Cytotoxic sigma-2 ligands trigger cancer cell death via cholesterol-induced-ER-stress.

Sigma-2-ligands (S2L) are characterized by high binding affinities to their cognate sigma-2 receptor, overexpressed in rapidly proliferating tumor cells. As such, S2L were developed as imaging probes (ISO1) or as cancer therapeutics, alone (SV119 [C6], SW43 [C10]) and as delivery vehicles for cytotoxic drug cargoes (C6-Erastin, C10-SMAC). However, the exact mechanism of S2L-induced cytotoxicity remains to be fully elucidated. A series of high-affinity S2L were evaluated regarding their cytotoxicity profiles across cancer cell lines. While C6 and C10 displayed distinct cytotoxicities, C0 and ISO1 were essentially non-toxic. Confocal microscopy and lipidomics analysis in cellular and mouse models revealed that C10 induced increases in intralysosomal free cholesterol and in cholesterol esters, suggestive of unaltered intracellular cholesterol trafficking. Cytotoxicity was caused by cholesterol excess, a phenomenon that contrasts the effects of NPC1 inhibition. RNA-sequencing revealed gene clusters involved in cholesterol homeostasis and ER stress response exclusively by cytotoxic S2L. ER stress markers were confirmed by qPCR and their targeted modulation inhibited or enhanced cytotoxicity of C10 in a predicted manner. Moreover, C10 increased sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR), both found to be pro-survival factors activated by ER stress. Furthermore, inhibition of downstream processes of the adaptive response to S2L with simvastatin resulted in synergistic treatment outcomes in combination with C10. Of note, the S2L conjugates retained the ER stress response of the parental ligands, indicative of cholesterol homeostasis being involved in the overall cytotoxicity of the drug conjugates. Based on these findings, we conclude that S2L-mediated cell death is due to free cholesterol accumulation that leads to ER stress. Consequently, the cytotoxic profiles of S2L drug conjugates are proposed to be enhanced via concurrent ER stress inducers or simvastatin, strategies that could be instrumental on the path toward tumor eradication.

Unraveling the CLCC1 interactome: Impact of the Asp25Glu variant and its interaction with SigmaR1 at the Mitochondrial-Associated ER Membrane (MAM).

The endoplasmic reticulum (ER) plays an indispensable role in cellular processes, including maintenance of calcium homeostasis, and protein folding, synthesized and processing. Disruptions in these processes leading to ER stress and the accumulation of misfolded proteins can instigate the unfolded protein response (UPR), culminating in either restoration of balanced proteostasis or apoptosis. A key player in this intricate balance is CLCC1, an ER-resident chloride channel, whose essential role extends to retinal development, regulation of ER stress, and UPR. The importance of CLCC1 is further underscored by its interaction with proteins localized to mitochondria-associated endoplasmic reticulum membranes (MAMs), where it participates in UPR induction by MAM proteins. In previous research, we identified a p.(Asp25Glu) pathogenic CLCC1 variant associated with retinitis pigmentosa (RP) (CLCC1 hg38 NC_000001.11; NM_001048210.3, c.75C > A; UniprotKB Q96S66). In attempt to decipher the impact of this variant function, we leveraged liquid chromatography-mass spectrometry (LC-MS) to identify likely CLCC1-interacting proteins. We discovered that the CLCC1 interactome is substantially composed of proteins that localize to ER compartments and that the Asp25Glu variant results in noticeable loss and gain of specific protein interactors. Intriguingly, the analysis suggests that the CLCC1Asp25Glu mutant protein exhibits a propensity for increased interactions with cytoplasmic proteins compared to its wild-type counterpart. To corroborate our LC-MS data, we further scrutinized two novel CLCC1 interactors, Calnexin and SigmaR1, chaperone proteins that localize to the ER and MAMs. Through microscopy, we demonstrate that CLCC1 co-localizes with both proteins, thereby validating our initial findings. Moreover, our results reveal that CLCC1 co-localizes with SigmaR1 not merely at the ER, but also at MAMs. These findings reinforce the notion of CLCC1 interacting with MAM proteins at the ER-mitochondria interface, setting the stage for further exploration into how these interactions impact ER or mitochondria function and lead to retinal degenerative disease when impaired.

