Role of Protein Interactions in Retina Development and Function

National Eye Institute

Investigators

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Abstract

A study to examine the contribution of PEDF-R to the phagocytosis process was completed. During phagocytosis, RPE cells ingest abundant phospholipids and protein in the form of photoreceptor outer segment (POS) tips, which are then hydrolyzed. Mice in which Pnpla2 was conditionally knocked out (cKO) in the RPE were generated. Expression of the Pnpla2 gene was determined by RT-PCR in dissected RPE/choroid from the mice. The RPE of the cKO mice accumulated lipid droplets as detected by transmission electron microscopy of retinal sections, as well as more abundant and larger rhodopsin particles detected by immunofluorescence of RPE/choroid flatmounts, compared to littermate controls. Determination of ketone body -hydroxybutyrate in media of cultures showed that upon POS exposure, RPE explants from cKO mice released less -hydroxybutyrate compared to controls. To inhibit PEDF-R, the phospholipase A2 inhibitor bromoenol lactone was used in ARPE-19 cell cultures. The ARPE-19 cells transfected with siPNPLA2 silencing duplexes and POSs isolated from bovine retinas were used. Rhodopsin in cell extracts from pulse-chase experiments with POS was resolved by western blots and showed that after POS ingestion during phagocytosis, rhodopsin degradation was stalled both in cells treated with bromoenol lactone and in PNPLA2-knocked-down cells relative to their corresponding controls. Phospholipase A2 inhibition lowered -hydroxybutyrate release from phagocytic RPE cells. Total phospholipids and fatty acids were determined from cell lysates of transiently transfected cells challenged with POS (collaboration with MP Agbaga). PNPLA2 knockdown also resulted in a decline in fatty acids and -hydroxybutyrate release from phagocytic RPE cells. The findings imply that the efficiency of RPE phagocytosis depends on PEDF-R, thus identifying a novel contribution of this protein to POS degradation in the RPE. A study to evaluate the neurotrophic effects of PEDF and its fragments in an in vitro model of cultured primary retinal neurons that die spontaneously in the absence of trophic factors was completed. Wistar albino rats were used. The cell types in the cultures were characterized by immunolabeling for photoreceptor and amacrine neuron markers, cone-rod homeobox (CRX) and HPC1 (syntaxin), respectively, and followed by confocal microscopy and flow cytometry of CRX-labeled cells. RT-PCR, and western blotting showed that the cells at 1 - 5 days in culture and in native retinas at postnatal ages between 2 6 days contained PEDF-R. Immunolabeling with PEDF-R antibodies in the cultured cells showed colocalization of PEDF-R with membrane markers wheat germ agglutinin and Na/K pump. PEDF and peptides with PEDF-R affinity (44-mer and 17-mer), and without PEDF-R affinity (34-mer), a PEDF-R enzymatic inhibitor and a peptide that blocks the PEDF ligand-receptor interaction were used. Cell death was assayed by immunofluorescence and flow cytometry by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, propidium iodide, MitoTrackerTM, and annexin V assays. Neurite outgrowth was evaluated by immunolabeling with anti--tubulin antibody and measured by confocal microscopy. Cytotoxicity was assayed by measuring the formazan formation due to lactate dehydrogenase activity released to the media by cells. PEDF protected photoreceptor precursors from apoptosis, preserved mitochondrial function and promoted polarization of opsin enhancing their developmental process, as well as induced neurite-outgrowth in amacrine neurons. The 44-mer and 17-mer peptides also promoted photoreceptor survival and amacrine neurite outgrowth, however the 34-mer peptide lacked such effects. These effects were abolished by both the PEDF-R inhibitor and blocking peptide. The findings illustrated the use of primary neuronal retinal cell cultures to gain understanding of the individual activities of the multimodal PEDF on the diverse cell population of the retina and highlight the potential of the PEDF peptides as promoters of retinal differentiation, and inhibitors of retina cell death. We continued to explore the potential cascade of signaling events triggered by the extracellular PEDF on the primary retinal cells in culture. Pilot experiments to determine the quality of samples for proteomics (in collaboration with L Jenkins) and lipidomics (in collaboration with B Schwarz) were performed. The effects of PEDF and derived peptides on regulation pro-apoptotic- and anti-apoptotic-related factors (Bcl2, Bax and RAC1 proteins) were examined by western blotting of cell lysates, cytosolic and mitochondrial fractions of the rat primary cultures treated with and without the effectors. Retinal explant cultures were prepared from adult wild type C57BL/6J mice. For mimicry of a retinal degeneration model, namely rd10, zaprinast was included in the medium of wild type retinal explants at different concentrations to optimize the effects on photoreceptor cell death ex vivo. Photoreceptor cell death was assessed in whole retinal mounts after DAPI staining, TUNEL, and anti-cleaved caspase-3 labeling, and visualized by fluorescence microscopy. Recombinant human PEDF was used as cytoprotectant and pilot experiments to determine the optimal timing of addition of zaprinast and PEDF concentration to the explants were performed. To define the molecular mechanisms of the photoreceptor cytoprotection, detection of cone-rod homeobox (CRX), lamin-A/C antibody, rhodopsin and peanut agglutinin (PNA, a marker of cone photoreceptors) in whole mounts of PEDF-treated explants was performed by immunofluorescence and confocal microscopy super resolution imaging. The changes in the subcellular distribution of CRX, PEDF-R, as well as BODIPY staining of neutral lipids in the photoreceptor cells of the explants each exposed to PEDF, zaprinast, or a combination of PEDF and zaprinast were examined relative to vehicle additions. To investigate the transcriptional regulation that is promoted by PEDF stimuli in photoreceptors ex vivo, the expression levels of several genes known to be regulated by CRX (Arr3, Pde6a, Rho, Opn1mw, Opn1sw, Thrb) were determined by quantitative real-time PCR (qPCR) of RNA isolated and its respective proteins by western blots of protein extracts from retinal explants treated without or with zaprinast, PEDF or a combination of PEDF plus zaprinast. We continued to purify large amounts of recombinant human PEDF protein versions from conditioned media of stably transfected Hek.Ebna 293 cells with PEDF expression plasmids. Cells expressing for a full-length human PEDF version with a single point alteration at H105A were cultured at large scale, and the secreted proteins from the cells into the serum-free media were collected, concentrated, and dialyzed (in collaboration with J Shiloach). The proteins were purified using a two-step ion-exchange column chromatography. The purified PEDF protein combined with a chemically synthesized peptide fragment of the ectodomain of PEDF-R was used in high-throughput crystallization screening (in collaboration with V Sagar). Expression and production of recombinant human PEDF-R versions in E.coli cells were performed. Conditions for overproduction, solubilization and purification were optimized. To investigate the effects of PEDF to inhibit the hallmark of COVID-19 cytokine storm, in particular regulation of interlukin-6 (IL-6), cells were cultured and treated with tumor necrosis factor alpha TNF alpha for 24 hours to induce IL-6. Following the treatment, culture media was collected for protein analyses (ELISA) and total RNA was isolated from harvested cells for RT-PCR. Serpinf1 mice were bred for a study of PEDF as potential biomarker for intervention in COVID-16 during cytokine storm (collaboration with P Yuen).