Abstract
Purpose :
The multifunctional PEDF protein promotes photoreceptor survival. Here we assessed the effects of neurotrophic PEDF fragments on neurite development, cell survival, mitochondrial potential and photoreceptor development in primary retinal neuronal cultures.
Methods :
Synthetic PEDF peptides (44-mer and 17-mer) and recombinant human PEDF were used as effectors, while synthetic PEDF receptor (PEDF-R)-derived P1 mimotope as blocking peptide, and atglistatin as a selective PEDF-R lipase inhibitor. Postnatal day 1 rats were used to obtain retinal neuron cultures. Cells incubated in a chemically defined medium for 2 days in vitro (DIV2) were treated with effectors, with or without mimotope or inhibitor and cultured up to DIV5. qPCR and Western blot were performed for PEDF-R. Subcellular distribution of PEDF-R and membrane markers was performed by immunofluorescence. Cell death was measured using propidium iodide, TUNEL and Annexin V by immunofluorescence and flow cytometry. Mitochondrial integrity and function were measured with MitoTracker and metramethylrhodamine ethyl ester, respectively. Immunofluorescence of β-III tubulin and rhodopsin was performed to evaluate neurite outgrowth and photoreceptor development, respectively.
Results :
Results: Rat retinal neurons expressed PEDF-R mRNA and produced protein throughout the culturing period (DIV1-5). PEDF-R immunostaining co-localized with that for plasma membrane markers, implying PEDF-R is localized in the cell membrane. Peptides 44-mer and 17-mer decreased cell death relative to non-treated conditions, which paralleled with mitochondrial function improvement and comparable to the effects of full-length PEDF. Both peptides promoted a polarized rhodopsin localization resembling the natural development of the retina, as did the full-length PEDF. They also stimulated neurite outgrowth, especially in amacrine neurons. In all assays performed in this study, the effects of PEDF and derived peptides were suppressed by both P1 mimotope and atglistatin.
Conclusions :
PEDF peptides acted as survival factors for retinal photoreceptors contributing to their development, differentiation and neurite outgrowth in vitro. Interactions between the P1 region of membrane PEDF-R and a neurotrophic domain within the 17-mer region (98-114) of human PEDF seemed to mediate these effects.
This is a 2020 ARVO Annual Meeting abstract.