-soluble compounds (e.g., vitamins and sterols). We evaluated the ability of systemically administered fat-soluble vitamins and/or Chol to ameliorate or prevent retinal degeneration in a rat model of SLOS.
Methods: Pregnant Sprague-Dawley rats (6 days sperm-positive; N=8) were given AY9944 (an inhibitor of the defective enzyme in SLOS) in their diet and their progeny were injected 3X per wk with AY9944 as an aqueous-olive oil emulsion (Fliesler et al., IOVS 40:1792, 1999). In parallel, 2 control groups received vehicle injections only and no drug. Four drug treatment groups were established, according to supplementation of the vehicle with: A) no additions; B) vitamins A, D, and E; C) 2% (w/v) Chol; D) vitamins plus Chol. Rats were maintained under dim cyclic light (12L:12D, 20-40 lux) and fed Chol-free chow and water ad lib. At 9 postnatal weeks, dark- and light-adapted ERGs were recorded; one eye from each rat was taken for histological and quantitative morphometric analysis, while the contralateral retina, as well as serum, liver, and brain, were harvested for sterol analysis.
Results: 7DHC/Chol mole ratio values for retinas from all treatment groups were comparable, and were >500X higher than for control retinas. Rod and cone function were markedly compromised (decreased amplitudes, increased implicit times), relative to controls, and retinal degeneration (particularly photoreceptor loss) was substantial and comparable in all treatment groups.
Conclusions: Under the given conditions, systemic administration of fat-soluble vitamins (A, D, E) and/or Chol does not ameliorate or prevent retinal degeneration in this animal model of SLOS. Higher concentrations of these supplements, in combination with dietary administration, may be required to overcome the cholesterol biosynthesis defect and reduce the severity of the associated retinal degeneration and dysfunction.

An Improved Adult Animal Model of Smith-Lemli-Opitz Syndrome
S.J. Fliesler^1A, M.J. Richards^1A, B.A. Nagel^1A, G.A. Vogler^1B, N.S. Peachey², D.K. Vaughan³. ^AOphthalmology, ^BComparative Med., ¹Saint Louis Univ School of Med, St Louis, MO; ²Cleveland VAMC and Cole Eye Inst./Cleveland Clinic Foundation, Cleveland, OH; ³Biology, Univ. of Wisconsin-Oshkosh, Oshkosh, WI.
Purpose: Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disease caused by a defect in cholesterol (Chol) biosynthesis, at the level of 3β-hydroxysterol-Δ⁷-reductase. Prior SLOS animal models have employed either feeding or bolus injection of inhibitors of the reductase (AY9944 or BM15.766). To provide a more controlled pharmacological model of SLOS, we tested the efficacy of continuous systemic AY9944 delivery in adult rats, using implanted osmotic pumps.

Methods: Alzet pumps (Model 2ML4, 28-day) containing AY9944 (2 ml, 50 mg/ml water, 2.5 l/h) were implanted into anesthetized adult (3-mo. old, ca. 250 g) female Sprague-Dawley rats (N=10) under the dorsal skin. Over a 3-mo. Period, new pumps were inserted and old pumps removed every 4 wk. Rats were maintained under dim cyclic light (12L:12D, 20-40 lux) and fed Chol-free chow and water ad lib. Serum sterol levels were monitored by HPLC bi-weekly. After dark- and light-adapted ERGs were recorded, one eye from each rat was taken for histological and quantitative morphometric analysis, while contralateral retinas, livers, and brains, were harvested for sterol analysis. The results were compared against those obtained previously from control rats.

Results: 7-dehydrocholesterol (7DHC) to Chol mole ratio in all tissues of treated rats was >5:1; by contrast, 7DHC/Chol < 0.01 in control tissues. Dark- and light-adapted ERG amplitudes were markedly reduced, and their implicit times were substantially elevated, relative to controls. Outer nuclear layer (ONL) thickness was reduced by 20-30%, relative to controls.

Conclusions: Osmotic pump delivery of AY9944 offers an improved SLOS animal model, obviating the tedium and inherent variability of daily drug administration by diet or injections, while providing reliable, uniform, and continuous drug delivery at pharmacologically effective levels. Retinal degeneration and functional deficits were even greater than those previously obtained by systemic administration of AY9944 during gestational and postnatal development in rats (Fliesler et al., ARVO 2002), and also are consistent with retinal dysfunction observed in SLOS patients (Boisvert et al, ARVO 2002).

