The tunicamycin biosynthetic gene cluster of Streptomyces chartreusis consists of 14 genes (tunA to tunN) with a high degree of apparent translational coupling. Transcriptional analysis revealed that all of these genes are likely to be transcribed as a single operon from two promoters, tunp1 and tunp2. In-frame deletion analysis revealed that just six of these genes (tunABCDEH) are essential for tunicamycin production in the heterologous host Streptomyces coelicolor, while five (tunFGKLN) with likely counterparts in primary metabolism are not necessary, but presumably ensure efficient production of the antibiotic at the onset of tunicamycin biosynthesis. Three genes are implicated in immunity, namely, tunI and tunJ, which encode a two-component ABC transporter presumably required for export of the antibiotic, and tunM, which encodes a putative S-adenosylmethionine (SAM)-dependent methyltransferase. Expression of tunIJ or tunM in S. coelicolor conferred resistance to exogenous tunicamycin. The results presented here provide new insights into tunicamycin biosynthesis and immunity.

The pulmonary endothelium is a dynamic semipermeable barrier that orchestrates tissue-fluid homeostasis; regulating physiological and immunological responses. Endothelial abnormalities are caused by inflammatory stimuli interacting with intracellular messengers to remodel cytoskeletal junctions and adhesion proteins. Those phenomena are associated with sepsis, acute lung injury, and acute respiratory distress syndrome. The molecular processes beyond those responses are the main interest of our group. Unfolded protein response (UPR) is a highly conserved molecular pathway resolving protein-folding defects to counteract cellular threats. An emerging body of evidence suggests that UPR is a promising target against lung and cardiovascular disease. In the present study, we reveal that Tunicamycin (TM) (UPR inducer) protects against lipopolysaccharide (LPS)-induced injury. The barrier function of the inflamed endothelium was evaluated in vitro (transendothelial and paracellular permeability); as well as in mice exposed to TM after LPS. Our study demonstrates that TM supports vascular barrier function by modulating actomyosin remodeling. Moreover, it reduces the internalization of vascular endothelial cadherin (VE-cadherin), enhancing endothelial integrity. We suggest that UPR activation may deliver novel therapeutic opportunities in diseases related to endothelial dysregulation.

Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca2+ homeostasis. The store-operated calcium (SOC) channel is the primary Ca2+ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca2+ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca2+ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.

How cells exposed to one stress are later able to better survive other types of stress is not well understood. In eukaryotic organisms, physiological and pathological stresses can disturb endoplasmic reticulum (ER) function, resulting in "ER stress." Here, we found that exposure to tunicamycin, an inducer of ER stress, resulted in the acquisition of a specific aneuploidy, chromosome 2 trisomy (Chr2x3), in Candida albicans. Importantly, the resulting aneuploidy also conferred cross-tolerance to caspofungin, a first-line echinocandin antifungal, as well as to hydroxyurea, a common chemotherapeutic agent. Exposure to a range of tunicamycin concentrations induced similar ER stress responses. Extra copies of one Chr2 gene, MKK2, affected both tunicamycin and caspofungin tolerance, while at least 3 genes on chromosome 2 (ALG7, RTA2, and RTA3) affected only tunicamycin and not caspofungin responses. Other Chr2 genes (RNR1 and RNR21) affected hydroxyurea tolerance but neither tunicamycin nor caspofungin tolerance. Deletion of components of the protein kinase C (PKC) or calcineurin pathways affected tolerance to both tunicamycin and caspofungin, supporting the idea that the ER stress response and echinocandin tolerance are regulated by overlapping stress response pathways. Thus, antifungal drug tolerance can arise rapidly via ER stress-induced aneuploidy. IMPORTANCE Candida albicans is a prevalent human fungal commensal and also a pathogen that causes life-threatening systemic infections. Treatment failures are frequent because few therapeutic antifungal drug classes are available and because drug resistance and tolerance limit drug efficacy. We found that C. albicans rapidly overcomes the cellular stress induced by the drug tunicamycin by duplicating chromosome 2. Also, chromosome 2 duplication confers tolerance not only to tunicamycin but also to the following two unrelated drugs: caspofungin, an antifungal drug, and hydroxyurea, a chemotherapeutic. Cross tolerance to the three drugs involves different sets of genes, although some genetic pathways affect the tolerance to two of these three drugs. This work highlights a serious concern, namely, that changes in whole chromosome copy number can occur in response to one type of stress, and yet, they may facilitate the emergence of tolerance to multiple drugs, including the few antifungal drug classes available to treat Candida infections.

Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes ER stress, resulting in the activation of the unfolded protein response (UPR). ER stress and UPR are associated with many neurodevelopmental and neurodegenerative disorders. The developing brain is particularly susceptible to environmental insults which may cause ER stress. We evaluated the UPR in the brain of postnatal mice. Tunicamycin, a commonly used ER stress inducer, was administered subcutaneously to mice of postnatal days (PDs) 4, 12 and 25. Tunicamycin caused UPR in the cerebral cortex, hippocampus and cerebellum of mice of PD4 and PD12, which was evident by the upregulation of ATF6, XBP1s, p-eIF2α, GRP78, GRP94 and MANF, but failed to induce UPR in the brain of PD25 mice. Tunicamycin-induced UPR in the liver was observed at all stages. In PD4 mice, tunicamycin-induced caspase-3 activation was observed in layer II of the parietal and optical cortex, CA1-CA3 and the subiculum of the hippocampus, the cerebellar external germinal layer and the superior/inferior colliculus. Tunicamycin-induced caspase-3 activation was also shown on PD12 but to a much lesser degree and mainly located in the dentate gyrus of the hippocampus, deep cerebellar nuclei and pons. Tunicamycin did not activate caspase-3 in the brain of PD25 mice and the liver of all stages. Similarly, immature cerebellar neurons were sensitive to tunicamycin-induced cell death in culture, but became resistant as they matured in vitro. These results suggest that the UPR is developmentally regulated and the immature brain is more susceptible to ER stress.

Disorders of hepatic energy metabolism, which can be regulated by endoplasmic reticulum (ER) stress, lead to metabolic diseases such as hepatic steatosis and hypoglycemia. Tunicamycin, a pharmacological ER stress inducer, is used to develop an anti-cancer drug. However, the effects of tunicamycin on hepatic energy metabolism have not been well elucidated. Mice were intraperitoneally injected with tunicamycin or vehicle. Twenty-four hours later, hepatic triglyceride and glycogen content and serum lipids profiles were analyzed, as well as the expression of lipogenic and gluconeogenic genes. Tunicamycin significantly induced hepatic a yellowish color and ER stress, as well as increasing serum levels of aspartate transaminase and alanine transaminase. Besides, tunicamycin remarkably increased hepatic triglyceride content and suppressed the expression of apolipoprotein B100. In addition, tunicamycin-treated mice had lower serum levels of triglyceride, apolipoprotein B, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol. Gene expression of peroxisome proliferator-activated receptor α was decreased by tunicamycin, but the protein level was increased. Furthermore, blood glucose level and hepatic glycogen content were decreased in tunicamycin-treated mice. Protein kinase B signaling was attenuated in the tunicamycin-treated liver, but the expression and activities of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were unchanged. Tunicamycin alters hepatic energy homeostasis by increasing triglyceride accumulation and decreasing glycogen content.

