In the eye, PEDF exerts its prosurvival function by binding to a cell surface receptor, named PEDF-R. PEDF-R is encoded by the Pnpla2 gene, which is expressed in the retina and RPE cells. To elucidate the contribution of PEDF-R to the PEDF biological activity in vivo, we continued to generate Pnpla2 conditional knockout mice and used homozygous Pnpla2/LoxP (Pnpla2f/f) mutant mice with LoxP sites flanking a 1.5 kb region (floxed region) of the Pnpla2 gene. The mice were crossed with Cre transgenic mouse lines that express the Cre recombinase in the photoreceptor (Crx-cre, generated by Dr. T. Glaser and received from Dr. A. Swaroop) or RPE (Best1-cre, shared by Dr. J. Dunaief) compartments of the retina. The Cre-mediated genomic deletion of the floxed region was evaluated by PCR using as a template the genomic DNA isolated from whole retinas and RPE. The sequence of the LoxP sites was determined. The expression of the Pnpla2 transcripts was evaluated in the whole retina and in the outer nuclear layer (ONL) by semi-quantitative Real-Time PCR. PEDF-R protein levels were determined by immunoblotting of retina extracts and by immunohistochemistry on eye sections using antibodies specific to PEDF-R. Electroretinography (ERG) was conducted on a larger cohort of conditional Pnpla2-null mice and control (Pnpla2f/f, Pnpla2+/+, Crx-cre+) littermates to assess the retina functionality. The morphology of the retinas was evaluated by H&E staining of retina sections. Desnutrin/ATGL are alternative names for the Pnpla2 genes. The homozygous Desnutrin/ATGLflox mice (Desnutrin/ATGLf/f, generated and given by Dr. H.S. Sul) were inter-crossed to expand the colony. The rd10 mouse strain, an established model for retinitis pigmentosa, carries a missense point mutation in the beta-subunit of the rod cGMP phosphodiesterase gene (Pde6b) resulting in genetically induced photoreceptor degeneration. The recombinant human PEDF protein was injected into the vitreous of the left eye (OS) of rd10 mice. The right eye (OD) was injected with the vehicle, becoming the control eye. We prepared cryosections from retinas of both OS and OD of rd10 mice and stained their nuclei with DAPI and photographed with Zeiss Imager. We measured the height of ONL of photoreceptors and compared the ONL height of the OS versus the control eye per each mouse as a way to assess the prosurvival effect of PEDF on photoreceptors of the rd10 mice. To generate rd10 mice in a PEDF-free background, rd10 mice were crossed with Serpinf1 knockout mice (Serpinf1-/-, generated and shared by Dr. S. Crawford), which does not express PEDF (coded by the Serpinf1 gene). We designed the generation of photoreceptor-specific Pnpla2-null mice with photoreceptors that are sensitive to damage induced by light, as a model of environment-induced retinal degeneration. Given that Pnpla2 null mice are on a C57BL/6J background, which carries methionine at codon 450 of Rpe65 gene conferring resistance to light damage, photoreceptor-specific Pnpla2 null mice were crossed with 129S strain, which has a leucine at codon 450 of Rpe65 and is sensitive to light. The cross generated photoreceptor-specific Pnpla2 null mice carrying the light sensitive variant of Rpe65 gene and therefore susceptible to light-induced retina degeneration. To develop long-term delivery systems, we constructed Adeno-associated virus (AAV) vectors carrying the cDNAs coding for human full length PEDF and 2 variants each with 1 single-amino acid substitution at R99 and H105 to alanine and cloned into a vector (provided by the Ocular Gene Therapy Core at NEI), which contains 2 Inverted Terminal Repeat (ITR) sequences from AAV.
| 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 |
