Pigment epithelium-derived factor (PEDF) prevents apoptosis and promotes apical localization of opsin in retina photoreceptors

BECERRA P; MICHELIS G; GERMAN O.L.; ROTSTEIN NP; POLITI L.E.

Seattle, Washington, EEUU

Congreso; ARVO Annual Meeting; 2016

ARVO

Pigment epithelium-derived factor (PEDF) prevents apoptosis and promotes apical localization of opsin in retina photoreceptorsPosterboard #: B0053Abstract Number: 188 - B0053Author Block: S Patricia Becerra1 , Germán Michelis2 , Lorena Germán2 , Nora P. Rotstein2 , Luis Politi2 1 NEI-NIH, Bethesda, Maryland, United States; 2 Instituto de Investigaciones Bioquímicas (INIBIBB), Universidad Nacional del Sur (UNS)-CONICET, Bahia Blanca, Argentina; 2 Instituto de Investigaciones Bioquímicas (INIBIBB), Universidad Nacional del Sur (UNS)-CONICET, Bahia Blanca, Argentina; 2 Instituto de Investigaciones Bioquímicas (INIBIBB), Universidad Nacional del Sur (UNS)-CONICET, Bahia Blanca, Argentina; 2 Instituto de Investigaciones Bioquímicas (INIBIBB), Universidad Nacional del Sur (UNS)-CONICET, Bahia Blanca, ArgentinaPurpose: PEDF plays a critical role in promoting survival of photoreceptors and in an R28 retinal progenitor cell line by binding to PEDF membrane receptors. However, the intracellular mechanisms it activates in retinal neurons remain to be established. In the present work, we investigated the effects of PEDF in developing retina neurons in culture.Methods: Pure neuronal cultures were prepared from retinas ofrats at post-natal day 1. Recombinant human PEDF was purified from media of BHK cells stably transfected with expression vectors containing the full-length PEDF cDNA. Rod photoreceptors and amacrine neurons were grown in chemically defined medium supplemented at day 3 in culture with either PEDF (10 nM); PEDF plus P1 blocking peptide (100nM); or vehicle (control) and fixed at days 5 or 6 in culture. Apoptosis, cell death, mitochondrial activity, opsin expression and localization and axonal outgrowth in vitro were then determined.Results: PEDF enhanced the survival of photoreceptors with time in culture compared to controls, as determined by Propidium Iodide and TUNEL assays. PEDF-mediated prevention of apoptosis involved the preservation of mitochondrial membrane potential. Pre-incubation of PEDF with P1, a PEDF receptor blocking peptide, completely abolished PEDF effects on survival. PEDF did not increase opsin expression but promoted apical localization of opsin, compared to its diffuse distribution in controls, resembling what occurs in mature retinas. Interestingly, PEDF also stimulated axon outgrowth in amacrine neurons, since axonal length doubled in these neurons in PEDF-supplemented cultures, compared to controls.Conclusions: In summary, this work suggests that PEDF is an effective survival factor for retinal photoreceptors during development in vitro. It also implies that PEDF plays different roles in neuronal differentiation, promoting the localization of opsin in apical processes and stimulating axonal outgrowth in amacrine neurons.