Endocannabinoid (eCB)-binding receptors can be modulated by several ligands and membrane environment, yet the effect of glycosylation remains to be assessed. In this study, we used human neuroblastoma SH-SY5Y cells to interrogate whether expression, cellular localization, and activity of eCB-binding receptors may depend on N-linked glycosylation. Following treatment with tunicamycin (a specific inhibitor of N-linked glycosylation) at the non-cytotoxic dose of 1 µg/mL, mRNA, protein levels and localization of eCB-binding receptors, as well as N-acetylglucosamine (GlcNAc) residues, were evaluated in SH-SY5Y cells by means of quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and confocal microscopy, respectively. In addition, the activity of type-1 and type-2 cannabinoid receptors (CB₁ and CB₂) was assessed by means of rapid binding assays. Significant changes in gene and protein expression were found upon tunicamycin treatment for CB₁ and CB₂, as well as for GPR55 receptors, but not for transient receptor potential vanilloid 1 (TRPV1). Deglycosylation experiments with N-glycosidase-F and immunoblot of cell membranes derived from SH-SY5Y cells confirmed the presence of one glycosylated form in CB₁ (70 kDa), that was reduced by tunicamycin. Morphological studies demonstrated the co-localization of CB₁ with GlcNAc residues, and showed that tunicamycin reduced CB₁ membrane expression with a marked nuclear localization, as confirmed by immunoblotting. Cleavage of the carbohydrate side chain did not modify CB receptor binding affinity. Overall, these results support N-linked glycosylation as an unprecedented post-translational modification that may modulate eCB-binding receptors' expression and localization, in particular for CB₁.

Since Inhibitor of Apoptosis (IAP) proteins have been implicated in cellular adaptation to endoplasmic reticulum (ER) stress, we investigated the regulation of ER stress-induced apoptosis by small-molecule second mitochondria-derived activator of caspase (Smac) mimetics that antagonize IAP proteins. Here, we discover that Smac mimetic suppresses tunicamycin (TM)-induced apoptosis via resolution of the unfolded protein response (UPR) and ER stress. Smac mimetics such as BV6 selectively inhibit apoptosis triggered by pharmacological or genetic inhibition of protein N-glycosylation using TM or knockdown of DPAGT1, the enzyme that catalyzes the first step of protein N-glycosylation. In contrast, BV6 does not rescue cell death induced by other typical ER stressors (i.e., thapsigargin (TG), dithiothreitol, brefeldin A, bortezomib, or 2-deoxyglucose). The protection from TM-triggered apoptosis is found for structurally different Smac mimetics and for genetic knockdown of cellular IAP (cIAP) proteins in several cancer types, underlining the broader relevance. Interestingly, lectin microarray profiling reveals that BV6 counteracts TM-imposed inhibition of protein glycosylation. BV6 consistently abolishes TM-stimulated accumulation of ER stress markers such as glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) and reduces protein kinase RNA-like ER kinase (PERK) phosphorylation and X box-binding protein 1 (XBP1) splicing upon TM treatment. BV6-stimulated activation of nuclear factor-κB (NF-κB) contributes to the resolution of ER stress, since NF-κB inhibition by overexpression of dominant-negative IκBα superrepressor counteracts the suppression of TM-stimulated transcriptional activation of CHOP and GRP78 by BV6. Thus, our study is the first to show that Smac mimetic protects from TM-triggered apoptosis by resolving the UPR and ER stress. This provides new insights into the regulation of cellular stress responses by Smac mimetics.

Tunicamycin (TM) induces endoplasmic reticulum (ER) stress and inhibits N-glycosylation in cells. ER stress is associated with neuronal death in neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease, and most patients complain of the impairment of olfactory recognition. Here we examined the effects of TM on aversive olfactory learning and the underlying synaptic plasticity in the main olfactory bulb (MOB). Behavioral experiments demonstrated that the intrabulbar infusion of TM disabled aversive olfactory learning without affecting short-term memory. Histological analyses revealed that TM infusion upregulated C/EBP homologous protein (CHOP), a marker of ER stress, in the mitral and granule cell layers of MOB. Electrophysiological data indicated that TM inhibited tetanus-induced long-term potentiation (LTP) at the dendrodendritic excitatory synapse from mitral to granule cells. A low dose of TM (250nM) abolished the late phase of LTP, and a high dose (1μM) inhibited the early and late phases of LTP. Further, high-dose, but not low-dose, TM reduced the paired-pulse facilitation ratio, suggesting that the inhibitory effects of TM on LTP are partially mediated through the presynaptic machinery. Thus, our results support the hypothesis that TM-induced ER stress impairs olfactory learning by inhibiting synaptic plasticity via presynaptic and postsynaptic mechanisms in MOB.

