The patatin-like phospholipase domain-containing 2 (Pnpla2) gene, coding for a lipase termed PEDF-R, mediates PEDF pro-survival and neurotrophic functions in the eye. We continued to investigate the role of PEDF-R in the RPE using RPE-specific Pnpla2 conditional knockout (RPE-Pnpla2-KO) mice. Given that phagocytosis is one of the main functions of the RPE, it was further investigated in RPE of wild-type (WT) mice. The number of rhodopsin-stained phagosomes was determined in WT RPE flatmounts isolated at the time that the light is turned on (light onset) in the mouse facility and at 2 more time points (after 2 and 5 hours from light onset). Furthermore, the expression of genes that code for known markers for phagocytosis (e.g., plasminogen activator inhibitor-1, PAI-1, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha, PGC1a) was measured by real-time PCR at the same time points in RPE from WT mice. The same markers were evaluated by measuring their expression by real-time PCR, in RNA from RPE/choroid explants exposed to bovine photoreceptor outer segments (POS) for 2 hours, and compared to those which did not receive POS. To determine whether PEDF-R has a role in phagocytosis, RPE/choroid flatmounts from RPE-Pnpla2-KO and littermate controls were isolated at 30 minutes before and at 2 and 5 hours after light onset in the facility. The RPE flatmounts were stained for Rhodopsin and Cre and analyzed by confocal microscopy. Rhodopsin-stained phagosomes were then counted, and numbers from KO and littermate controls were compared. To correlate the results with Pnpla2 expression, RNA was isolated from the RPE/choroid of the contralateral eyes, and the expression of Pnpla2 was determined by real-time PCR. To explore the role of Pnpla2 in the entire eye, we characterized a newly generated mouse strain in which the gene has been constitutively knocked out using CRISPR-CAS-9 technology. After verification of the genetic deletion of a portion of Pnpla2 gene in the mouse genome by sequencing, the lack of PEDF-R protein in the KO mice was confirmed by immunoblot in adipose tissue. The absence of Pnpla2 expression in ocular tissues from KO mice was also tested by real-time PCR. Furthermore, given that PEDF-R is a lipase, the levels of free fatty acids and triglycerides were determined in the serum from Pnpla2-KO mice and their littermate controls. The ultrastructure of the retina and RPE was visualized in Pnpla2-KO mice by electron microscopy and compared to control mice. The ocular structure and morphology of KO mice were also assessed by Optical Coherence Tomography and compared to littermate controls. The functionality of their retinas was measured by electroretinography. We completed a study on the evaluation of the protective properties of PEDF peptides on rd10 mouse models of retinal degeneration ex vivo and in vivo. Human recombinant PEDF and synthetic peptides were used. Rd10 retinal explants, as well as wild-type retinal explants treated with zaprinast to mimic the rd10 photoreceptor cell death, were employed. PEDF protein was intravitreally administered into rd10 mice. PEDF administration increased the outer nuclear layer thickness of rd10 retinas in vivo and decreased the number of TUNEL+ nuclei of photoreceptors in rd10 retinal explant cultures, both relative to untreated controls. Peptides containing the PEDF neurotrophic region reduced the number of TUNEL+ photoreceptors in both rd10 and zaprinast-induced cell death ex vivo models, while peptides without the neurotrophic region and/or lacking affinity for PEDF-R were ineffective in protecting photoreceptors. PEDF protein levels in the RPE of rd10 mice, assessed by western blot, decreased with age (P15-P25). To promote degeneration of the outer retina, oxidative stress was experimentally induced by intraperitoneal sodium iodate (NaIO3) administration. The course of retinal degeneration was initially followed in C57BL/6J by imaging the mouse fundi at 1-8 days post-injection. At given time points, mice were euthanized, eyes were enucleated and processed for Hematoxylin and Eosin staining for histopathological evaluations of retinal degeneration. The effects of intravitreal injections of PEDF protein in the retina of C57BL/6J mice treated with NaIO3 were evaluated. The SERPINF1 gene encodes the neurotrophic and antiangiogenic PEDF. Mutations in SERPINF1 cause osteogenesis imperfecta (OI) type VI in humans and patients with OI type VI have very low levels of circulating PEDF, a marker of this disease. The Serpinf1 null mouse is a model of OI. PEDF protein levels and distribution were compared in Serpinf1 null and wildtype controls by immunoreactions with antibodies to PEDF in plasma and retina sections, by western blotting and immunofluorescence. Comparison of the retinal damage progression by sodium iodate-induced oxidative stress was performed in Serpinf1 null mice and littermate controls. The NaIO3-mediated glial cell stress was also tested by performing immunofluorescence staining of glial cell marker proteins such as Glial fibrillary acidic protein (GFAP) and S100 beta in Serpinf1 null mice and littermate controls. We used an inhibitor of the lipase activity of PEDF-R to assess the course of retinal damage induced by oxidative stress in Serpinf1 null and control mice. The autosomal recessive retinitis pigmentosa (RP) mouse model, i.e., rd10 and rd10/Serpinf1 null double mutant mouse model were tested for the time-dependent progression of retinal degeneration in the absence of PEDF protein.
| Polato, Federica; Becerra, S Patricia (2016) Pigment Epithelium-Derived Factor, a Protective Factor for Photoreceptors in Vivo. Adv Exp Med Biol 854:699-706 |
| Kenealey, Jason; Subramanian, Preeti; Comitato, Antonella et al. (2015) Small Retinoprotective Peptides Reveal a Receptor Binding Region on Pigment Epithelium-derived Factor. J Biol Chem : |
| Amaral, Juan; Becerra, S Patricia (2010) Effects of human recombinant PEDF protein and PEDF-derived peptide 34-mer on choroidal neovascularization. Invest Ophthalmol Vis Sci 51:1318-26 |
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