Convolamine, a tropane alkaloid extracted from Convolvulus plauricalis, is a potent sigma-1 receptor-positive modulator with cognitive and neuroprotective properties.

BACKGROUND AND AIM: By using an in vivo phenotypic screening assay in zebrafish, we identified Convolamine, a tropane alkaloid from Convulvus plauricalis, as a positive modulator of the sigma-1 receptor (S1R). The wfs1abKO zebrafish larva, a model of Wolfram syndrome, exhibits an increased visual-motor response due to a mutation in Wolframin, a protein involved in endoplasmic reticulum-mitochondria communication. We previously reported that ligand activating S1R, restored the cellular and behavioral deficits in patient fibroblasts and zebrafish and mouse models. EXPERIMENTAL PROCEDURES: We screened a library of 108 repurposing and natural compounds on zebrafish motor response. KEY RESULTS: One hit, the tropane alkaloid Convolamine, restored normal mobility in wfs1abKO larvae without affecting wfs1abWT controls. They did not bind to the S1R agonist/antagonist binding site nor dissociated S1R from BiP, an S1R activity assay in vitro, but behaved as a positive modulator by shifting the IC50 value of the reference agonist PRE-084 to lower values. Convolamine restored learning in Wfs1∆Exon8 , Dizocilpine-treated, and Aß25-35 -treated mice. These effects were observed at low ~1 mg/kg doses, not shared by Convolvine, the desmethyl metabolite, and blocked by an S1R antagonist. CONCLUSION AND IMPLICATIONS: Convolamine therefore acts as an S1R positive modulator and this pharmacological action is relevant to the traditional use of Shankhpushpi in memory and cognitive protection.

BS148 Reduces the Aggressiveness of Metastatic Melanoma via Sigma-2 Receptor Targeting.

The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer.

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 co-immunoprecipitation (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.

Overview of Sigma-1R Subcellular Specific Biological Functions and Role in Neuroprotection.

For the past several years, fundamental research on Sigma-1R (S1R) protein has unveiled its necessity for maintaining proper cellular homeostasis through modulation of calcium and lipid exchange between the endoplasmic reticulum (ER) and mitochondria, ER-stress response, and many other mechanisms. Most of these processes, such as ER-stress response and autophagy, have been associated with neuroprotective roles. In fact, improving these mechanisms using S1R agonists was beneficial in several brain disorders including neurodegenerative diseases. In this review, we will examine S1R subcellular localization and describe S1R-associated biological activity within these specific compartments, i.e., the Mitochondrion-Associated ER Membrane (MAM), ER-Lipid Droplet (ER-LD) interface, ER-Plasma Membreane (ER-PM) interface, and the Nuclear Envelope (NE). We also discussed how the dysregulation of these pathways contributes to neurodegenerative diseases, while highlighting the cellular mechanisms and key binding partners engaged in these processes.

Sigma-1 Receptor Signaling: In Search of New Therapeutic Alternatives for Cardiovascular and Renal Diseases.

Cardiovascular and renal diseases are among the leading causes of death worldwide, and regardless of current efforts, there is a demanding need for therapeutic alternatives to reduce their progression to advanced stages. The stress caused by diseases leads to the activation of protective mechanisms in the cell, including chaperone proteins. The Sigma-1 receptor (Sig-1R) is a ligand-operated chaperone protein that modulates signal transduction during cellular stress processes. Sig-1R interacts with various ligands and proteins to elicit distinct cellular responses, thus, making it a potential target for pharmacological modulation. Furthermore, Sig-1R ligands activate signaling pathways that promote cardioprotection, ameliorate ischemic injury, and drive myofibroblast activation and fibrosis. The role of Sig-1R in diseases has also made it a point of interest in developing clinical trials for pain, neurodegeneration, ischemic stroke, depression in patients with heart failure, and COVID-19. Sig-1R ligands in preclinical models have significantly beneficial effects associated with improved cardiac function, ventricular remodeling, hypertrophy reduction, and, in the kidney, reduced ischemic damage. These basic discoveries could inform clinical trials for heart failure (HF), myocardial hypertrophy, acute kidney injury (AKI), and chronic kidney disease (CKD). Here, we review Sig-1R signaling pathways and the evidence of Sig-1R modulation in preclinical cardiac and renal injury models to support the potential therapeutic use of Sig-1R agonists and antagonists in these diseases.