Identification of Retinal Proteins that are Nitrated in Elevated Glucose Concentration
Y.Du^1A, M.Miyagi^2A, K.West^2A, J.W. Crabb^2B, T.S. Kern^1B. ^ADepartments of Medicine, ^BDepartments of Medicine, Ophthalmology, Center for Diabetes Research, ¹Case Western Reserve Univ, Cleveland, OH; ^ACole Eye Institute, ^BCole Eye Institute and Lerner Research Institute, ²Cleveland Clinic Foundation, Cleveland, OH.
Purpose: Inhibition of diabetic retinopathy in animals by aminoguanidine is accompanied by a reduction in retinal protein nitration. We sought the identity of retinal proteins that are nitrated in diabetes as an approach to better understanding the pathogenic mechanisms of this retinopathy.
Methods: In vivo studies utilized retinas collected from STZ-diabetic rats (2 month duration), and in vitro studies used a transformed Muller cell line (rMC-1) incubated in normal (5mM) and high (25 mM) glucose. Retinas or cells were homogenized and subjected to 2D Western analysis using an anti-nitrotyrosine antibody. Immunoreactive spots were excised from the 2D gel, digested in situ with trypsin and analyzed by MALDI-TOF MS and/or LC MS/MS.
Results: Nitrotyrosine immunoreactivity in Western blots of retinas from diabetic rats was greater than that from nondiabetic rats or from diabetic rats treated with aminoguanidine. Likewise, rMC-1 cells incubated in 25 mM glucose exhibited greater nitrotyrosine immunoreactivity in Western blots than cells incubated in 5 mM glucose or in 25 mM glucose + aminoguanidine. The same nitrotyrosine immunoreactive proteins were identified from retinal homogenates and cultured rMC-1 cells, and include alpha enolase, glyceraldehyde 3-phosphate dehydrogenase, voltage-dependent anion-selective channel protein, aldolase, triosephosphate isomerase. Many of the identified nitrotyrosine immunoreactive proteins appear to be glycolytic enzymes. Alpha enolase exhibited the most dramatic change in apparent nitrotyrosine content.
Conclusions: Hyperglycemia appears to increase nitration of glycolytic enzymes in retina and retinal cells. NO or nitration can alter enzyme activities of proteins. The present findings justify further consideration of nitration and nitric oxide as possible mediators in the mechanisms of diabetic retinopathy.

Optical Coherence Tomographic Patterns of Diabetic Macular Edema
B.Y. Kim, P.K. Kaiser, J.E. Sears. Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH.
Purpose: To describe the various morphologic patterns of diabetic macular edema demonstrated by optical coherence tomography (OCT).
Methods: Retrospective chart review of 144 patients with clinically evident, diabetic macular edema. A total of 199 OCT scans (99 right eyes, 100 left eyes) were reviewed for the study. In addition to OCT evaluation, all patients underwent comprehensive ophthalmic history and examination.
Results: All patients had diabetic retinopathy and diabetic macular edema. OCT demonstrated five distinct morphologic subgroups of diabetic macular edema: diffuse retinal thickening (192 [96%] of 199 scans), cystoid macular edema (114 [57%]), subretinal fluid without posterior hyaloidal traction (21 [11%]), posterior hyaloidal traction without traction retinal detachment (28 [14%]), and posterior hyaloidal traction with traction retinal detachment (4 [2%]). Including all morphologic subgroups, mean retinal thickness measured at the central fovea was 411 microns.
Conclusions: Diabetic macular edema exhibits at least five different morphologic patterns. These include diffuse retinal thickening (96%), cystoid macular edema (57%), posterior hyaloidal traction without traction retinal detachment (14%), subretinal fluid without posterior hyaloidal traction (11%), and posterior hyaloidal traction with traction retinal detachment (2%).

