355. AGONISM HETEROGENEITY: ANTI-AMNESIC SIGMAR1 AGONISTS PRE-084 AND (+)PENTAZOCINE DIFFERENTIALLY TRANSLOCATE SIGMAR1 TO SUBSTRUCTURES OF ENDOPLASMIC RETICULUM IN NEURO2A CELLS

Abstract Background Sigma-1 receptor (SIGMAR1) is a ligand-operated CNS transmembrane chaperone implicated in psychiatric diseases such as addiction, depression, amnesia, and schizophrenia. SIGMAR1 normally co-locates with another chaperone BiP at the endoplasmic reticulum (ER)-mitochondrion contact site (termed the MAM). Upon stimulation by SIGMAR1 agonists, with diverse structures, SIGMAR1 dissociates from BiP at the MAM however with the molecular sequalae to be fully clarified. Recently, SIGMAR1 was reported to express solely in the ER sheet structure where it may shape the homeostasis of two ER substructures, vis-a-viz, sheet verse tubule which are important for proper neuronal functionality. However, the effects of SIGMAR1 activation by its ligands in this regard have never been tested. Here we utilized two structurally diverse anti-amnesic SIGMAR1 agonists PRE084 and (+)pentazocine and examined the SIGMAR1 translocation to the ER substructures. Aims & Objectives The aim of this study is to use structurally distinct anti-amnesic agonists to activate SIGMAR1 in Neuro2a cells and investigate the pattern and dynamics of SIMGAR1 translocation between cellular organelles and within ER substructures. Objectives are: (a) Understand the role of SIGMAR1 as it relates to differential structural or functional integrity of the ER; (2) Results from structurally distinct SIGMAR1 agonists may guide future drug designs to target different structural and eventually functional aspects of the ER; (3) Provide data for potential behavioral readouts of ER substructure, if any. Method We treated the cultured Neuro2a cells with 10 mM of structurally distinct (+)-Pentazocine or PRE-084 for 30 min and then immune-stained endogenous SIGMAR1 and other specific organelle marker proteins including those of the ER substructures, i.e., ER sheet or ER tubule. Images were captured using Zeiss confocal microscopy with Airyscan and the SIMGAR1 colocalization with organelle or ER substructure markers was quantitatively analyzed. Results The PRE-084 treatment decreased the SIGMAR1 co-localization with the mitochondrial outer membrane protein Tom20 while concomitantly increased the colocalization with an ER tubule-specific protein RTN3, but not with another ER tubule protein RTN4. This result suggests SIMGAR1 targeting RTN3-enriched loci in the ER tubules after the PRE-084 treatment. The structurally distinct (+)-pentazocine however did not alter the pattern of SIGMAR1 in ER tubules but caused instead changes of SIGMAR1 in the ER sheets. A slight decrease and significant increase of SIGMAR1 colocalization with ER sheet protein LRRC59 and CLIMP63 respectively were found. Discussion & Conclusions Our results indicate that an agonist treatment causes SIGMAR1 migration within the peripheral ER structures rather than leaving the ER for other cellular compartments in Neuo2a cells. Besides, the agonist treatment causes SIGMAR1 to interact with certain specific ER substructure proteins, implying that SIMGAR1 may be involved in separate functional activities of the ER. Those results, together with our previous report showing that (+)-pentazocine and PRE-084 causes distinct effect on the SIGMAR1 oligomerization, suggest subcategories or agonism heterogeneity in the action of SIMGAR1 agonists. How or whether current results on ER substructure modifications may relate to the behavioral/anti-amnesic action of those two drugs remains a challenge to be realized in the future.