Sigma-1 receptor is involved in modification of ER-mitochondria proximity and Ca2+ homeostasis in cardiomyocytes.

The Sigma-1 receptor (Sigmar1) is downregulated in heart failure model mice with mitochondrial dysfunction. However, the mechanism in detail has not been investigated. In this study, we investigated the role of Sigmar1 in ER-mitochondria proximity using Sigmar1-knockdown or -overexpressed neonatal rat ventricular myocytes (NRVMs). The endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy was aggravated with the dysregulation of mitochondrial function and ER-mitochondrial junctional formation in Sigmar1-knockdown NRVMs, whereas improved in Sigmar1 overexpressed NRVMs. Our data suggests that the reduction of the cardiac Sigmar1 results in decrease mitochondrial Ca2+ influx and promotes mitochondrial fission, followed by reduced ER-mitochondria proximity, exacerbating ET-1-induced cardiomyocyte injury.

An investigation of Sigma-1 receptor expression and ligand-induced endoplasmic reticulum stress in breast cancer.

Targeted therapeutic options and prognostic biomarkers for hormone receptor- or Her2 receptor-negative breast cancers are severely limited. The sigma-1 receptor, a stress-activated chaperone, is frequently dysregulated in disease. However, its significance in breast cancer (BCa) has not been adequately explored. Here, we report that the sigma-1 receptor gene (SIGMAR1) is elevated in BCa, particularly in the aggressive triple-negative (TNBC) subtype. By examining several patient datasets, we found that high expression at both the gene (SIGMAR1) and protein (Sig1R) levels associated with poor survival outcomes, specifically in ER-Her2- groups. Our data further show that high SIGMAR1 was predictive of shorter survival times in patients treated with adjuvant chemotherapy (ChT). Interestingly, in a separate cohort who received neoadjuvant taxane + anthracycline treatment, elevated SIGMAR1 associated with higher rates of pathologic complete response (pCR). Treatment with a Sig1R antagonist, 1-(4-iodophenyl)-3-(2-adamantyl)guanidine (IPAG), activated the unfolded protein response (UPR) in TNBC (high-Sig1R expressing) and ER + (low-Sig1R expressing) BCa cell lines. In tamoxifen-resistant LY2 cells, IPAG caused Sig1R to aggregate and co-localise with the stress marker BiP. These findings showcase the potential of Sig1R as a novel biomarker in TNBC as well as highlight its ligand-induced interference with the stress-coping mechanisms of BCa cells.

Specific detection and deletion of the sigma-1 receptor widely expressed in neurons and glial cells in vivo.

The chaperon protein sigma-1 receptor (S1R) has been discovered over 40 years ago. Recent pharmacological studies using S1R exogenous ligands demonstrated a promising therapeutical potential of targeting the S1R in several neurological disorders. Although intensive in vitro studies have revealed S1Rs are mainly residing at the membrane of the endoplasmic reticulum (ER), the cell-specific in vivo expression pattern of S1Rs is still unclear, mainly because of the lack of a reliable detection method which also prevented a comprehensive functional analysis. Here, first, we identified a highly specific antibody using S1R knockout (KO) mice and established an immunohistochemical protocol involving a 1% sodium dodecyl sulphate (SDS) antigen retrieval step. Second, we characterized the S1R expression in the mouse brain and can demonstrate that the S1R is widely expressed: in principal neurons, interneurons and all glial cell types. In addition, unlike reported in previous studies, we showed that the S1R expression in astrocytes is not colocalized with the astrocytic cytoskeleton protein GFAP. Thus, our results raise concerns over previously reported S1R properties. Finally, we generated a Cre-dependent S1R conditional KO mouse (S1R flox) to study cell-type-specific functions of the S1R. As a proof of concept, we successfully ablated S1R expressions in neurons or microglia employing neuronal and microglial Cre-expressing mice, respectively. In summary, we provide powerful tools to cell-specifically detect, delete and functionally characterize S1R in vivo.