Induction of acute ER (endoplasmic reticulum) stress using thapsigargin contributes to complex I damage in mouse hearts. Thapsigargin impairs complex I by increasing mitochondrial calcium through inhibition of Ca2+-ATPase in the ER. Tunicamycin (TUNI) is used to induce ER stress by inhibiting protein folding. We asked if TUNI-induced ER stress led to complex I damage.

Endoplasmic reticulum (ER) stress, a dysfunction in protein-folding capacity, is involved in many pathological and physiological responses, including embryonic development. This study aims to determine the developmental competence, apoptosis, and stress-induced gene expression in mouse preimplantation embryos grown in an in vitro culture medium supplemented with different concentrations of the ER stress inducer tunicamycin (TM) and the antioxidant glutathione (GSH). Treatment of zygotes with 0.5 µg/ml TM significantly decreased (P < 0.05) the rate of blastocyst formation, whereas 1 mM GSH supplementation improved the developmental rate of blastocysts. Furthermore, TM treatment significantly increased (P < 0.05) the apoptotic index and reduced the total number of cells, whereas GSH significantly increased the total number of cells and decreased the apoptotic index. The expression levels of ER chaperones, including immunoglobulin-binding protein, activating transcription factor 6, double-stranded activated protein kinase-like ER kinase, activating transcription factor 4, and C/EBP homologous protein were significantly increased (P < 0.05) by TM, but significantly decreased (P < 0.05) by GSH treatment. A similar pattern was observed in the case of the pro-apoptotic gene, B cell lymphoma-associated X protein. The expression level of the anti-apoptotic gene B cell lymphoma 2, was decreased by TM, but significantly increased after co-treatment with GSH. In conclusion, GSH improves the developmental potential of mouse embryos and significantly alleviates ER stress.

In higher eukaryotic cells, pertubations in ER environment, called ER stress, usually activate unfolded protein response (UPR) pathway in an attempt to re-stablish the ER homeostasis and prevent cell death. Because trypanosomatids appear to lack the classical UPR, it is not clear how these parasites respond to ER stress. Thus, the aim of this work was to evaluate the effects of ER stressors tunicamycin (TM) or dithiothreitol (DTT) on Trypanosoma cruzi. The TM treatment showed strong trypanostatic effect. At 2.5 μg/mL of TM, the mRNA levels of both binding protein (BiP) and calreticulin (CRT) increased significantly, whereas the protein levels of BiP remained stable. TM treatment induced ultrastructural changes compatible with an autophagic process. The DTT treatment inhibited the cell growth, induced drastic morphological changes, mitochondrial membrane depolarization and increased ROS production. The expression of BiP apparently was not affected by DTT, whereas the mRNA levels of BiP and CRT were significantly reduced. Our results suggest that TM induces autophagy/ER-phagy without causing substantial injury to the parasite. Conversely, the DTT treatment seems to rupture the mitochondrion homeostasis leading to parasite death. The comprehension of the mechanisms behind the susceptibility of T. cruzi to ER stress open perspectives for the development of chemotherapeutic agents addressed to these pathways.

