Evaluation of the role of Sigma 1 receptor and Cullin3 in retinal photoreceptor cells

Sigma 1 receptor (Sig1R), a pluripotent modulator of cell survival, is neuroprotective in models of retinal degeneration when activated by the high-affinity, high-specificity ligand (+)-pentazocine ((+)-PTZ). The molecular mechanisms of Sig1R-mediated retinal neuroprotection are under investigation. We previously reported that the antioxidant regulatory transcription factor Nrf2 may be involved in Sig1R-mediated retinal photoreceptor cell (PRC) rescue. Cullin 3 (Cul3) is a component of the Nrf2-Keap1 antioxidant pathway and facilitates Nrf2 ubiquitination. Our earlier transcriptome analysis revealed decreased Cul3 in retinas lacking Sig1R. Here, we asked whether Sig1R activation can modulate Cul3 expression in 661 W cone PRCs. Proximity ligation and coimmunoprecipitation (co-IP) showed that Cul3 resides closely to and co-IPs with Sig1R. Activation of Sig1R using (+)-PTZ significantly increased Cul3 at the gene/protein level; silencing Sig1R decreased Cul3 gene/protein levels. Experiments in which Cul3 was silenced in cells exposed to tBHP resulted in increased oxidative stress, which was not attenuated with Sig1R activation by (+)-PTZ, whereas cells transfected with scrambled siRNA (and incubated with tBHP) responded to (+)-PTZ treatment by decreasing levels of oxidative stress. Assessment of mitochondrial respiration and glycolysis revealed significantly improved maximal respiration, spare capacity and glycolytic capacity in oxidatively-stressed cells transfected with scrambled siRNA and treated with (+)-PTZ, but not in (+)-PTZ treated, oxidatively-stressed cells in which Cul3 had been silenced. The data provide the first evidence that Sig1R co-localizes/interacts with Cul3, a key player in the Nrf2-Keap1 antioxidant pathway. The data suggest that the preservation of mitochondrial respiration/glycolytic function and reduction of oxidative stress observed upon activation of Sig1R occur in part in a Cul3-dependent manner.

Automated summary

Sigma 1 receptor (Sig1R) activation is neuroprotective in retinal degeneration models, and the molecular mechanisms involve the antioxidant pathway mediated by the transcription factor Nrf2. This study demonstrates that Sig1R interacts with Cul3, a component of the Nrf2-Keap1 antioxidant pathway, and activation of Sig1R increases Cul3 levels, leading to preserved mitochondrial function and reduced oxidative stress.