Differential Responses to Sigma-1 or Sigma-2 Receptor Ablation in Adiposity, Fat Oxidation, and Sexual Dimorphism.

Obesity is increasing at epidemic rates across the US and worldwide, as are its co-morbidities, including type-2 diabetes and cardiovascular disease. Thus, targeted interventions to reduce the prevalence of obesity are of the utmost importance. The sigma-1 receptor (S1R) and sigma-2 receptor (S2R; encoded by Tmem97) belong to the same class of drug-binding sites, yet they are genetically distinct. There are multiple ongoing clinical trials focused on sigma receptors, targeting diseases ranging from Alzheimer's disease through chronic pain to COVID-19. However, little is known regarding their gene-specific role in obesity. In this study, we measured body composition, used a comprehensive laboratory-animal monitoring system, and determined the glucose and insulin tolerance in mice fed a high-fat diet. Compared to Sigmar1+/+ mice of the same sex, the male and female Sigmar1-/- mice had lower fat mass (17% and 12% lower, respectively), and elevated lean mass (16% and 10% higher, respectively), but S1R ablation had no effect on their metabolism. The male Tmem97-/- mice exhibited 7% lower fat mass, 8% higher lean mass, increased volumes of O2 and CO2, a decreased respiratory exchange ratio indicating elevated fatty-acid oxidation, and improved insulin tolerance, compared to the male Tmem97+/+ mice. There were no changes in any of these parameters in the female Tmem97-/- mice. Together, these data indicate that the S1R ablation in male and female mice or the S2R ablation in male mice protects against diet-induced adiposity, and that S2R ablation, but not S1R deletion, improves insulin tolerance and enhances fatty-acid oxidation in male mice. Further mechanistic investigations may lead to translational strategies to target differential S1R/S2R regulations and sexual dimorphism for precision treatments of obesity.

Sigma-1 receptor attenuates osteoclastogenesis by promoting ER-associated degradation of SERCA2.

Sigma-1 receptor (Sigmar1) is a specific chaperone located in the mitochondria-associated endoplasmic reticulum membrane (MAM) and plays a role in several physiological processes. However, the role of Sigmar1 in bone homeostasis remains unknown. Here, we show that mice lacking Sigmar1 exhibited severe osteoporosis in an ovariectomized model. In contrast, overexpression of Sigmar1 locally alleviated the osteoporosis phenotype. Treatment with Sigmar1 agonists impaired both human and mice osteoclast formation in vitro. Mechanistically, SERCA2 was identified to interact with Sigmar1 based on the immunoprecipitation-mass spectrum (IP-MS) and co-immunoprecipitation (co-IP) assays, and Q615 of SERCA2 was confirmed to be the critical residue for their binding. Furthermore, Sigmar1 promoted SERCA2 degradation through Hrd1/Sel1L-dependent ER-associated degradation (ERAD). Ubiquitination of SERCA2 at K460 and K541 was responsible for its proteasomal degradation. Consequently, inhibition of SERCA2 impeded Sigmar1 deficiency enhanced osteoclastogenesis. Moreover, we found that dimemorfan, an FDA-approved Sigmar1 agonist, effectively rescued bone mass in various established bone-loss models. In conclusion, Sigmar1 is a negative regulator of osteoclastogenesis, and activation of Sigmar1 by dimemorfan may be a potential treatment for osteoporosis in clinical practice.

Knocking Out Sigma-1 Receptors Reveals Diverse Health Problems.

