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Shannon Renee Barwick, Mevish Siddiq, Brendan Marshall, Elizabeth Perry, Jing Wang, Sylvia B Smith; Investigation of the interaction of Sigma1 Receptor (S1R) and NRF2 in cone photoreceptor cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4867.
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© ARVO (1962-2015); The Authors (2016-present)
S1R, a modulator of cell survival, has emerged as a novel target in retinal degenerations. Recent studies from our lab showed that activation of S1R (using the high affinity ligand (+)-pentazocine ((+)-PTZ)) improves cone function in a retinopathy model. The improvement was accompanied by normalization of levels of NRF2, a transcription factor that regulates the antioxidant response. While the interaction of S1R with several proteins (e.g. IP3R3, ankyrin, BiP) has been investigated, its interaction with NRF2 is not known. Here, we investigated S1R-NRF2 co-localization as a first step in addressing whether these proteins interact.
661W cells (cone PRC line) were cultured 24h in DMEM in the presence/absence of (+)-PTZ [20 or 50µM]. They were incubated with anti-S1R polyclonal & anti-NRF2 monoclonal antibodies and subsequently with (-)-mouse & (+)-rabbit proximity ligation assay (PLA) probes using Duolink® PLA In Situ Orange Starter Kit (Mouse/Rabbit). Proteins located ≤40nm of each other fluoresce orange; intensity was quantified using NIH-ImageJ. The subcellular location of S1R & NRF2 was determined by electron microscopy (EM) in cells cultured 24h in DMEM in the presence/absence of 20µM (+)-PTZ. Immuno-EM-gold labeling was performed using Colloidal Gold AffiniPure Goat 6nm anti-mouse/18nM anti-rabbit antibodies. S1R & NRF2 were considered co-localized when distances between 6nm & 18nm particles were ≤ 50nm. The incidence of co-localized proteins was quantified in the cytoplasm & nucleus. Data for PLA & EM-immunodetection were analyzed by one-way ANOVA.
PLA assays performed 3X in duplicate detected orange fluorescence in cones indicating that S1R & NRF2 were within 40nm of each other. Fluorescence increased significantly in cells treated with 50uM PTZ (p<0.0001). EM data confirmed that S1R is within 50nm of NRF2 in the nucleus & cytoplasm. Upon (+)-PTZ treatment, co-localization of S1R-NRF2 was limited to the cytoplasm & was not observed in the nucleus (p<0.05).
This study represents the first investigation of S1R-NRF2 interaction. Using two immunodetection methods we observed that S1R & NRF2 co-localize in cones, in both the nucleus & cytoplasm. The co-localization may be modulated by S1R activation. Future studies will analyze whether S1R works directly through NRF2 to mediate robust retinal neuroprotection.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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