Proteomic Analysis of Vitreous in the Presence of Diabetic Macular Edema
M.Ouchi¹, M.Kamei², K.West³, T.Yasuhara¹, M.Tei¹, H.Komori¹, T.Yamamoto¹, S.Kinoshita¹, J.W. Crabb³. ¹Ophthalmology, Kyoto Prefectural Univ of Medicine, Kyoto, Japan; ²Ophthalmology, Osaka University, Osaka, Japan; ³Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH.
Purpose: Even patients with posterior vitreous detachment show improvement in their diabetic macular edema (DME) after vitreous surgery. This may be attributable to the removal of chemical mediators present in the posterior vitreous cortex. Earlier studies of DEM-related proteins have focused on single proteins such as interleukin-6 (IL-6) and vascular endothelial growth factor (VEGE). We are pursuing a global approach to identifying DEM-related proteins using 2D electrophoresis and mass-spectrometry (MS).
Methods: We divided 44 patients who had undergone vitreous surgery into 4 groups; those with a macular hole/epiretinal membrane (MH/ERM) (n=11, 11 eyes); patients with non-DME pre-proliferative diabetic retinopathy (PPDR) (n=4, 4 eyes); those with DME (n=14, 16 eyes); and patients with proliferative diabetic retinopathy (PDR) (n=15, 15 eyes). Vitreous (~300 ul) were collected from the pre-macular vitreous body, total protein determined by the blood-fold method, and ~15 ug samples subjected to 2D electrophoresis and stained with SYPRO-Ruby. Proteins unique to DME vitreous were determined by comparsion of gel patterns using image analysis software, excised from the gel, digested in situ with trypsin and identified by LC MS/MS sequence analysis.
Results: Compared with the PPDR group, the DME group exhibited many 2D gel spots with significantly greater staining intensities. Furthermore, three of the prominently demarcated spots from the DME group were identified as pigment epithelium derived factor (PEDF), apoliprotein A-4 (ApoA-4), and thyroid hormone receptor-interacting protein-11 (Trip-11).
Conclusions: These findings suggest that PEDF, Apo-4 and Trip-11 may play a role in the pathogenesis of DME. The cytokine PEDF is involved in several retinal diseases, and high levels of the ApoA-4 are found in sera from diabetics and patients with renal failure. Further study of the possible relationships between these endogenous factors and DME is warranted.

Evaluation of Inner Retinal Activity in Mutant Mice Using c-fos Immunhistochemistry
B.W. Hanzlicek¹, N.S. Peachey², S.L. Ball³. ¹Research Service, Cleveland VA Medical Center, Cleveland, OH; ²Ophthalmic Research, Cleveland VA Medical Center; Cole Eye Institute, CCF, Cleveland, OH; ³Psychology, Cleveland VA Medical Center; Case Western Reserve University, Cleveland, OH.
Purpose: Visual information is mediated by multiple pathways in the mouse retina. Mice with functional defects in these pathways provide the opportunity to study their contribution to various aspects of visual function. For example, the nob mouse lacks communication between photoreceptors and depolarizing bipolar cells (DBC) while transducin null mice (Tr-/-) lack rod-mediated function. We have examined how these mutations alter inner retinal activity, by monitoring light-induced expression of an immediate early gene, c-fos, in a subpopulation of amacrine cells.
Methods: After overnight dark adaptation, mice were exposed to a strobe light stimulus presented at 2 Hz for 60 min. In different trials, light intensity varied from –2.7 to 0.4 log cd/sec m². For each stimulus condition, at least 3 wild-type (WT), nob, Tr-/- and Tr+/- mice were studied. Eyes were removed immediately following light exposure and processed for immunohistochemistry with c-fos anti-serum (Santa Cruz Biotechnology). Using light microscopy, c-fos-positive cells were counted in the inner nuclear layer.
Results: In all mouse lines, the number of cells labeled for c-fos increased with increasing stimulus intensity. Fewer cells labeled for c-fos in Tr-/- or nob mice than in WT or Tr+/- retinas. In mice exposed to the lowest flash intensities, more cells were labeled in nob than in Tr-/- retinas. At the highest flash intensity more cells were labeled in Tr-/- than in nob retinas.
Conclusions: These results indicate that c-fos activation can be used as an assay of inner retinal function and, when applied to mice lacking particular pathways, to elucidate inner retinal circuitry. Our results also indicate that c-fos activation is predominantly mediated by rod DBCs. Differences between Tr-/- and nob mice may reflect the use of an alternative rod pathway spared by the nob defect.