Sigma-1 receptor (Sig-1R) is a protein present in several organs such as brain, lung, and heart. In a cell, Sig-1R is mainly located across the membranes of the endoplasmic reticulum and more specifically at the mitochondria-associated membranes. Despite numerous studies showing that Sig-1R could be targeted to rescue several cellular mechanisms in different pathological conditions, less is known about its fundamental relevance. In this review, we report results from various studies and focus on the importance of Sig-1R in physiological conditions by comparing Sig-1R KO mice to wild-type mice in order to investigate the fundamental functions of Sig-1R. We note that the Sig-1R deletion induces cognitive, psychiatric, and motor dysfunctions, but also alters metabolism of heart. Finally, taken together, observations from different experiments demonstrate that those dysfunctions are correlated to poor regulation of ER and mitochondria metabolism altered by stress, which could occur with aging.

Sigma-1 receptor activity in primary sensory neurons is a critical driver of neuropathic pain.

The Sigma-1 receptor (σ1R) is highly expressed in the primary sensory neurons (PSNs) that are the critical site of initiation and maintenance of pain following peripheral nerve injury. By immunoblot and immunohistochemistry, we observed increased expression of both σ1R and σ1R-binding immunoglobulin protein (BiP) in the lumbar (L) dorsal root ganglia (DRG) ipsilateral to painful neuropathy induced by spared nerve injury (SNI). To evaluate the therapeutic potential of PSN-targeted σ1R inhibition at a selected segmental level, we designed a recombinant adeno-associated viral (AAV) vector expressing a small hairpin RNA (shRNA) against rat σ1R. Injection of this vector into the L4/L5 DRGs induced downregulation of σ1R in DRG neurons of all size groups, while expression of BiP was not affected. This was accompanied by attenuation of SNI-induced cutaneous mechanical and thermal hypersensitivity. Whole-cell current-clamp recordings of dissociated neurons showed that knockdown of σ1R suppressed neuronal excitability, suggesting that σ1R silencing attenuates pain by reversal of injury-induced neuronal hyperexcitability. These findings support a critical role of σ1R in modulating PSN nociceptive functions, and that the nerve injury-induced elevated σ1R activity in the PSNs can be a significant driver of neuropathic pain. Further understanding the role of PSN-σ1R in pain pathology may open routes to exploit this system for DRG-targeted pain therapy.

The molecular role of Sigmar1 in regulating mitochondrial function through mitochondrial localization in cardiomyocytes.

Sigmar1 is a widely expressed molecular chaperone protein in mammalian cell systems. Accumulating research demonstrated the cardioprotective roles of pharmacologic Sigmar1 activation by ligands in preclinical rodent models of cardiac injury. Extensive biochemical and immuno-electron microscopic research demonstrated Sigmar1's sub-cellular localization largely depends on cell and organ types. Despite comprehensive studies, Sigmar1's direct molecular role in cardiomyocytes remains elusive. In the present study, we determined Sigmar1's subcellular localization, transmembrane topology, and function using complementary microscopy, biochemical, and functional assays in cardiomyocytes. Quantum dots in transmission electron microscopy showed Sigmar1 labeled quantum dots on the mitochondrial membranes, lysosomes, and sarcoplasmic reticulum-mitochondrial interface. Subcellular fractionation of heart cell lysates confirmed Sigmar1's localization in purified mitochondria fraction and lysosome fraction. Immunocytochemistry confirmed Sigmar1 colocalization with mitochondrial proteins in isolated adult mouse cardiomyocytes. Sigmar1's mitochondrial localization was further confirmed by Sigmar1 colocalization with Mito-Tracker in isolated mouse heart mitochondria. A series of biochemical experiments, including alkaline extraction and proteinase K treatment of purified heart mitochondria, demonstrated Sigmar1 as an integral mitochondrial membrane protein. Sigmar1's structural requirement for mitochondrial localization was determined by expressing FLAG-tagged Sigmar1 fragments in cells. Full-length Sigmar1 and Sigmar1's C terminal-deletion fragments were able to localize to the mitochondrial membrane, whereas N-terminal deletion fragment was unable to incorporate into the mitochondria. Finally, functional assays using extracellular flux analyzer and high-resolution respirometry showed Sigmar1 siRNA knockdown significantly altered mitochondrial respiration in cardiomyocytes. Overall, we found that Sigmar1 localizes to mitochondrial membranes and is indispensable for maintaining mitochondrial respiratory homeostasis in cardiomyocytes.