Different forms of acute kidney injury (AKI) have been associated with endoplasmic reticulum (ER) stress; these include AKI caused by acetaminophen, antibiotics, cisplatin, and radiocontrast. Tunicamycin (TM) is a nucleoside antibiotic known to induce ER stress and is a commonly used inducer of AKI. 4-phenylbutyrate (4-PBA) is an FDA approved substance used in children who suffer from urea cycle disorders. 4-PBA acts as an ER stress inhibitor by aiding in protein folding at the molecular level and preventing misfolded protein aggregation. The main objective of this study was to determine if 4-PBA could protect from AKI induced by ER stress, as typified by the TM-model, and what mechanism(s) of 4-PBA's action were responsible for protection. C57BL/6 mice were treated with saline, TM or TM plus 4-PBA. 4-PBA partially protected the anatomic segment most susceptible to damage, the outer medullary stripe, from TM-induced AKI. In vitro work showed that 4-PBA protected human proximal tubular cells from apoptosis and TM-induced CHOP expression, an ER stress inducible proapoptotic gene. Further, immunofluorescent staining in the animal model found similar protection by 4-PBA from CHOP nuclear translocation in the tubular epithelium of the medulla. This was accompanied by a reduction in apoptosis and GRP78 expression. CHOP(-/-) mice were protected from TM-induced AKI. The protective effects of 4-PBA extended to the ultrastructural integrity of proximal tubule cells in the outer medulla. When taken together, these results indicate that 4-PBA acts as an ER stress inhibitor, to partially protect the kidney from TM-induced AKI through the repression of ER stress-induced CHOP expression.

Severity or duration of endoplasmic reticulum (ER) stress leads to two different cellular events: cell survival and apoptosis. Drug-induced ER stress or neurotoxicity has been observed as one of the main side effects. However, how ER stress affects cellular signaling cascades leading to neuronal damage is still not well understood. In this study, the toxicological mechanisms of two typical ER stress inducers, tunicamycin (Tm) and dithiothreitol (DTT), were investigated by cell viability, unfolded protein response, apoptosis and proteomic responses in mouse neuro-2a cells. A large portion of differentially expressed proteins (DEPs) that participate in protein synthesis and folding were identified in the Tm treated group, indicating adaptive cellular responses like the unfolded protein response were activated, which was not the case in the DTT treated group. Interestingly, KEGG pathway analysis and validation experiments revealed that proteins involved in proteasomal degradation were down-regulated by both inducers, while proteins involved in ubiquitination were up-regulated by Tm and down-regulated by DTT. A protein responsible for delivering ubiquitinated proteins to the proteasome, the UV excision repair protein RAD23 homolog A (HR23 A), was discovered as a DEP altered by both Tm and DTT. This protein was down-regulated in the Tm treated group and up-regulated in the DTT treated group, which explained the differences we observed in the ubquintination and proteasomal degradation pathways. Autophagy was activated in the Tm treated group, suggesting that it may serve as a compensatory effect to proteasomal degradation. Our work provides new insights into the neurotoxicity generated by various ER stress inducers and the underlying mechanisms.

The TempO-Seq S1500+ platform(s), now available for human, mouse, rat, and zebrafish, measures a discrete number of genes that are representative of biological and pathway co-regulation across the entire genome in a given species. While measurement of these genes alone provides a direct assessment of gene expression activity, extrapolating expression values to the whole transcriptome (~26 000 genes in humans) can estimate measurements of non-measured genes of interest and increases the power of pathway analysis algorithms by using a larger background gene expression space. Here, we use data from primary hepatocytes of 54 donors that were treated with the endoplasmic reticulum (ER) stress inducer tunicamycin and then measured on the human S1500+ platform containing ~3000 representative genes. Measurements for the S1500+ genes were then used to extrapolate expression values for the remaining human transcriptome. As a case study of the improved downstream analysis achieved by extrapolation, the "measured only" and "whole transcriptome" (measured + extrapolated) gene sets were compared. Extrapolation increased the number of significant genes by 49%, bringing to the forefront many that are known to be associated with tunicamycin exposure. The extrapolation procedure also correctly identified established tunicamycin-related functional pathways reflected by coordinated changes in interrelated genes while maintaining the sample variability observed from the "measured only" genes. Extrapolation improved the gene- and pathway-level biological interpretations for a variety of downstream applications, including differential expression analysis, gene set enrichment pathway analysis, DAVID keyword analysis, Ingenuity Pathway Analysis, and NextBio correlated compound analysis. The extrapolated data highlight the role of metabolism/metabolic pathways, the ER, immune response, and the unfolded protein response, each of which are key activities associated with tunicamycin exposure that were unrepresented or underrepresented in one or more of the analyses of the original "measured only" dataset. Furthermore, the inclusion of the extrapolated genes raised "tunicamycin" from third to first upstream regulator in Ingenuity Pathway Analysis and from sixth to second most correlated compound in NextBio analysis. Therefore, our case study suggests an approach to extend and enhance data from the S1500+ platform for improved insight into biological mechanisms and functional outcomes of diseases, drugs, and other perturbations.