Nyctalopin Is Required for Signaling Through Depolarizing Bipolar Cells in the Murine Retina
R.G. Gregg^1A, P.D. Lukasiewicz², N.S. Peachey³, B.T. Sagdullaev^1B, M.A. McCall^1C. ^ABiochem & Molecular Biology / Opthalmology & Visual Sciecnes, ^BPsychological & Brain Sciences, ^CPsychological & Brain Sciences/ Ophthalmology and Visual Sciences, ¹University of Louisville, Louisville, KY; ²Ophthalmology & Visual science, Washington University, St. Louis, MO; ³Cole Eye Institute, CCF, Cleveland VAMC, Cleveland, OH.
Purpose: The nob mouse mutant, which lacks nyctalopin, has no b-wave. We investigated whether this defect was attributed to the inability of depolarizing bipolar cells (DBCs) to respond to glutamate. To determine whether glutamate release from photoreceptors was compromised, visual response properties of nob retinal ganglion cells were examined.
Methods: Whole cell recordings from bipolar cells were made in retinal slices from adult normal and nob mice. Current responses to puffs of glutamate were obtained under voltage clamp conditions. Each bipolar cell was filled with Lucifer yellow and classified by its morphology and the laminar location of its cell body and axon terminals. Ganglion cell recordings were made in vivo from the optic nerve, using tungsten electrodes. Computer driven visual stimuli were used to characterize their response properties.
Results: In retinas from normal mice, glutamate puffs produced a robust outward current in DBCs and an inward current in hyperpolarizing bipolar cells (HBCs). By contrast, in nob retinas, only HBCs responded to glutamate. The inability to respond to glutamate suggests the nob defect resides in the DBCs. In normal mice, visual responses from ON and OFF center ganglion cells were recorded in similar proportions. In nob retinas only OFF center cells responded to light stimulation.
Conclusion: These data indicate nyctalopin is required for modulation by glutamate of the cation current in DBCs and visual processing through the ON pathway.

Possible TULP1 Protein Interactions
Q.Xi, K.A. West, J.W. Crabb, S.A. Hagstrom. Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH.
Purpose: TULP1, a member of a family of four proteins with unknown function designated tubby-like proteins or TULPs, is expressed specifically in the inner segments and connecting cilium of photoreceptor cells. Mutations in TULP1 are associated with autosomal recessive retinitis pigmentosa and Tulp1 knockout mice develop an early-onset, progressive photoreceptor degeneration involving both rods and cones. To explore the physiologic function of TULP1, we are pursuing the identification of interacting proteins.
Methods: Immunoprecipitation experiments were performed with bovine retinal homogenates and a polyclonal anti-TULP1 antibody. Immunoprecipitation products were seperated by SDS-PAGE, protein bands excised, digested in situ with trypsin and identified by LC MS/MS.
Results: The following proteins were immunoprecipitated: Microtubule Associated Protein 1B, Clathrin Heavy Chain, Interphotoreceptor Retinoid Binding Protein, Dynamin-1, Rab Gerynl Gerynl Transferase, Dynein Intermediate Chain, Tubulin and Actin. In vitro pull-down experiments and in vivo co-immunoprecipitation experiments are in progress to further evaluate these possible TULP1 interactions.
Conclusions: Several of the identified proteins are involved in various aspects of vesicle transport or protein movement. TULP1 may function in intracellular trafficking of proteins synthesized in the inner segment to the outer segment of photoreceptor cells. These results provide a first step toward defining the mechanism underlying photoreceptor degeneration caused by mutations in TULP1.

Analysis of Phosphorylation of Connexin 36 in Bovine Retina
A.Sitaramayya¹, J.W. Crabb², A.Margulis¹, D.F. Matesic³, V.Singh¹, S.Pulukuri¹. ¹Eye Research Institute, Oakland University, Rochester, MI; ²Cleveland Clinic Foundation, Cleveland, OH; ³Mercer University, Atlanta, GA.
Purpose: Gap junction-mediated intercellular communication between specific retinal neurons is known to be regulated by cyclic nucleotide-dependent protein kinases. Connexin 36 is a major gap junction protein in retina. We attempted here to demonstrate phosphorylation of connexin 36 by kinases responsive to cyclic nucleotides and calcium.
Methods: Retinal homogenate was phosphorylated using ³²P-ATP in the presence of cyclic AMP, cyclic GMP or calcium. Proteins of the membrane fraction and membranes enriched in gap junctions were resolved by electrophoresis, and phosphoproteins were detected by autoradiography. Phosphorylated membrane proteins were also dissolved in detergent and connexin 36 was immunoprecipitated with an anti-connexin 36 antibody. Immunoprecipitated proteins were separated by 1- or 2-D electrophoresis and phosphorylated proteins were detected by autoradiography. Proteins of interest were excised from 1- or 2-D gels, digested in situ with trypsin and identified by capillary LC MS/MS.
Results: Specific retinal membrane proteins were phosphorylated in response to activation by cyclic AMP, cyclic GMP or calcium. Of them, a protein of about 36 kDa was phosphorylated only in incubations with calcium. However, immunoprecipitated connexin 36 was found not to be phosphorylated. The immunoprecipitate did contain other phosphorylated proteins, and the most prominent of them, a 58 kDa protein, was identified as beta tubulin. Alpha tubulin and trypsin inhibitor were also detected in the immunoprecipitate.
Conclusions: These results suggest that connexin 36 may not be directly phosphorylated in response to changes in cyclic nucleotides or calcium. Proteins associated with connexin 36, though not necessarily the ones we identified so far, might be targets of phosphorylation and could be involved in the regulation of gap junction intercellular communication.

