Sigma 1 Receptor Co-Localizes with NRF2 in Retinal Photoreceptor Cells
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 accompani...
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| Format: | Article |
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MDPI AG
2021-06-01
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| Series: | Antioxidants |
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| Online Access: | https://www.mdpi.com/2076-3921/10/6/981 |
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| author | Shannon R. Barwick Mevish S. Siddiq Jing Wang Haiyan Xiao Brendan Marshall Elizabeth Perry Sylvia B. Smith |
| author_facet | Shannon R. Barwick Mevish S. Siddiq Jing Wang Haiyan Xiao Brendan Marshall Elizabeth Perry Sylvia B. Smith |
| author_sort | Shannon R. Barwick |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T10:15:33Z |
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| id | doaj.art-5b4bafe863b1477c854967782a42bc36 |
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| issn | 2076-3921 |
| language | English |
| last_indexed | 2024-03-10T10:15:33Z |
| publishDate | 2021-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antioxidants |
| spelling | doaj.art-5b4bafe863b1477c854967782a42bc362023-11-22T00:51:40ZengMDPI AGAntioxidants2076-39212021-06-0110698110.3390/antiox10060981Sigma 1 Receptor Co-Localizes with NRF2 in Retinal Photoreceptor CellsShannon R. Barwick0Mevish S. Siddiq1Jing Wang2Haiyan Xiao3Brendan Marshall4Elizabeth Perry5Sylvia B. Smith6Department of Cellular Biology and Anatomy, Medical College of Georgia at Augusta University, Augusta, GA 30912, USADepartment of Cellular Biology and Anatomy, Medical College of Georgia at Augusta University, Augusta, GA 30912, USADepartment of Cellular Biology and Anatomy, Medical College of Georgia at Augusta University, Augusta, GA 30912, USADepartment of Cellular Biology and Anatomy, Medical College of Georgia at Augusta University, Augusta, GA 30912, USADepartment of Cellular Biology and Anatomy, Medical College of Georgia at Augusta University, Augusta, GA 30912, USADepartment of Cellular Biology and Anatomy, Medical College of Georgia at Augusta University, Augusta, GA 30912, USADepartment of Cellular Biology and Anatomy, Medical College of Georgia at Augusta University, Augusta, GA 30912, USASigma 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.https://www.mdpi.com/2076-3921/10/6/981sigma receptorNRF2retinal degenerationoxidative stresselectron microscopy (EM) immunodetectionretinal neuroprotection |
| spellingShingle | Shannon R. Barwick Mevish S. Siddiq Jing Wang Haiyan Xiao Brendan Marshall Elizabeth Perry Sylvia B. Smith Sigma 1 Receptor Co-Localizes with NRF2 in Retinal Photoreceptor Cells Antioxidants sigma receptor NRF2 retinal degeneration oxidative stress electron microscopy (EM) immunodetection retinal neuroprotection |
| title | Sigma 1 Receptor Co-Localizes with NRF2 in Retinal Photoreceptor Cells |
| title_full | Sigma 1 Receptor Co-Localizes with NRF2 in Retinal Photoreceptor Cells |
| title_fullStr | Sigma 1 Receptor Co-Localizes with NRF2 in Retinal Photoreceptor Cells |
| title_full_unstemmed | Sigma 1 Receptor Co-Localizes with NRF2 in Retinal Photoreceptor Cells |
| title_short | Sigma 1 Receptor Co-Localizes with NRF2 in Retinal Photoreceptor Cells |
| title_sort | sigma 1 receptor co localizes with nrf2 in retinal photoreceptor cells |
| topic | sigma receptor NRF2 retinal degeneration oxidative stress electron microscopy (EM) immunodetection retinal neuroprotection |
| url | https://www.mdpi.com/2076-3921/10/6/981 |
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