Therapeutics for the chorioretina

National Eye Institute

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Abstract

To examine the contribution of PEDF-R to the visual process, we continued to characterize a mouse strain 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 and Pnpla2 expression was followed by RT-PCR in dissected mouse retinas. 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 than of littermate Pnpla2+/+ controls. Fundoscopy was performed and revealed that the Pnpla2-/- and Pnpla2+/- mice had white spots in the ocular fundus of one line and an excess of pigment in the optic nerve area 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+/+. Angiography was also performed. Enucleated whole eyes were processed for histology, immunofluorescence, confocal microscopy, and transmission electron microscopy. The retinal pigment epithelial cells of Pnpla2-/- mice accumulated lipid droplets in their cytoplasm, and their retinas had abnormalities in several layers with deformities in the photoreceptors. Retinas and RPE/choroid of Pnpla2+/+, Pnpla2+/- and Pnpla2-/- mice were dissected, and proteins were extracted and prepared for mass spectrometry for proteomics and protein profiles by were obtained (in collaboration with L Jenkins). Similarly, retinas and RPE/choroid from enucleated eyes of Pnpla2+/+, Pnpla2+/- and Pnpla2-/- mice were dissected in preparation of lipid profiling by lipidomics (in collaboration with MP Agbaga). To investigate the potential participation of a regulator of lipid metabolism-related genes associated with retinal degeneration protection, we determined the expression levels of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1-alpha) in the retina. RT-PCR of RNA from Pnpla2+/+, Pnpla2+/- and Pnpla2-/- retinas of showed lower pgc-1-alpha gene expression levels associated with decreasing Pnpla2, both before and after light damage of photoreceptors. The observed defect in photoreceptor function of retinas missing the Pnpla2 gene led us to cross-bred of Serpinf1-/- mice with selected Pnpla2-KO mice for investigations of retina function in mice without PEDF and PEDF-R. To develop a method to evaluate photoreceptor cell death in mice, we performed non-invasive in vivo fluorescence imaging of the apoptotic retinal photoreceptors of rd10 mouse models of retinal degeneration. Phosphatidylserine (PS) externalization, an early molecular signature for apoptosis, 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 fluorescent fundoscopy. Administration of eye drops of PSVue were performed in mice at ages varying between 18-24 days old to determine a time point for the maximum PS externalization in the fundus of the retina in this model. To screen cytoprotective PEDF peptides retinal explant cultures were prepared from adult wild type C57BL/6J mice. Zaprinast was included in the medium of wild type retinal explants to obtain a mimicry of a retinal degeneration model, namely rd10, to induce photoreceptor cell death ex vivo. Photoreceptor cell death was assessed in whole retinal mounts after DAPI staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and visualized by fluorescence microscopy. Evaluation of the effects of synthetic peptides 44-mer, 17-mer and 17-merH105A were performed in a dose-response fashion for cytoprotection of photoreceptors in the optimized model ex vivo. We also used the zaprinast-retinal explant model for screening a set of PEDF protein versions with modifications that affect glycosaminoglycan affinity (collaboration with H Chu). Photoreceptor cell death was determined by TUNEL and confocal super resolution microscopy. The modified PEDF versions retained photoreceptor cytoprotective activity ex vivo.