Measurement of Ganglion Cell Layer and Inner Plexiform Layer Thickness with Optical Coherence Tomography
O.Tan¹, Y.Li², D.Huang¹. ¹Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH; ²Dept Biomedical Engineering, Case Western Reserve University, Cleveland, OH.
Purpose: To investigate direct measurement of the retinal ganglion cell layer (GCL) and inner plexiform layer (IPL) thickness with the commercial available optical coherence tomography (OCT).
Method: The OCT3 system (Carl Zeiss, OCT3000, Dublin, CA) was capable of acquiring 400 axial-scans (A-scan) per second with axial resolution of 10 micron in tissue. 2, 2.5, 3, 3.5, and 4 mm diameter circular retinal scans (centered at fovea) were obtained from normal volunteers with an OCT3 system. Each circular scan comprised 256 A-scans. An automatic computer algorithm was developed to process the resulting OCT images. Firstly, A-scans in each image were aligned according to the vitreo-retinal boundary. Second, a Gaussian low-pass filtering was used to smooth the image and suppress the speckle noise. Then a progressive segmentation was performed to detect the boundaries based on the gradient of the filtered OCT image.
Result: The thickness of the retinal layers were measured from each of the images and statistical analysis was performed. As expected, the GCL thickness decreases with increasing distance from the fovea. The thickness of the IPL is relatively constant in the area centralis. The SD of the combined GCL+IPL thickness is smaller (3-5 microns) than that of the GCL alone (4-6 microns).
Conclusion: This is the first demonstration of the direct GCL and IPL thickness measurements on a clinical OCT3 system. The only other report of segmentation of these retinal sublayers are based on images from an ultrahigh resolution (1-3 microns FWHM) OCT system that uses femtosecond laser light source, which is too bulky and expensive for routine clinical use. We have achieved the same desirable end result by image processing on moderately high-resolution images.

Analysis of the Inner Retina following Implantation of a Subretinal Artificial Silicon Retina in RCS Rats
B.D. Sippy¹, H.Yin², S.L. Ball³, M.J. Phillips², M.Blum⁴, A.Y. Chow⁵, M.T. Pardue⁶. ¹Emory University, Atlanta, GA; ²Atlanta VA Medical Center, Decatur, GA; ³Case Western Reserve University, Cleveland VA Medical Center, Cole Eye Institute CCF, Cleveland, OH; ⁴Cleveland VA Medical Center, Cleveland, OH; ⁵Optobionics, Naperville, IL; ⁶Emory University, Atlanta VA Medical Center, Atlanta, GA.
Purpose: An artificial silicon retina (ASR) implanted in the subretinal space may provide a possible treatment for photoreceptor degeneration by activating inner retinal circuitry to generate a visual signal. Here we evaluate retinal morphology in an animal model of photoreceptor degeneration, the RCS rat, after ASR implantation to determine if subretinal electrical stimulation has any beneficial or deleterious effects on inner retinal cell layers or bipolar cell populations.

Methods: The ASR was implanted into the subretinal space of RCS rats at 3-4 weeks of age. In each rat, one eye was implanted with an active implant while the other eye served as an unoperated control, underwent a sham procedure or was implanted with an inactive ASR. At 8 to 52 weeks post-implantation, the eyes were enucleated and fixed in mixed aldehydes. Eye cups were processed into plastic and sectioned vertically at 0.5μm or frozen and processed for immunohistochemistry using antibodies for PKC and recoverin. For evaluation of retinal thickness, digital images of plastic sections were taken and each retinal layer measured at ten locations across the retina using an image analysis program.

