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Sigma 1 Receptor Co-Localizes with NRF2 in Retinal Photoreceptor Cells.

ABSTRACT: Sigma 1 receptor (Sig1R), a modulator of cell survival, has emerged as a novel target for retinal degenerative disease. Studies have shown that activation of Sig1R, using the high affinity ligand (+)-pentazocine ((+)-PTZ), improves cone function in a severe retinopathy model. The rescue is accompanied by normalization of levels of NRF2, a key transcription factor that regulates the antioxidant response. The interaction of Sig1R with a number of proteins has been investigated; whether it interacts with NRF2, however, is not known. We used co-immunoprecipitation (co-IP), proximity ligation assay (PLA), and electron microscopy (EM) immunodetection methods to investigate this question in the 661W cone photoreceptor cell line. For co-IP experiments, immune complexes were precipitated by protein A/G agarose beads and immunodetected using anti-NRF2 antibody. For PLA, cells were incubated with anti-Sig1R polyclonal and anti-NRF2 monoclonal antibodies, then subsequently with (-)-mouse and (+)-rabbit PLA probes. For EM analysis, immuno-EM gold labeling was performed using nanogold-enhanced labeling with anti-NRF2 and anti-Sig1R antibodies, and data were confirmed using colloidal gold labeling. The co-IP experiment suggested that NRF2 was bound in a complex with Sig1R. The PLA assays detected abundant orange fluorescence in cones, indicating that Sig1R and NRF2 were within 40 nm of each other. EM immunodetection confirmed co-localization of Sig1R with NRF2 in cells and in mouse retinal tissue. This study is the first to report co-localization of Sig1R-NRF2 and supports earlier studies implicating modulation of NRF2 as a mechanism by which Sig1R mediates retinal neuroprotection.

SUBMITTER: Barwick SR

PROVIDER: S-EPMC8234060 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Oxidative stress figures prominently in retinal diseases, including diabetic retinopathy, and glaucoma. Ligands for ?1R, a unique transmembrane protein localized to the endoplasmic reticulum, mitochondria, and nuclear and plasma membranes, have profound retinal neuroprotective properties in vitro and in vivo. Studies to determine the mechanism of ?1R-mediated retinal neuroprotection have focused mainly on neurons. Little is known about the effects of ?1R on Müller cell function, yet these radial glial cells are essential for homeostatic support of the retina. Here we investigated whether ?1R mediates the oxidative stress response of Müller cells using wild-type (WT) and ?1R-knockout (?1RKO) mice. We observed increased endogenous reactive oxygen species (ROS) levels in ?1RKO Müller cells compared to WT, which was accompanied by decreased expression of Sod1, catalase, Nqo1, Hmox1, Gstm6, and Gpx1. The protein levels of SOD1, CAT, NQO1, and GPX1 were also significantly decreased. The genes encoding these antioxidants contain an antioxidant response element (ARE), which under stress is activated by NRF2, a transcription factor that typically resides in the cytoplasm bound by KEAP1. In the ?1RKO Müller cells Nrf2 expression was decreased significantly at the gene (and protein) level, whereas Keap1 gene (and protein) levels were markedly increased. NRF2-ARE binding affinity was decreased markedly in ?1RKO Müller cells. We investigated system xc(-), the cystine-glutamate exchanger important for synthesis of glutathione (GSH), and observed decreased function in ?1RKO Müller cells compared to WT as well as decreased GSH and GSH/GSSG ratios. This was accompanied by decreased gene and protein levels of xCT, the unique component of system xc(-). We conclude that Müller glial cells lacking ?1R manifest elevated ROS, perturbation of antioxidant balance, suppression of NRF2 signaling, and impaired function of system xc(-). The data suggest that the oxidative stress-mediating function of retinal Müller glial cells may be compromised in the absence of ?1R. The neuroprotective role of ?1R may be linked directly to the oxidative stress-mediating properties of supportive glial cells.

Project description:Sigma receptors (sigmaRs) are nonopioid, nonphencyclidine binding sites with robust neuroprotective properties. Previously, the authors induced death in the RGC-5 cell line using very high concentrations (1 mM) of the excitatory amino acids glutamate (Glu) and homocysteine (Hcy) and demonstrated that the sigmaR1 ligand (+)-pentazocine ((+)-PTZ) could protect against cell death. The purpose of the present study was to establish a physiologically relevant paradigm for testing the neuroprotective effect of (+)-PTZ in retinal ganglion cells (RGCs).Primary ganglion cells (GCs) were isolated by immunopanning from retinas of 1-day-old mice, maintained in culture for 3 days, and exposed to 10, 20, 25, or 50 microM Glu or 10, 25, 50, or 100 microM Hcy for 6 or 18 hours in the presence or absence of (+)-PTZ (0.5, 1, 3 microM). Cell viability was measured using the viability and apoptosis detection fluorescein in situ assays. Expression of sigmaR1 was assessed by immunocytochemistry, RT-PCR, and Western blotting. Morphologic appearance of live ganglion cells and their processes was examined over time (0, 3, 6, 18 hours) by differential interference contrast (DIC) microscopy after exposure to excitotoxins in the presence or absence of (+)-PTZ.Primary GCs showed robust sigmaR1 expression. The cells were exquisitely sensitive to Glu or Hcy toxicity (6-hour treatment with 25 or 50 microM Glu or 50 or 100 microM Hcy induced marked cell death). Primary GCs pretreated for 1 hour with (+)-PTZ followed by 18-hour cotreatment with 25 microM Glu and (+)-PTZ showed a marked decrease in cell death: 25 microM Glu alone, 50%; 25 microM Glu/0.5 microM (+)-PTZ, 38%; 25 microM Glu/1 microM (+)-PTZ, 20%; 25 microM Glu/3 microM (+)-PTZ, 18%. Similar results were obtained with Hcy. sigmaR1 mRNA and protein levels did not change in the presence of the excitotoxins. DIC examination of cells exposed to excitotoxins revealed substantial disruption of neuronal processes; cotreatment with (+)-PTZ revealed marked preservation of these processes. The stereoselective effect of (+)-PTZ for sigmaR1 was established in experiments in which (-)-PTZ, the levo-isomer form of pentazocine, had no neuroprotective effect on excitotoxin-induced ganglion cell death.Primary GCs express sigmaR1; their marked sensitivity to Glu and Hcy toxicity mimics the sensitivity observed in vivo, making them a highly relevant model for testing neuroprotection. Pretreatment of cells with 1 to 3 microM (+)-PTZ, but not (-)-PTZ, affords significant protection against Glu- and Hcy-induced cell death. sigmaR1 ligands may be useful therapeutic agents in retinal diseases in which ganglion cells die.

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Sigma 1 Receptor Co-Localizes with NRF2 in Retinal Photoreceptor Cells.

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