Abstract
Purpose :
Ring finger protein 146 (Iduna) facilitates DNA repair and protects against cell death induced by NMDA receptor-mediated glutamate excitotoxicity or by cerebral ischemia. NPD1 promotes cell survival under UOS. The purpose of this study is to define the underlying molecular mechanism of NPD1-mediated Iduna neuroprotection against UOS-induced cell death.
Methods :
ARPE-19 cells were grown and maintained in a T-75mM flask. Transient transfection of ARPE-19 cells by Iduna-siRNAs constructs were done by transfecting with 5 µg of constructs using Fugene-6 (Roach, NJ, USA). In addition to the Iduna-siRNA constructs, we also used shRNA-Iduna or YRAA-Iduna, an Iduna mutant lacking PAR binding sequences. A GFP-Iduna construct was used as transfection control. Transfected cells were incubated 24h at 37°C, then serum starved for 8h at 37°C, exposed to UOS (600 µM H2O2) and treated with NPD1 (50-100 nM) for 6h followed by Western blot analysis and 16h for apoptotic cell death assessment. Iduna protein abundance was detected by Western blot analysis using either anti-Iduna antibody (RNF146) or clone N201/35 containing anti-Iduna antibody (UC Davis/NIH Neiro Mab Facility, CA).
Results :
Our data demonstrates that NPD1 potently upregulates Iduna expression and provides remarkable protection against UOS. NPD1 induction of Iduna expression requires interaction binding at the poly (ADP-ribose) (PAR) sites. Use of Caspase and PARP inhibitors indicates that NPD1 induced Iduna-mediated cytoprotection involved in apoptotic and parthanatos signaling cascades. Iduna-specific and non-specific siRNAs established as controls for Iduna off-target effects. Western blot analysis of the Iduna protein in whole cell, cytoplasmic and nuclear preparations indicated that NPD1 triggered Iduna translocation into the nucleus.
Conclusions :
NPD1 enhances Iduna expression under disruptive neuro-homeostasis. These findings provide a conceptual advancement for survival of neural cells undergoing challenges to homeostasis because a lipid mediator (NPD1), made “on demand,” modulates abundance of a critically important protein (Iduna) for cell survival. Further unraveling of the molecular details of DHA-NPD1-Iduna expression signaling may contribute to therapeutic interventions for retinal degenerations.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.