Understanding how neural cells handle proteostasis stress in the endoplasmic reticulum (ER) is important to decipher the mechanisms that underlie the cell death associated with neurodegenerative diseases and to design appropriate therapeutic tools. Here we have compared the sensitivity of a human neuroblastoma cell line (SH-SY5H) to the ER stress caused by an inhibitor of protein glycosylation with that observed in human embryonic kidney (HEK-293T) cells. In response to stress, SH-SY5H cells increase the expression of mRNA encoding downstream effectors of ER stress sensors and transcription factors related to the unfolded protein response (the spliced X-box binding protein 1, CCAAT-enhancer-binding protein homologous protein, endoplasmic reticulum-localized DnaJ homologue 4 and asparagine synthetase). Tunicamycin-induced death of SH-SY5H cells was prevented by terminal aromatic substituted butyric or valeric acids, in association with a decrease in the mRNA expression of stress-related factors, and in the accumulation of the ATF4 protein. Interestingly, this decrease in ATF4 protein occurs without modifying the phosphorylation of the translation initiation factor eIF2α. Together, these results show that when short chain phenyl acyl acids alleviate ER stress in SH-SY5H cells their survival is enhanced.

We have assessed amyloid beta protein (Abeta)-induced neurotoxicity, with and without added tunicamycin (TM), an inhibitor of N-glycosylation in the endoplasmic reticulum (ER), in rat organotypic hippocampal slice cultures (OHCs). In the rat OHCs cultured for 3 weeks, there was little neurotoxicity after treatment with Abeta(25-35) (25 microM) alone for 48 h. However, with TM alone, concentration-dependent neuronal death was observed at concentrations between 20 and 80 microg/mL. When amyloid-beta protein was combined with tunicamycin (Abeta+TM), cell death was more acute than with TM alone. Western blot analysis revealed that calpain activity and the active forms of caspase-12 and caspase-3 was increased after exposure to Abeta+TM as compared with exposure to TM alone. In contrast, the levels of glucose regulated protein (GRP)94, GRP78 and C/EBP homologous protein (CHOP) were not changed in the presence of Abeta. Abeta potentiation of TM neurotoxicity was reversibly blocked by S-allyl-L-cysteine (SAC), an organosulfur compound purified from aged garlic extract, and the L-type calcium channel blocker, nifedipine, in a restricted neuronal area of the OHCs. Simultaneously applied SAC also reversed the increases in calpain activity and the active forms of caspase-12 and caspase-3 by Abeta+TM with no change in the increased levels of GRP94, GRP78 and CHOP. These data indicate that Abeta facilitates the calpain-caspase-12-caspase-3 pathway, thus potentiating TM-induced neuronal death in the hippocampus.