Results: The thickness of the ganglion cell layer, inner nuclear layer and inner plexiform layer were similiar between eyes implanted with active implants vs. inactive implants, sham operated or unoperated eyes. Antibodies to PKC and recoverin labeled rod and cone bipolar cells, respectively, with a similar distribution in all eyes.

Conclusions: The presence of an electrically active ASR within the subretinal space of RCS rats causes no apparent loss of inner retinal cells. In addition, ASR stimulation does not appear to induce reorganization of inner retinal circuitry.

Neuroprotective Effect of the Subretinal Artificial Silicon Retina
M.T. Pardue¹, S.L. Ball², H.Yin³, M.J. Phillips³, N.S. Peachey², B.Hanzlicek⁴, B.Sippy⁵, A.Y. Chow⁶. ¹Atlanta VA Medical Center, Emory University, Decatur, GA; ²Case Western Reserve University, Cleveland VA Medical Center, Cole Eye Institute CCF, Cleveland, OH; ³Atlanta VA Medical Center, Decatur, GA; ⁴Cleveland VA Medical Center, Cleveland, OH; ⁵Emory University, Decatur, GA; ⁶Optobionics, Naperville, IL.
Purpose: To evaluate possible neuroprotective effects of the subretinal artificial silicon retina (ASR) by measuring retinal function and photoreceptor preservation in RCS rats implanted at an early stage of degeneration.

Methods: Three week old RCS rats were implanted with an active ASR in one eye while the other eye was implanted with an inactive ASR, underwent a sham surgery, or served as an unoperated control. Retinal function was measured with ERGs before and after implantation. The number of photoreceptor nuclei were measured in plastic sections obtained from a subset of rats that were euthanized at 8 weeks post-implantation.

Results: While retinal function declined in all groups, preservation of retinal function occurred in eyes implanted with the ASR. Eyes implanted with the active ASRs showed significantly larger dark-adapted b-wave amplitudes than eyes implanted with inactive ASRs, sham operated or unoperated. In comparison, the dark-adapted a-wave decreased in amplitude equally in all groups. Counts of photoreceptor nuclei revealed a significantly larger number of photoreceptors directly overlying the implant in eyes implanted with an active or inactive ASR compared to unoperated or sham controls. However, no significant differences in photoreceptor numbers were detected between the eyes implanted with the active and inactive ASR.

Conclusions: These results indicate that the subretinal ASR may have a temporary protective effect on the RCS retina. While photoreceptor cell counts at 8 weeks did not reveal a significant difference between active and inactive ASR implanted retinas, the ERG data suggest that electrical stimulation provides a protective effect.

Hydrodynamic Properties of Porcine Bestrophin in Triton X-100
B.Stanton¹, A.F. X. Goldberg², A.D. Marmorstein¹. ¹Ophthalmic Research, i31, Cleveland Clinic Foundation, Cleveland, OH; ²Eye Research Institute, Oakland University, Rochester, MI.
Purpose: To determine the hydrodynamic properties of bestrophin and the stoichiometry of the detergent solubilized bestrophin complex under non-denaturing conditions.
Methods: Using porcine RPE detergent lysates or a solubilized membrane fraction derived from porcine RPE, the Stokes radius of bestrophin was determined using size exclusion chromatography on a Sephacryl S-300HR column. The Svedberg coefficient (S_20,w) and partial specific volume (ν) of the bestrophin-detergent complex were determined by velocity sedimentation through 5% - 20% (W/V) sucrose gradients prepared in either H₂O or D₂O containing 0.2% Triton X-100 (TX-100). An estimation of molecular mass of the protein portion of the bestrophin / TX-100 complex was made assuming additivity of ν for TX-100 (0.94 ml/g) and protein (0.74 ml/g).
Results: The Stokes radius of bestrophin was determined to be ~7.25nm. Velocity sedimentation experiments indicate a S_20,w for bestrophin of 4.9 ± 0.4 and ν of the bestrophin / TX-100 complex of 0.80 ± .02 (ml/g). Based on these data we estimate a mass of ~206,000 kDa for the bestrophin / TX-100 complex. Assuming ν = 0.74 ml/g for the protein portion of the complex, we calculate that bestrophin binds~0.31 g of Triton X-100 / g protein and is present as a single major species with an estimated mass of ~ 138 kDa.
Conclusions: We have determined the hydrodynamic properties of porcine bestrophin solubilized in TX-100. The combined data indicate a protein mass, measured under non-denaturing conditions, of approximately twofold that of monomeric bestrophin (138 kDa / 68 kDa = 2.03). We consider it likely that the dimeric form is the minimal unit of function for bestrophin in the porcine RPE plasma membrane.

