EXAMINING THE EFFECTS OF SIGMA-1 RECEPTOR AGONISTS ON THE INTRACELLULAR LOCALIZATION OF THE ENDOGENOUS RECEPTOR IN NEURO2A CELLS

Abstract

Background

Sigma-1 receptor (SIGMAR1) is a ligand-operated transmembrane protein highly expressed in the central nervous system, implicated in various psychiatric diseases such as addiction, depression and schizophrenia. Several psychostimulants and drugs for psychiatric diseases have affinity to SIGMAR1.

Pharmacological studies using animal models show that SIGMAR1 ligands have treatment effects on the disease conditions suggesting that SIGMAR1 can be a promising target for therapeutic approaches.

However, how SIGMAR1 ligands influence the molecular function of SIGMAR1 remains ambiguous. One potential aspect of SIGMAR1 ligand-mediated regulation lies on the localization of SIGMAR1. SIGMAR1 is enriched in the endoplasmic reticulum (ER), mostly at the membrane contact sites between ER and mitochondria, where SIGMAR1 associates with GRP78. Currently, there are contradicting reports on whether SIGMAR1 dissociates from GRP78 at the ER and translocates to other cellular compartments, such as the plasma membrane (PM), upon agonist stimulation. Also, many studies were conducted using overexpression of SIGMAR1 to examine the localization in cells, which may not represent the behavior of the endogenous protein.

Aims & Objectives

The aim of this study is to investigate the effects of agonist stimulation on SIGMAR1 intracellular localization at the endogenous level in the neuroblastoma cell line Neuro2a.

Methods

We utilized a novel immunocytochemistry protocol which can effectively label endogenous SIGMAR1 in cells. We fixed the cells and labeled SIGMAR1 and organelle marker proteins by using specific antibodies. Images were captured using confocal microscopy and analyzed to obtain Pearson’ s correlation co- efficiency. To test the agonist effect, we treated the cells with 10 mM of (+)-Pentazocine or PRE-084 for 30 min and performed the immunocytochemistry as described above.

Results

In untreated conditions, most of SIGMAR1 co-localizes with ER marker proteins followed by mitochondria markers, as expected. On the other hand, co-localization with other organelle markers, such as PM and ER-PM contact sites, was insignificant. The presence of SIGMAR1 in the nuclear envelope (NE) was negligible as well. With agonist stimulation, we found that SIGMAR1 co-staining with marker proteins for PM, ER-PM contact sites and NE did not increase. Nor was there any effect on the co-localization of SIGMAR1 with GRP78.

Discussion & Conclusion

In summary, our results confirm that SIGMAR1 localizes primarily in the ER in Neruo2a cells. When SIGMAR1 is stimulated with an agonist, it does not apparently translocate to PM, PM-ER contact site or NE. Nevertheless, it is possible that SIGMAR1 shows cell-type dependent dynamics. We speculate that SIGMAR1 stays in the ER and moves along within the peripheral ER structure after agonist stimulation and currently plan to study the dynamics of SIGMAR1 within the ER structures.