The silencing of thyroid-related genes presents difficulties in radioiodine therapy for anaplastic thyroid cancers (ATCs). Tunicamycin (TM), an N-linked glycosylation inhibitor, is an anticancer drug. Herein, we investigated TM-induced restoration of responsiveness to radioiodine therapy in radioiodine refractory ATCs. 125I uptake increased in TM-treated ATC cell lines, including BHT101 and CAL62, which was inhibited by KClO4, a sodium-iodide symporter (NIS) inhibitor. TM upregulated the mRNA expression of iodide-handling genes and the protein expression of NIS. TM blocked pERK1/2 phosphorylation in both cell lines, but AKT (protein kinase B) phosphorylation was only observed in CAL62 cells. The downregulation of glucose transporter 1 protein was confirmed in TM-treated cells, with a significant reduction in 18F-fluorodeoxyglucose (FDG) uptake. A significant reduction in colony-forming ability and marked tumor growth inhibition were observed in the combination group. TM was revealed to possess a novel function as a redifferentiation inducer in ATC as it induces the restoration of iodide-handling gene expression and radioiodine avidity, thereby facilitating effective radioiodine therapy.

Background: Major depressive disorder (MDD) was reported to be associated with endoplasmic reticulum stress (ERS) combined with oxidative stress (OS) (ERS/OS). Here, we aimed to investigate the effects of escitalopram (ESC) on blood-brain barrier (BBB) permeability and ERS/OS-related pathways in brain microvascular endothelial cells (bEnd.3 cells) induced by tunicamycin (TM). Methods: bEnd.3 cells were divided into four groups: control, TM, ESC, and ESC + TM groups. CCK-8 and flow cytometry were used to detect cell survival and apoptosis, respectively. The expression levels of proteins involved in cell permeability and ERS/OS-related pathways were assessed by western blot and immunofluorescence. Malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity were determined by commercial kits. Results: We revealed that TM-induced bEnd.3 cells exhibited remarkably decreased viability and increased apoptosis rate, while ESC treatment reversed these changes. Additionally, TM treatment resulted in markedly increased PERK, GRP78, ATF6, XBP1, and CHOP protein expression levels. On the contrary, the expression of PERK, GRP78, XBP1, and CHOP was obviously reduced in TM-induced bEnd.3 cells after ESC treatment. Moreover, TM significantly reduced the expression of p-eNOS and P-gp and increased the expression of CaMKII and MMP9 compared with the control group. However, ESC reversed these changes in TM-induced bEnd.3 cells. Furthermore, the expression of SOD was significantly decreased, while MDA was significantly increased by TM treatment. In contrast, the expression of SOD was dramatically increased, while MDA was remarkably decreased by ESC treatment. Conclusion: Our results demonstrated that ESC can inhibit ERS/OS and BBB permeability of TM-induced bEnd.3 cells. ESC may alleviate cognitive impairment and prevent comorbidities in MDD patients through ERS/OS.

Efficient mitochondrial Ca2+ uptake takes place at contact points between the ER and mitochondria, and represents a key regulator of many cell functions. In a previous study with HeLa cells, we showed that ER-to-mitochondria Ca2+ transfer increases during the early phase of ER stress induced by tunicamycin as an adaptive response to stimulate mitochondrial bioenergetics. It remains unknown whether other types of stress signals trigger similar responses. Here we observed that rapamycin, which inhibits the nutrient-sensing complex mTORC1, increased ER-mitochondria coupling in HeLa cells to a similar extent as did tunicamycin. Interestingly, although global responses to both stressors were comparable, there were notable differences in the spatial distribution of such changes. While tunicamycin increased organelle proximity primarily in the perinuclear region, rapamycin increased organelle contacts throughout the entire cell. These differences were paralleled by dissimilar alterations in the distribution of regulatory proteins of the ER-mitochondria interface, heterogeneities in mitochondrial Ca2+ uptake, and the formation of domains within the mitochondrial network with varying mitochondrial transmembrane potential. Collectively, these data suggest that while increasing ER-mitochondria coupling appears to represent a general response to cell stress, the intracellular distribution of the associated responses needs to be tailored to meet specific cellular requirements.