Role of Protein Interactions in Retina Development and Function

National Eye Institute

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Abstract

To explore the regulatory role played by the PEDF/PEDF-R axis in photoreceptor survival and lipid metabolism, we continued characterizing photoreceptors deficient in PEDF and PEDF-R. We used Pnpla2 knock-out mice crossbred with Serpinf1 null mice to generate wild type, Serpinf1+/-/Pnpla2-/- and Serpinf1-/-/Pnpla2-/- genotypes. Evaluations were performed on 3-month-old mice of mixed sexes. Whole eyes were enucleated and processed for histology, immunofluorescence, and confocal microscopy. Retinal samples were prepared for lipid profiling (collaboration with K Schey) and ultrastructural assessment by transmission electron microscopy (collaboration with R Fariss). Ablation of PEDF and PEDF-R led to early and pronounced defects in photoreceptor morphology, lipid accumulation and function, surpassing effects seen in single knockout models. The findings support the idea that PEDF/PEDF-R links retinal lipid metabolism to photoreceptor survival and function. The biochemistry of PEDF-R continued to be investigated. A truncated form, PEDF-R[1-288], was expressed in E. coli Rosetta (DE3) using an N-terminal His-, SUMO-tag, and C-terminal Strep-tag fusion, and purified by ammonium sulfate precipitation followed by His- and Strep-affinity chromatography. This yielded a detergent-soluble protein forming complexes >120 kDa. PEDF-R[1-288] catalyzed PLA1 and PLA2 reactions, revealing PLA1 activity not previously reported and establishing PEDF-R as a phospholipase B (PLB). The enzyme remained stable after shock-freezing and required no calcium. PLA1 was observed over a broad pH range, while PLA2 had optimal activity at basic pH. Atglistatin selectively inhibited PLA1, whereas bromoenol lactone inhibited PLA2. Both activities were stimulated by the 17-mer peptide and by the 21-mer or 29-mer[H105A] peptides, with the 25-mer and 29-mer selectively enhancing PLA2. PEDF-R[1-288] also exhibited triglyceride lipase activity, stimulated by the 17-mer peptide. This work establishes a robust method for producing active recombinant PEDF-R, and broadens the functional scope of PEDF-R, likely contributing to its neurotrophic effects and promotion of cell survival. We continued evaluating modified PEDF peptides for their ability to stimulate PEDF-R enzymatic activity. Peptides derived from the neurotrophic 17-mer region with N- and C-terminal modifications, amino acid substitutions, truncations, or incorporation of non-alpha-amino acid linkers, were tested for effects on PLA1 and PLA2 activities of PEDF-R[1-288]. Peptides showing extreme effects were further assessed for neurotrophic activity in the rd10 mouse model of retinal degeneration via eye drop administration. The effects of these peptides on PLA1 and PLA2 activities were measured and evaluated for potential correlation with their neurotrophic properties, providing a basis for future screening. We continued investigating the effects of comparative gene identification-58 (CGI-58) on PEDF-R phospholipase activity. CGI-58, encoded by the ABHD5 gene, is an α/β-hydrolase-fold protein essential for lipid metabolism. Recombinant human CGI-58 was expressed in E. coli Rosetta (DE3) from ABHD5 cDNA cloned into pE-SUMOstar with an N-terminal His6-tag, and purified using His-Trap affinity, ion exchange, and size exclusion chromatography, followed by protease cleavage. CGI-58 remained soluble in buffers containing zwitterionic detergents and formed complexes >242 kDa. Biochemical assays revealed that CGI-58 did not stimulate the PLA1 or PLA2 activities of the PEDF-R[1-288], which retains the active site and PEDF-binding region. However, CGI-58 itself exhibited intrinsic PLA1 and PLA2 activities, consistent with the esterase/lipase/thioesterase subfamily. A CGI-58[N153A] mutant retained similar activity, identifying a novel intrinsic PLA activity and providing a method for producing active recombinant protein for future mechanistic studies. These findings indicate that CGI-58 is unsuitable as selective co-activator in PEDF-R activity assays. We continued studying CRX expression regulation by PEDF in retinal degeneration models. CRX is a transcription factor that plays a key role in photoreceptor development and gene expression. Wild-type (C57BL/6J), Serpinf1-/-, Pnpla2-/-, rd10, and rd10/Serpinf1-/- mice were used. Retinal explants were prepared and treated with zaprinast to induce photoreceptor cell death. Treatments included PEDF[H105A], PEDF-derived peptides (H105A, R99A), and atglistatin (PEDF-R inhibitor); H105A was also administered as eye drops. Crx transcript levels and CRX protein were assessed by qRT-PCR and immunofluorescence. PEDF or PEDF-R deficiency reduced CRX levels in photoreceptor nuclei, whereas PEDF[H105A] restored CRX levels and mitigated zaprinast-induced changes. H105A eye drops transiently increased Crx in rd10 and rd10/Serpinf1-/- mice. R99A, lacking PEDF-R affinity, had no effect, and atglistatin abolished H105A-mediated CRX upregulation, indicating PEDF modulates CRX via PEDF-R. We continued studies on the role of PEDF in inhibiting IL-6 production in RPE cells. iPSC-RPE were treated with PEDF, PEDF[H105A] protein, and the cytokines IFN-γ and TNF-α. IL-6 levels were measured by ELISA and RT-PCR; cell viability was assessed using crustal violet. IFN-γ or TNF-α increased IL-6 secretion and decreased cell viability, which were mitigated by PEDF or PEDF[H105A], suggesting PEDF prevented inflammatory signaling. Finally, we examined whether Pnpla2 deficiency induces ferroptosis in RPE. Wild-type and Pnpla2-/- mice (12 weeks) were analyzed for ferroptosis hallmarks. Lipid peroxidation was assessed by C11 BODIPY staining, showing marked increases in Pnpla2-/- RPE. Reactive oxygen species generation was evaluated by DHE staining. Mitochondrial abnormalities were observed in electron micrographs, showing shrunken mitochondria with reduced cristae, and decreased mitochondrial signal was observed by MitoTracker fluorescence staining. These findings suggest that Pnpla2 deficiency promotes ferroptosis in RPE, linking lipid dysregulation and retinal degeneration.