In the eye, a cell-surface receptor encoded by the PNPLA2 gene and termed PEDF-R mediates the prosurvival and neurotrophic functions of PEDF. PEDF-R exhibits lipase activities and is found in the RPE and retina. We generated RPE and photoreceptor-specific Pnpla2 mouse models to investigate the role of PEDF-R in those tissue. We assessed the lipid content in the RPE and photoreceptors that do not express Pnpla2 by staining paraformaldehyde-fixed eye cryosections from RPE-specific and photoreceptor-specific Pnpla2-KO mice respectively, with Oil Red O. We analyzed electron microscopy micrographs of RPE from Pnpla2-KO mice, photoreceptors from Pnpla2-KO mice and relative control mice. We evaluated retina functionality of RPE-specific Pnpla2-KO and photoreceptor-specific Pnpla2-KO mice by electroretinography (ERG) measuring the amplitude of scotopic a- and b-waves, and photopic b-wave. The light response of the RPE of Pnpla2-KO mice and controls was also evaluated by dcERG to measure the amplitude of the c-wave. Immunohistochemistry was performed with antibodies specific for Pnpla2 protein on eye cryosections of wild-type mice. We measured beta-hydroxybutyrate levels, as a derivative of fatty acid ketogenesis that can be produced after phagocytosis of outer segments by RPE/choroid explants from RPE-specific Pnpla2-KO mice and control littermates. To characterize the activity of PEDF-R and its role on the survival of retina cells in vivo, we used the state of the art technology of matrix assisted laser desorption imaging mass spectrometry (MALDI-IMS), for direct analysis of lipids from RPE and retina tissue sections of WT mice. To implement the therapeutic use of PEDF as a neurotrophic and neuroprotective factor for the retina, we constructed PEDF vectors for delivery of PEDF in vivo by nanoparticles. In preparation for in vivo testing of retinoprotective proteins and peptides, we developed degenerative models in vitro using ARPE-19 cells and cone-type photoreceptors 661W cells exposed to hydrogen peroxide and sodium iodate. Characterization of the degenerative course was performed using cytotoxicity and cell viability assays. The concentration-dependent effects of the toxic agents were assayed. The protective effects of PEDF, PN-1 R346A PN-1 proteins, and 44mer and 17mer peptides were tested against oxidative stress agents in the cells. We compared the expression of the Serpinf1 gene in the eyecup (RPE/choroid) of rd10, a mouse model for retinal degeneration, and wild-type mice by real-time PCR at different ages (postnatal day 15- 21). Similarly, from the same mice we measured PEDF protein levels in the eyecup (RPE/choroid) by ELISA and PEDF-R transcript and protein levels in the retina by real-time PCR and western blot, respectively.
| 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 |
| Samtani, Sonia; Amaral, Juan; Campos, Maria M et al. (2009) Doxycycline-mediated inhibition of choroidal neovascularization. Invest Ophthalmol Vis Sci 50:5098-106 |