Sigma-1 Receptor Is Critical for Mitochondrial Activity and Unfolded Protein Response in Larval Zebrafish.

The sigma-1 receptor (S1R) is a highly conserved transmembrane protein highly enriched in mitochondria-associated endoplasmic reticulum (ER) membranes, where it interacts with several partners involved in ER-mitochondria Ca2+ transfer, activation of the ER stress pathways, and mitochondria function. We characterized a new S1R deficient zebrafish line and analyzed the impact of S1R deficiency on visual, auditory and locomotor functions. The s1r+25/+25 mutant line showed impairments in visual and locomotor functions compared to s1rWT. The locomotion of the s1r+25/+25 larvae, at 5 days post fertilization, was increased in the light and dark phases of the visual motor response. No deficit was observed in acoustic startle response. A critical role of S1R was shown in ER stress pathways and mitochondrial activity. Using qPCR to analyze the unfolded protein response genes, we observed that loss of S1R led to decreased levels of IRE1 and PERK-related effectors and increased over-expression of most of the effectors after a tunicamycin challenge. Finally, S1R deficiency led to alterations in mitochondria bioenergetics with decreased in basal, ATP-linked and non-mitochondrial respiration and following tunicamycin challenge. In conclusion, this new zebrafish model confirmed the importance of S1R activity on ER-mitochondria communication. It will be a useful tool to further analyze the physiopathological roles of S1R.

The Sigma-1 receptor is an ER-localized type II membrane protein.

The Sigma-1 receptor (S1R) is a transmembrane protein with important roles in cellular homeostasis in normal physiology and in disease. Especially in neurodegenerative diseases, S1R activation has been shown to provide neuroprotection by modulating calcium signaling, mitochondrial function and reducing endoplasmic reticulum (ER) stress. S1R missense mutations are one of the causes of the neurodegenerative Amyotrophic Lateral Sclerosis and distal hereditary motor neuronopathies. Although the S1R has been studied intensively, basic aspects remain controversial, such as S1R topology and whether it reaches the plasma membrane. To address these questions, we have undertaken several approaches. C-terminal tagging with a small biotin-acceptor peptide and BirA biotinylation in cells suggested a type II membrane orientation (cytosolic N-terminus). However, N-terminal tagging gave an equal probability for both possible orientations. This might explain conflicting reports in the literature, as tags may affect the protein topology. Therefore, we studied untagged S1R using a protease protection assay and a glycosylation mapping approach, introducing N-glycosylation sites. Both methods provided unambiguous results showing that the S1R is a type II membrane protein with a short cytosolic N-terminal tail. Assessments of glycan processing, surface fluorescence-activated cell sorting, and cell surface biotinylation indicated ER retention, with insignificant exit to the plasma membrane, in the absence or presence of S1R agonists or of ER stress. These findings may have important implications for S1R-based therapeutic approaches.

Sigma-1 Receptor Agonists Acting on Aquaporin-Mediated H2O2 Permeability: New Tools for Counteracting Oxidative Stress.

Sigma1 Receptor (S1R) is involved in oxidative stress, since its activation is triggered by oxidative or endoplasmic reticulum stress. Since specific aquaporins (AQP), called peroxiporins, play a relevant role in controlling H2O2 permeability and ensure reactive oxygen species wasted during oxidative stress, we studied the effect of S1R modulators on AQP-dependent water and hydrogen peroxide permeability in the presence and in the absence of oxidative stress. Applying stopped-flow light scattering and fluorescent probe methods, water and hydrogen peroxide permeability in HeLa cells have been studied. Results evidenced that S1R agonists can restore water permeability in heat-stressed cells and the co-administration with a S1R antagonist totally counteracted the ability to restore the water permeability. Moreover, compounds were able to counteract the oxidative stress of HeLa cells specifically knocked down for S1R. Taken together these results support the hypothesis that the antioxidant mechanism is mediated by both S1R and AQP-mediated H2O2 permeability. The finding that small molecules can act on both S1R and AQP-mediated H2O2 permeability opens a new direction toward the identification of innovative drugs able to regulate cell survival during oxidative stress in pathologic conditions, such as cancer and degenerative diseases.

