BENZOMORPHAN AND NON-BENZOMORPHAN AGONISTS DIFFERENTIALLY ALTER SIGMA-1 RECEPTOR QUATERNARY STRUCTURE, AS DOES TYPES OF CELLULAR STRESS

Abstract Background Sigma-1 receptor (S1R) is a calcium-sensitive, ligand-operated receptor chaperone protein present on the endoplasmic reticulum (ER) membrane and more specifically at the mitochondria- associated endoplasmic reticulum membrane (MAM)[1, 2]. At the MAM, S1R plays an important role in ER- mitochondrial inter-organelle calcium signaling and cell survival[3, 4]. Moreover, S1R and its agonists confer resilience against various neurodegenerative diseases[5-8]. At resting state, S1R is either in a monomeric or oligomeric state but the ratio of these concentrations seems to change upon activation of S1R[9-12]. S1R is activated by either cellular stress, such as ER-calcium depletion, or ligand binding[1]. While the effect of ligand binding on S1R quaternary structure remains unclear, the effect of cellular stress has not been studied. Aims & Objectives The objective of this study is to shed the light on the effects of cellular stress and ligand binding on the quaternary structure of S1R. Methods In this study we utilize cellular and an in-vivo models to study changes in quaternary structure of S1R upon activation. We incubated cells with cellular stressors (H2O2 and thapsigargin) or exogenous ligands, then quantified monomeric and oligomeric forms of S1R. Results We observed that benzomorphan-based S1R agonists induce monomerization of S1R and decrease oligomerization, which was confirmed with in-vivo experiments by measuring the ratio monomers/oligomers in the liver of mice injected with (+)-Pentazocine. Meanwhile with non- benzomorphan S1R agonists we did not observe effect on oligomerization. We also noted that antagonists block the effect of benzomorphan-based ligands but do not induce any changes when used alone. Oxidative stress (H2O2) increases the monomeric/oligomeric S1R ratio whereas ER calcium depletion (thapsigargin) has no effect. We also analyzed the oligomerization ability of various truncated S1R fragments and identified the fragments that promote oligomerization. Discussion & Conclusion In this publication we demonstrate that quaternary structural changes differ according to the mechanism of S1R activation. Therefore, we offer a novel perspective on S1R activation as a nuanced phenomenon dependent on the type of stimulus. References 1.Hayashi, T. and T.P. Su, Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2+) signaling and cell survival. Cell, 2007. 131(3): p. 596-610 DOI: 10.1016/j.cell.2007.08.036. 2.Mori, T., et al., Sigma-1 receptor chaperone at the ER-mitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival. PLoS One, 2013. 8(10): p. e76941 DOI: 10.1371/journal.pone.0076941. 3.Goguadze, N., et al., Sigma-1 Receptor Agonists Induce Oxidative Stress in Mitochondria and Enhance Complex I Activity in Physiological Condition but Protect Against Pathological Oxidative Stress. Neurotox Res, 2019. 35(1): p. 1-18 DOI: 10.1007/s12640-017-9838-2. 4.Tagashira, H., et al., Sigma-1 receptor is involved in modification of ER-mitochondria proximity and Ca(2+) homeostasis in cardiomyocytes. J Pharmacol Sci, 2023. 151(2): p. 128-133 DOI: 10.1016/j.jphs.2022.12.005. 5.Ionescu, A., et al., Targeting the Sigma-1 Receptor via Pridopidine Ameliorates Central Features of ALS Pathology in a SOD1(G93A) Model. Cell Death Dis, 2019. 10(3): p. 210 DOI: 10.1038/s41419-019-1451- 2. 6.Ryskamp, D.A., et al., Neuronal Sigma-1 Receptors: Signaling Functions and Protective Roles in Neurodegenerative Diseases. Front Neurosci, 2019. 13: p. 862 DOI: 10.3389/fnins.2019.00862. 7.Couly, S., et al., Exposure of R6/2 mice in an enriched environment augments P42 therapy efficacy on Huntington's disease progression. Neuropharmacology, 2021. 186: p. 108467 DOI: 10.1016/j.neuropharm.2021.108467. 8.Maurice, T., et al., Neuroprotection in non-transgenic and transgenic mouse models of Alzheimer's disease by positive modulation of σ(1) receptors. Pharmacol Res, 2019. 144: p. 315-330 DOI: 10.1016/j.phrs.2019.04.026. 9.Mishra, A.K., et al., The sigma-1 receptors are present in monomeric and oligomeric forms in living cells in the presence and absence of ligands. Biochem J, 2015. 466(2): p. 263-271 DOI: 10.1042/bj20141321. 10.Yano, H., et al., Pharmacological profiling of sigma 1 receptor ligands by novel receptor homomer assays. Neuropharmacology, 2018. 133: p. 264-275 DOI: 10.1016/j.neuropharm.2018.01.042. 11.Abramyan, A.M., et al., The Glu102 mutation disrupts higher-order oligomerization of the sigma 1 receptor. Comput Struct Biotechnol J, 2020. 18: p. 199-206 DOI: 10.1016/j.csbj.2019.12.012. 12.Yano, H., et al., The Effects of Terminal Tagging on Homomeric Interactions of the Sigma 1 Receptor. Front Neurosci, 2019. 13: p. 1356 DOI: 10.3389/fnins.2019.01356.