In the eye, a cell-surface receptor encoded by the PNPLA2 gene and termed PEDF-R mediates the prosurvival and neutrophic functions of PEDF. We assessed the expression of the Pnpla2 gene in the retina and eyecup of wild-type mice at different ages after birth (from post-natal day 2 to 3.5 months) by measuring its transcript levels by real-time PCR. PEDF-R protein was detected by immunofluorescence in flat mounts of retinal pigment epithelium (RPE)/choroid and eye cryosections of adult wild-type mice to reveal its spatial distribution and layer localization. To decipher the function of PEDF-R in vivo, mouse lines with a cyclization recombinase (Cre)-mediated knockdown of Pnpla2 in photoreceptors (Pnpla2(flox/flox);Crx-Cre), and in the RPE (Pnpla2(flox/flox);Best1-Cre) were generated. Specifically, homozygous Pnpla2/LoxP (Pnpla2(flox/flox)) mutant mice were crossed with transgenic mouse lines that express the Cre recombinase in the photoreceptor (Crx-Cre) to generate photoreceptor-specific Pnpla2 knockout (KO) mice. To generate RPE-specific Pnpla2-KO mice, Best1-Cre were crossed with homozygous Pnpla2/Lox. The Pnpla2-KO mice were characterized at the genomic level. The Cre-mediated genomic deletion of the floxed region (exon 1) was verified by PCR using as a template the genomic DNA isolated from whole retinas of photoreceptor-specific Pnpla2-KO mice and RPE/choroid eyecup of RPE-specific Pnpla2-KO mice. We evaluated the level of Pnpla2 transcripts in RPE/choroid samples of RPE-Pnpla2 specific KO mice and in retinas from photoreceptor-specific Pnpla2-KO mice by real-time PCR. PEDF-R protein was assessed by immunoblotting of retina extracts from photoreceptor-specific Pnpla2-KO mice and by immunofluorescence on eye sections with antibodies specific to PEDF-R. The functionality and the structure of the retina were evaluated in the mice by Electroretinography (ERG) and Optical Coherence Tomography (OCT), respectively. The morphology of the retinas was evaluated by hematoxylin and eosin staining of retina sections. To implement the therapeutic use of PEDF as a neurotrophic and neuroprotective factor for the retina we evaluated the efficacy and safety of PEDF delivery by replication deficient adenovirus-associated vectors (AAV) and possible effect in vivo. The AAV-derived constructs carrying the cDNAs for human full-length PEDF and 2 variants each with a single-amino acid substitution at arginine position 99 (R99) or histidine position 105 (H105) to alanine were transfected into the COS-7 cell line. Recombinant PEDF production was followed by immunoblotting of the culture media. The wild-type human PEDF (PEDFWT) cDNA and those encoding its variants PEDFH105A and PEDFR99A were then packaged into replication deficient AAV (provided by the Ocular Gene Therapy Core at NEI). To evaluate the effects of AAV-PEDF vectors in a retinal degeneration mouse model, AAV-PEDFWT was administered by subretinal injection in the right eye of rd10, rd10/Serpinf1-/- double mutant, and wild-type mice. Rd10/Serpinf1-/- double mutant mice develop retinal degeneration and do not have PEDF (Serpinf1 is the gene coding for PEDF). The contralateral eye was instead injected with empty AAV-vectors or left untreated. Evaluation of the retinas was performed by Electroretinography for retina function and by Optical Coherence Tomography to image the structure of the retina at 3, 6 and 9 weeks post-injection (in collaboration with the Visual Function Core, NEI). At 7-10 weeks post-injection, the eyes were collected, the RNA and proteins were isolated, and the levels of human SERPINF1 transcripts and PEDF protein were determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. We also assessed the expression of the Serpinf1 gene in the retina and eyecup (RPE/choroid) of a mouse model for albinism, the OCA1B mice (in collaboration with the laboratory of Dr. Brian Brooks, NEI) by real-time PCR. These mice carry an alteration of the tyrosinase gene identical to that causing Oculocutaneous Albinism type 1 (OCA1) in humans. OCA1 individuals can experience vision loss. PEDF protein levels in the eyecup (RPE/choroid) of the same mice were measured by western blot and ELISA. In the same mice, PEDF-R protein levels in retina and eyecup protein extracts were assayed by immunoblot.
| 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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