Intestinal inflammation-associated hypersensitivity is attenuated in a DSS model of colitis in Sigma-1 knockout C57BL/6 mice.

Sigma-1 receptors (σ1R) have been implicated in several pain pathways. We assessed the implication of σ1Rs in the development of intestinal inflammation and inflammation-associated referred hypersensitivity in a model of colitis in σ1R knockout (KO) mice. Colitis was induced with dextran sulfate sodium (DSS) in wild type (WT) and σ1R KO mice. The development of referred mechanical hypersensitivity (von Frey test) was assessed. Colonic and spinal changes in expression of immune- and sensory-related markers were also investigated (RT-qPCR/Western blot). Absence of σ1Rs had little impact in colitis generation and progression, although during the chronic phase a reduction in edema and a down-regulation of iNOS gene expression was observed. In σ1R KO mice, inflammation-associated hypersensitivity was significantly attenuated (paw) or completely prevented (abdomen). During colitis, in WT mice, changes in the colonic expression of nociceptive markers were observed during the acute and chronic phases of inflammation. Although σ1R KO mice showed similar regulation in the acute phase, an attenuated response was observed during the chronic phase of colitis. These differences were especially relevant for CB2 and TRPV1 receptors, which could play an important role in σ1-mediated regulation of sensitivity. No changes were detected on ERK phosphorylation at the level of the lumbosacral spinal cord. In summary, intestinal inflammation-associated referred hyperalgesia was reduced (paw) or absent (abdomen) in σ1R KO mice, thus confirming an important role for σ1R in the development of colitis-associated hypersensitivity. These results identify σ1Rs as a possible therapeutic target for the treatment of hypersensitivity associated to intestinal inflammation.

Sigma-1R Protects Retinal Ganglion Cells in Optic Nerve Crush Model for Glaucoma.

Purpose: The purpose of this study was to determine the effects of the Sigma-1R (σ-1r) on retinal ganglion cell (RGC) survival following optic nerve crush (ONC) and the signaling mechanism involved in the σ-1r protection. Methods: The overall strategy was to induce injury by ONC and mitigate RGC death by increasing σ-1r expression and/or activate σ-1r activity in σ-1r K/O mice and wild type (WT) mice. AAV2-σ-1r vector was used to increase σ-1r expression and σ-1r agonist used to activate the σ-1r and RGCs were counted. Immunohistochemical and Western blot analysis determined phosphorylated (p)-c-Jun, c-Jun, and Caspase-3. Pattern electroretinography (PERG) determined RGC activity. Results: RGC counts and function were similar in pentazocine-treated WT mice when compared to untreated mice and in WT mice when compared with σ-1r K/O mice. Pentazocine-induced effects and the effects of σ-1r K/O were only observable after ONC. ONC resulted in decreased RGC counts and activity in both WT and σ-1r K/O mice, with σ-1r K/O mice experiencing significant decreases compared with WT mice. The σ-1r transgenic expression resulted in increased RGC counts and activity following ONC. In WT mice, treatment with σ-1r agonist pentazocine resulted in increased RGC counts and increased activity when compared with untreated WT mice. There were time-dependent increases in c-jun, p-c-jun, and caspase-3 expression in ONC mice that were mitigated with pentazocine-treatment. Conclusions: These findings suggest that the apoptotic pathway is involved in RGC losses seen in an ONC model. The σ-1r offers neuroprotection, as activation and/or transgenic expression of σ-1r attenuated the apoptotic pathway and restored RGCs number and function following ONC.