Role of Protein Interactions in Retina Development and Function

National Eye Institute

Investigators

Linked publications, trials & patents

Abstract

The survival effects of PEDF in retinal photoreceptors during development in vitro were investigated (collaboration with the Politi laboratory). Pure neuronal cultures were prepared from retinas of rats at postnatal day 1. Expression of Pnpla2, and PEDF-R protein was detected in the cells. We explored the subcellular localization of PEDF-R in the cells using specific antibodies to the protein and plasma membranes markers, and confocal microscopy (Bioimaging core, NEI). The effects of the PEDF peptides 34-mer, 44-mer and 17-mer in developing retina neurons in culture were evaluated using and Annexin V/Mitotracker Flow Cytometry assays in vitro (Flow cytometry core, NEI). The effects of PEDF-R derived peptide containing a ligand binding site and Atglistatin, an enzymatic inhibitor, were used to block and inhibit PEDF-R. Polarization of rhodopsin in cells treated with PEDF peptides was evaluated. We also measured axon outgrowth of photoreceptor and amacrine cells in cultures treated with PEDF peptides. We completed studies describing how PEDF hinders photoreceptor cell death by reducing intracellular calcium in the degenerating retina (in collaboration with the Marigo laboratory). We optimized the protective effects of PEDF peptides in 661W photoreceptor cells induced to die by light. 661W cells were exposed to damaging light for increasing amounts of time and analyzed for cell viability. The levels of Pnpla2 mRNA and PEDF-R protein in cells following light damage were determined. Concentration response curves of full-length PEDF and a neurotrophic peptide derived from PEDF were performed. Atglistatin, an inhibitor of PEDF-R activity, was used to assess the involvement of PEDF-R in PEDF-mediated protective effects. Cell viability assays of 661W cells following light damage in the presence of PEDF were performed in multiwell plates in preparation for the development of a high-throughput screen. To continue exploring the transcriptome profile of retina R28 cells upon promoting neurotrophic activity, a gene expression profiling real-time PCR-based array technology for rat cell death pathways was used. Quantitative PCR and western blots to validate genes and proteins selected from the profiler were performed. We described studies on protection of RPE cells against cytotoxicity in vitro by PEDF using a method of chronic sodium iodate-mediated injury on RPE cells, which may serve to evaluate protective factors against oxidative stress. Cytotoxicity and cell viability curves of RPE cells with sodium iodate were generated. The antioxidant PEDF decreased sodium iodate-mediated cytotoxicity without affecting RPE cell viability. A cell culture system to evaluate protection against oxidative stress injury with PEDF was discussed. Subcellular localization of PEDF-R was investigated using antibodies to the predicted extracellular and intracellular domains of the polypeptide in ARPE-19 cells under permeabilized and non-permeabilized conditions, and images were collected using confocal microscopy as well as immunoelectron microscopy (Bioimaging and Histology Cores, NEI). Topology of PEDF-R polypeptide was investigated in silico and full-length sequence of human PEDF-R was entered in the Constrained Consensus TOPology prediction server (CCTOP) for transmembrane prediction. Insertion of PEDF-R polypeptide in membranes begun to be investigated in collaboration with the Hessa laboratory. To explore the role of PEDF-R in regulation of lipid metabolism and phagocytosis in the RPE, we generated ARPE-19 cell lines stably selected for silencing PNPLA2. Attenuation of PNPLA2 and PEDF-R levels were confirmed at the gene and protein level. The cells were loaded with oleic acid to induce lipid droplets, which were visualized using BODIPY stain. The role of PEDF-R under oxidative stress was evaluated by measuring cell viability using crystal violet, reactive oxygen species, toxicity, and mitochondrial function. Oxidative stress was induced by NaIO3 and H2O2. LC-MS/MS based methodology was used to detect and measure lipids. The cells were also challenged with photoreceptor outer segments (POS) and assayed for binding and internalization of POS. POS degradation was measured by beta-hydroxybutyrate production and rhodopsin content by immunofluorescence and western blot. Intracellular lipid accumulation following POS addition in stable cell lines was assessed by BODIPY staining. PEDF-derived 17 mer peptides containing the neurotrophic region of PEDF, were synthesized with an Alexa 488 fluorophore and attached to the 17 mer via a linker (peptides generated by Imaging Probe and Development Center, NIH). Peptides were tested for binding affinity to cell-surface PEDF-R in ARPE-19 and R28 cell lines, and competition of binding assays were performed in the presence of excess unlabeled peptide. Peptide-peptide interactions were probed using MicroScale Thermophoresis (Biophysics Core, NHLBI). We continued to purify large amounts of PEDF versions from conditioned media of stably transfected mammalian cells with PEDF expression vectors. Serum-free media collected from Hek.Ebna 293 cells harboring expressing vectors for full-length human SERPINF1 gene and versions with single point alterations (H105A) were concentrated and purified using ion-exchange chromatography.