Correlation of Bestrophin Protein Expression in the Basolateral Plasma Membrane of the Mouse RPE with the Onset of Photoreceptor Activity in the Retina
B.Bakall¹, N.S. Peachey², L.Y. Marmorstein², C.Wadelius¹, A.D. Marmorstein². ¹Genetics and Pathology, Uppsala University, Uppsala, Sweden; ²Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH.
Purpose: Best macular dystrophy is an autosomal dominant disease, caused by mutations in the VMD2 gene that encodes the protein bestrophin. To increase our understanding of the function of bestrophin, the onset of bestrophin expression was followed in mouse eyes during development.
Methods:BALBc mice were examined at different embryonic (E) and postnatal (P) ages. Bestrophin mRNA was quantified using Taq-man quantitative PCR. Immunohistochemistry for mouse bestrophin was performed on paraffin-embedded sections, using a polyclonal mouse bestrophin antibody produced by immunizing rabbits with a peptide corresponding to the mouse bestrophin C-terminus. Electroretinograms were recorded from the corneal surface of mice beginning at P8, to strobe flashes after overnight dark adaptation.
Results: Bestrophin mRNA was detected as early as E15. Bestrophin protein expression in the RPE was not observed until P10 at which time it was observed to localize to the basolateral membrane of the RPE. The earliest age at which an a-wave could be recorded from mouse eyes was P10, coincident with the onset of bestrophin protein expression in the RPE.
Conclusions: Despite the presence of mRNA for Bestrophin during embryonic development, the onset of protein expression in RPE is late, beginning at P10. This coincides with the onset of photoreceptor electrical activity, supporting the hypothesis that bestrophin plays a role in the RPE electrical responses to phototransduction. In addition, we conclude that bestrophin protein is a late marker for RPE differentiation.

Effects of Overexpression of Bestrophin and Best Macular Dystrophy Associated Mutants of Bestrophin on the Rat DC-ERG
A.D. Marmorstein¹, J.Yocom¹, B.Stanton¹, B.Bakall², P.J. McLaughlin¹, M.T. Schiavone¹, C.Wadelius², L.Y. Marmorstein¹, N.S. Peachey¹. ¹The Cole Eye Institute, i31, The Cleveland Clinic Foundation, Cleveland, OH; ²Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden.
Purpose: To determine the effects of wild type and mutant bestrophin overexpression on the light peak of the rat DC-ERG.
Methods: Adenovirus-mediated gene transfer was used to express wild type bestrophin (wt-best), or two bestrophin mutants (W93C, R218C) associated with Best macular dystrophy (BMD) in the RPE of adult Long-Evans rats. Two weeks after subretinal injection, the retina and RPE were analyzed by fundus exam, conventional and DC-ERG recordings, histology, and immunofluorescence.
Results: Using doses between 0.2 x 10⁷ and 5 x 10⁷ pfu, wt-best and both the W93C and R218C mutants correctly localized to the basolateral plasma membrane of the RPE. No defects were noted on fundus exams and the morphology of the retina, RPE, and choroid was typically preserved. While an effect of viral load was noted on conventional ERG amplitudes, these effects did not differ from a null virus control. DC-ERGs recorded in response to a 5-min flash stimulus displayed consistent changes in the light peak (LP) when compared against sham- or null virus-injected controls. While LP amplitude did not change in eyes overexpressing wt-best, the LP time constant (τ) was significantly accelerated. In eyes overexpressing W93C or R218C, LP amplitude was significantly lowered vs. null controls. In addition, τ was significantly slowed in W93C animals but unaltered in animals receiving R218C. In normal, control, or wt-best rats, LP onset occurred ~112 sec after stimulus presentation. In eyes receiving R218C, LP onset was delayed, to 130-135 sec. No defects in LP sensitivity were noted, although the LP continued to increase with longer stimulus durations in W93C transduced eyes only.
Conclusions: Overexpression of wt-best accelerates the LP kinetics, but does not affect LP amplitude or the other components of the DC-ERG. BMD associated mutants exhibit LP reductions and delays that mimic the electrooculographic abnormalities seen in BMD patients. Differences between the effects induced by W93C and R218C suggest a complex mechanism behind bestrophin activity.

