June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Sigma 1 receptor (SIG1R) modulates Cullin3 (CUL3) in retinal cone photoreceptor cells.
Author Affiliations & Notes
  • Sylvia B Smith
    Cellular Biology & Anatomy, Augusta University, Augusta, Georgia, United States
    Culver Vision Discovery Institute, Augusta University, Augusta, Georgia, United States
  • Jing Wang
    Cellular Biology & Anatomy, Augusta University, Augusta, Georgia, United States
    Culver Vision Discovery Institute, Augusta University, Augusta, Georgia, United States
  • Shannon Barwick
    Cellular Biology & Anatomy, Augusta University, Augusta, Georgia, United States
    Culver Vision Discovery Institute, Augusta University, Augusta, Georgia, United States
  • Haiyan Xiao
    Cellular Biology & Anatomy, Augusta University, Augusta, Georgia, United States
    Culver Vision Discovery Institute, Augusta University, Augusta, Georgia, United States
  • Footnotes
    Commercial Relationships   Sylvia Smith None; Jing Wang None; Shannon Barwick None; Haiyan Xiao None
  • Footnotes
    Support  NIH/NEI Grants R01EY028103, P30EY031631
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 2582 – F0465. doi:
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    • Get Citation

      Sylvia B Smith, Jing Wang, Shannon Barwick, Haiyan Xiao; Sigma 1 receptor (SIG1R) modulates Cullin3 (CUL3) in retinal cone photoreceptor cells.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2582 – F0465.

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

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Abstract

Purpose : Retinal degenerative diseases frequently involve photoreceptor cell (PRC) death. We have observed dramatic rescue of cone PRCs in the rd10 mouse model of retinopathy when administered (+)-pentazocine ((+)-PTZ), a high affinity SIG1R ligand. The molecular mechanisms of SIG1R-mediated neuroprotection are under investigation. We previously reported that NRF2 may be central to SIG1R-mediated PRC rescue in rd10 mice. CUL3 is a component of the NRF2-KEAP1 pathway and facilitates NRF2 ubiquitination. CUL3 is critical to caspase-8 ubiquitination which regulates cellular apoptosis. Here, we asked whether SIG1R co-localizes with CUL3 and whether its activation alters CUL3 expression.

Methods : 661W cone cells were treated with (+)-PTZ. We used qRT-PCR to investigate Cul3 gene expression, immunoblotting to detect protein levels, CellROX assay to assess oxidative stress and reactive oxygen species (ROS) levels. Small interfering RNA (siRNA), targeting Sig1R or Cul3, was transfected into cells using lipofectamine RNAiMAX reagent to knockdown protein expression. Proximity ligation assay (PLA), co-immuno-EM and co-immunoprecipitation (co-IP) were used to detect the extent to which SIG1R co-localizes and interacts with CUL3.

Results : Treatment of 661W cells with (+)-PTZ [20 µM] significantly increased the expression of Cul3. With increasing concentrations of (+)-PTZ, CUL3 protein levels increased in a dose-dependent manner (~5 fold [3 µM], ~13 fold [10 µM], 18 fold [20 µM] & 18 fold [50 µM]). After silencing SIG1R using siRNA, Cul3 expression decreased dramatically. In Müller cells isolated from Sig1R-/- mice, we observed a significant decrease of CUL3, similar to that observed in 661W cells. In Cul3-siRNA-transfected 661W cells, cellular ROS levels increased significantly. Using PLA and EM immunogold labeling we observed co-localization of SIG1R and CUL3 in 661W cells. The proteins were co-localized in mouse retina tissue as well. Co-IP verified the interaction of SIG1R with CUL3.

Conclusions : Our data provide first evidence that SIG1R co-localizes/interacts with CUL3, a key player in the NRF2-KEAP1 antioxidant pathway. Furthermore, activation of SIG1R modulates Cul3 expression, which may be relevant to its retinal neuroprotective properties. Future studies will investigate comprehensively the role of CUL3 in SIG1R-mediated cone PRC rescue.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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