Therapeutics for the chorioretina

National Eye Institute

Investigators

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Abstract

To develop a method to evaluate photoreceptor cell death in mice, we continued to optimize non-invasive in vivo fluorescence imaging of dying photoreceptor cells using the rd10 mouse, which is an animal model of retinal degeneration. Phosphatidylserine (PS) externalization, an early molecular signature for cell death by apoptosis and necrosis, was followed transiently with the phosphatidylserine-binding conjugate of Bis(zinc(II)-dipicolylamine (Zn-DPA) with Texas-red (PSVue-550) in the fundus of mice by fluorescence fundoscopy. To determine a time point for the maximum PS externalization in the fundus, the fluorescent probe was administered daily as eyedrops to rd10 and rd10 x Serpinf1-/- mice of 15-25 days of age followed by evaluation of fluorescence in their fundi. To determine the effects of neurotrophic factors on photoreceptor survival, eyedrops of PEDF peptide 17-merH105A were administered daily to rd10 and rd10 x Serpinf1-/- mice at 15-24 days of age. At end point, the probe was administered as eyedrops to evaluate fluorescence in their fundi. Electroretinography (ERG) was performed to evaluate visual function of the treated animals. Enucleated whole eyes were processed for histology. Detection of anti-apoptotic B-cell lymphoma 2 (Bcl2) and pro-apoptotic BCL2 associated X (BAX) markers was performed by immunofluorescence and confocal microscopy. We continued optimizing retinal explant cultures prepared from adult wild type C57BL/6J mice to screen retino-protective PEDF peptides. Zaprinast, an inhibitor of phosphodiesterases, was included in the medium of the explant cultures to obtain a mimicry of retinal cell death ex vivo as in the retinal degeneration rd10 animal model. Photoreceptor cell death was assessed in whole retinal mounts by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and PSVue 550 staining followed by fluorescence and confocal microscopy visualization. We used the zaprinast-retinal explant model for screening a set of recombinant PEDF protein versions with modifications of selected neutral or anionic residues into cationic residues in positions that affect glycosaminoglycan affinity (collaboration with H Chu). The modified PEDF versions were assayed for retinal survival activity ex vivo and had comparable cytoprotective properties as the unmodified PEDF in the assay. To investigate the role of PEDF-R in photoreceptors, we continued to characterize mouse strains in which the gene was constitutively knocked out using CRISPR-CAS-9 technology (in collaboration with L Dong). CRISPR Pnpla2-/- knock-out mouse lines were generated. Pnpla2 expression was followed by RT-PCR in dissected mouse retinas. PEDF-R protein was detected by immunofluorescence of retina tissue sections. Phospholipase A2 activity was determined in extracts from dissected mouse retina. Three- and seven-month-old mice and their littermate controls of two lines were evaluated. Organs of the thorax were dissected and imaged using a LEICA microscope for anatomical evaluation revealing that mice from the two Pnpla2-/- mouse lines had hearts that were enlarged and whiter color than its control littermates. Plasma was collected for the determination of free fatty acids, triglycerides, cholesterol levels. Plasma free fatty acids and triglycerides of Pnpla2-/- mice were lower and cholesterol was higher than of littermate Pnpla2+/+ controls. Fundoscopy was performed on the Pnpla2-/- and Pnpla2+/- mice of both lines. Electroretinography (ERG) showed attenuation in the a-wave in the Pnpla2-/- and Pnpla2+/- mice for both lines relative to littermate controls Pnpla2+/+. Optical coherence tomography angiography (OCT-A) was also performed. Enucleated whole eyes were processed for histology, immunofluorescence, confocal microscopy, and transmission electron microscopy. The retinas had abnormalities with deformities in the photoreceptors. Protein extracts from dissected retinas and RPE/choroid of Pnpla2+/+, Pnpla2+/- and Pnpla2-/- mice prepared for mass spectrometry for proteomics and protein profiles were analyzed in collaboration with L Jenkins. Retinal flat mounts were prepared and stained for lipids using with BODIPY. Retinas and RPE/choroid from enucleated eyes of Pnpla2+/+, Pnpla2+/- and Pnpla2-/- mice were dissected, and extracts were prepared for analyses of phospholipid and fatty acid composition by mass spectrometry (in collaboration with MP Agbaga). We administered eyedrops of the fluorescent probe PSVue to identify cell death in the retina of the Pnpla2-KO mice in vivo. The fluorescent stain was evaluated by fluorescence fundoscopy. Dissected retinas from the mice were sectioned and cell death was evaluated by TUNEL using fluorescence microscopy. We cross-bred of Serpinf1-/- mice with selected Pnpla2-KO mice to generate double Serpinf1-/- x Pnpla2-KO mice. The organs of the thorax of the double KO mice were dissected and imaged using a LEICA microscope for anatomical evaluation generated. The retinas were evaluated by fundoscopy, ERG, and OCT-A. Enucleated whole eyes of the double KO mice were processed for and evaluated by histology, and transmission electron microscopy.