Mechanism of Induction of Choroidal Neovascularization in Sorsby Fundus Dystrophy
B.Anand-Apte¹, J.Hua Qi¹, Q.Ebrahem¹, G.Murphy², L.Claesson-Welsh³. ¹Cole Eye Inst/Cleveland Clinic F, Cleveland, OH; ²Oncology, Inst. for Medical Research/Cambridge University, Cambridge, United Kingdom; ³Genetics and Pathology, Uppsala University, Uppsala, Sweden.
Purpose: Sorsby fundus dystrophy (SFD) is a dominantly inherited condition characterized by the development of choroidal neovascularization, subretinal hemorrhages and changes consistent with disciform degeneration. Mutations in the Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) gene, all of which introduce an extra cysteine residue into exon 5, cause Sorsby Fundus Dystrophy (SFD). TIMP-3, a regulator of matrix metalloproteinases (MMPs) is deposited by retinal pigment epithelial (RPE) cells into Bruch's membrane (BM) where it is a component of the extracellular matrix. In this study we set out to elucidate the mechanism by which TIMP-3 mutations induce the angiogenic phenotype.

Methods: We have introduced TIMP-3 into porcine aortic endothelial cells expressing VEGFR-2 as well as human retinal pigment epithelial cells. In vitro, VEGF induced proliferation and migration of endothelial cells were analyzed by Coulter particle counting and modified Boyden chamber assays respectively. VEGF induced autophosphorylation of VEGFR-2 and activation of p44/p42 MAP kinase was evaluated by western blot analysis. Conditioned medium from RPE cells was tested for their ability to induce angiogenesis in an in vivo chick chorio-allantoic membrane (CAM) assay.

Results: TIMP-3 over-expression in endothelial cells resulted in an inhibition of the proliferative and migratory response of these cells to VEGF. VEGF-induced autophosphorylation of VEGFR-2 and p44/p42 MAP kinase activation were also suppressed. These effects appear to be independent of its MMP inhibitory activity. Conditioned medium from RPE cells expressing SFD mutant TIMP-3 demonstrated increased MMP activity as well as increased angiogenic activity on CAM assay.

Conclusions: Our data indicate that expression of SFD mutant TIMP-3 in RPE cells results in increased MMP activity and induction of angiogenesis.

Light Induced Protein Modifications and Lipid Oxidation Products in Rat Retina
K.Renganathan^1A, M.Sun^1B, R.Darrow², L.Shan^1B, X.Gu^1A, R.G. Salomon³, S.Hazen^1B, D.Organisciak², J.W. Crabb^1A. ^AOphthalmology, Cole Eye Institute, ^BLerner Research Institute, ¹Cleveland Clinic Foundation, Cleveland, OH; ²Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH; ³Department of Chemistry, Case Western Reserve University, Cleveland, OH.
Purpose: To better understand the mechanisms of oxidative damage in retinal pathology, we have sought the identity of lipid oxidation products and protein adducts in rat retina after in vivo exposure to damaging light
Methods: Albino rats maintained in a dark environment for 2 months were exposed to intense green light (1500 lux) for 1 or 4 hours and sacrificed immediately following light treatment. Retinas were isolated and immediately protected with antioxidants. Lipids were extracted with chloroform/methanol and analyzed by LC MS. Proteins were extracted with SDS-PAGE sample buffer and analyzed by Western blotting.
Results: Lipid oxidation products in rat retina from docosahexaenoyl phosphatidylcholine (DHA-PC), arachidonoyl (AA)-PC, and linoleyl (LA)-PC were more abundant after 4 h of light exposure than after 1h or no light. Anti-carboxyethylpyrrole, anti-argpyrimidine and anti-nitrotyrosine immunreactivities were significantly greater after 4h light exposure compared with control animals maintained in the dark. Anti-opsin immunoreactivity was also significantly greater after light treatment.
Conclusions: Current results are consistent with our recent observation that light modulates protein nitration in rat retina (2002 Mol. & Cell. Proteomics 1, 293). Intense light also generates lipid oxidation products in rat retina in vivo that result in oxidative protein modifications such as carboxyethylpyrrole from DHA containing lipids. Argpyrimidine, derived from methylglyoxal, appears to be another protein modification induced by light. The apparent increase in opsin after light may be due to modifications that increase the solubility and extractability of this integral membrane protein. These findings justify further consideration of lipid oxidation products and protein modifications as mediators in the light-induced biochemical sequel leading to photoreceptor cell death.

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