Role of Protein Interactions in Retina Development and Function

National Eye Institute

Investigators

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Abstract

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. We used Wistar albino rats. Cell death was assayed by immunofluorescence and flow cytometry using TUNEL, BrDU, propidium iodide, mitotracker, and annexin V. Immunofluorescence of cells for visualizing rhodopsin, CRX, and antisyntaxin under confocal microscopy was performed. Neurite outgrowth was also quantified. Results show that 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. These effects were abolished by an inhibitor of the PEDF receptor or receptor-derived peptides that block ligand/receptor interactions. While all the activities were specifically conferred by short peptide fragments (17 amino acid residues) derived from the PEDF neurotrophic domain, no effects were triggered by peptides from the PEDF antiangiogenic region. The observed effects on retinal neurons imply a specific activation of the PEDF receptor by a small neurotrophic region of PEDF. In addition, cytotoxicity assay was performed measuring the formazan formation due to lactate dehydrogenase activity released to the media by cells. Bcl2 and Bax proteins were also examined by western blotting of cell lysates and mitochondrial fractions of the rat primary cultures treated with and without the effectors. A study on expression and production of recombinant human PEDF and PEDF-R variants by mammalian and bacterial cells was completed. To obtain large amounts of recombinant PEDF proteins, we subcloned the coding sequence of human SERPINF1 mutated versions into the pCEP4 vector and generated stably transfected HEK.Ebna cells. Recombinant PEDF protein production and secretion was detected by SDS-PAGE and western blotting of the culturing media. The recombinant PEDF proteins were purified by ion-exchange column chromatography and milligram amounts of highly purified protein were recovered. Recombinant PEDF-R truncated versions were obtained from Escherichia coli containing expression vectors with human PNPLA2 cDNAs with 3'end deletions and by induction with isopropyl -d-1-thiogalactopyranoside. The bacterially derived PEDF-R proteins in insoluble inclusion bodies were solubilized with urea and purified by cation-exchange column chromatography. PEDF binding assays of the recombinant PEDF-R versions to bind PEDF were performed. Cells expressing for a full-length human PEDF version with a single point alteration at H105A were cultured at large scale, and the proteins secreted from the cells into the serum-free media were collected, concentrated, and dialyzed (in collaboration with the Y Shiloach laboratory). 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 in the Wistow laboratory). Expression and production of recombinant human PEDF-R versions in E. coli cells were performed. Conditions for overproduction, solubilization and purification were optimized. A study to examine the effects of PEDF deletion on the induction of cellular senescence in RPE cells was completed. We determined the expression of senescence-associated genes in the RPE of 3-month-old mice that lack the Serpinf1 gene by RT-PCR and found that Serpinf1 expression induced H2ax for histone H2AX protein, Cdkn1a for p21 protein, and Glb1 gene for -galactosidase. Senescence-associated -galactosidase activity increased in the Serpinf1 null RPE when compared with wild-type RPE in RPE/choroid flatmounts. Evaluation of the RPE subcellular morphology showed that ablation of Serpinf1 increased the volume and the number of the nuclei of RPE cells, implying chromatin reorganization. Given that the RPE phagocytic function declines with aging, we assessed the expression of the Pnpla2 gene by RT-PCR and PEDF-R protein levels by immunofluorescence and found that both declined with the Serpinf1 gene ablation. Determination of rhodopsin and lipid accumulation by immunofluorescence and BODIPY staining in the RPE flatmounts was performed. The levels of both rhodopsin and lipids in the RPE of the Serpinf1 null mice accumulated compared to littermate controls, implying an association of PEDF deficiency with RPE phagocytosis dysfunction. The findings established PEDF loss as a cause of senescence-like changes in the RPE, highlighting PEDF as both a retinoprotective and a regulatory protein of aging-like changes associated with defective degradation of the photoreceptor outer segment in the RPE. We examined the polarization of PEDF secretion and functionality of induced-primary RPE (ipRPE) monolayers of cells in transwells cultured for 15-50 days (collaboration with the laboratory of Valeria Canto-Soler, U Colorado). The PEDF concentration was measured in the apical and basal compartments of the transwells by ELISA and western blotting. The capacity of ipRPE monolayers to internalize and degrade photoreceptor outer segments during phagocytosis was assessed. We determined the amount of rhodopsin in cell lysates and the amount of hydroxybutyrate secreted to the culturing media of cells that had been exposed to bovine outer segments. We continued investigating the regulation of CRX by PEDF during photoreceptor survival. Serpinf1-/-, Serpinf1+/- and Serpinf1+/+ mice at 3 months of age were used. We prepared cultures of mouse retinal explants and treated them with recombinant human PEDF protein. The transcriptional levels of Crx, Pde6a, Opn1mw and Opn1sw were assessed using RT-PCR. Subcellular distribution of CRX, PDE6A and pan-acetylation in the outer nuclear layer of the retinal explants from Serpinf1-/- and Serpinf1+/+ mice was detected by immunofluorescence. Zaprinast was added to the retinal explant cultures to induce photoreceptor death. A fluorescent probe that targets anionic phospholipids such as phosphatidylserine, was used to identify dying photoreceptor cells in the retinal explants from Serpinf1-/-, and Serpinf1+/+ mice treated with combinations of zaprinast and PEDF. Subcellular distribution of CRX was visualized and evaluated by immunofluorescence in the outer nuclear layer of Serpinf1-/- and Serpinf1+/+ retinal explants treated with combinations of zaprinast and PEDF. To study the effects of PEDF to inhibit the cytokine induction ARPE-19 cell cultures were incubated with recombinant human tumor necrosis factor alpha (TNF-) to induce interlukin-6 (IL-6). Cell viability was determined and followed by microscopy. The culture media was collected to determine the IL-6 protein concentration secreted by the cells. Cells were collected to isolate RNA for RT-PCR. To challenge the stimulation of IL-6, we added recombinant human PEDF protein to the cultures. Concentration-response relationships were assessed for TNF--mediated induction of IL-6 and for the PEDF blocking effects. We used synthetic peptides designed from the pro-death 34-mer (positions 44-77), and the pro-survival 44-mer (78-121) and 17-merH105A (98-114) of PEDF to challenge the IL-6 overproduction. Serpinf1-/- mice were bred for a study of PEDF as potential biomarker for intervention in COVID-16 during cytokine storm (collaboration with R Star and P Yuen, NIDDK).