June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Neuroprotectin D1 (NPD1) upregulates Iduna expression and provides protection against uncompensated oxidative stress (UOS) in Human Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • Pranab K Mukherjee
    Neuroscience Cntr/Ophthalmology, LSU Health Sciences Center, New Orleans, Louisiana, United States
  • Veronica Bender
    Neuroscience Cntr/Ophthalmology, LSU Health Sciences Center, New Orleans, Louisiana, United States
  • Jorgelina Muriel Calandria
    Neuroscience Cntr/Ophthalmology, LSU Health Sciences Center, New Orleans, Louisiana, United States
  • Nicolas Guillermo Bazan
    Neuroscience Cntr/Ophthalmology, LSU Health Sciences Center, New Orleans, Louisiana, United States
  • Footnotes
    Commercial Relationships   Pranab Mukherjee, None; Veronica Bender, None; Jorgelina Calandria, None; Nicolas Bazan, None
  • Footnotes
    Support  Supported by NEI grant EY005121, NIGMS grant GM103340, the Eye, Ear, Nose and Throat Foundation (NGB) and Research to Prevent Blindness.
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5352. doi:
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      Pranab K Mukherjee, Veronica Bender, Jorgelina Muriel Calandria, Nicolas Guillermo Bazan; Neuroprotectin D1 (NPD1) upregulates Iduna expression and provides protection against uncompensated oxidative stress (UOS) in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5352.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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