The bioenergetics of traumatic brain injury and its long-term impact for brain plasticity and function.

Mitochondria provide the energy to keep cells alive and functioning and they have the capacity to influence highly complex molecular events. Mitochondria are essential to maintain cellular energy homeostasis that determines the course of neurological disorders, including traumatic brain injury (TBI). Various aspects of mitochondria metabolism such as autophagy can have long-term consequences for brain function and plasticity. In turn, mitochondria bioenergetics can impinge on molecular events associated with epigenetic modifications of DNA, which can extend cellular memory for a long time. Mitochondrial dysfunction leads to pathological manifestations such as oxidative stress, inflammation, and calcium imbalance that threaten brain plasticity and function. Hence, targeting mitochondrial function may have great potential to lessen the outcomes of TBI.

Salubrinal alleviates cartilage degradation in a rabbit temporomandibular joint osteoarthritis model.

OBJECTIVE: To investigate and explain the beneficial effects of local intra-articular injection of Salubrinal on temporomandibular joint osteoarthritis (TMJOA) using a rabbit model of anterior disc displacement (ADD). METHODS: Rabbits were divided and subjected to surgical ADD. Salubrinal was administered by intra-articular injection in the TMJ every other day for 2 and 4 weeks after operation. Histological examination and TUNEL staining were then performed. Immunohistochemistry, quantitative real-time PCR, and Western blot analysis were employed to evaluate the expression of endoplasmic reticulum (ER) stress-related markers, catabolic factors, extracellular matrix proteins, inflammatory factors, and nuclear factor-kappa B (NF-κB) activation. RESULTS: In the ADD groups, we found that Salubrinal partly reversed condylar cartilage deterioration according to the histological analysis. Salubrinal reduced chondrocytes apoptosis while increased matrix components including Collagen II and Aggrecan. Meanwhile, Salubrinal downregulated the catabolic expression of MMP13, ADAMTS5, VEGF, TNF-α, and IL-1ß. We also observed that Salubrinal inhibited ER stress activation by reducing the expression of GRP78, CHOP, ATF4, and Caspase-12. Additionally, Salubrinal suppressed the phosphorylation of NF-κB. CONCLUSION: These results indicate that Salubrinal alleviates cartilage degradation following ADD, suggesting that intra-articular injection of Salubrinal is a potential therapeutic approach for preventing TMJOA.

The Preventive Effects of Salubrinal against Pyrethroid-Induced Disruption of Adult Hippocampal Neurogenesis in Mice.

Environmental factors, including pesticide exposure, have been identified as substantial contributors to neurodegeneration and cognitive impairments. Previously, we demonstrated that repeated exposure to deltamethrin induces endoplasmic reticulum (ER) stress, reduces hippocampal neurogenesis, and impairs cognition in adult mice. Here, we investigated the potential relationship between ER stress and hippocampal neurogenesis following exposure to deltamethrin, utilizing both pharmacological and genetic approaches. To investigate whether ER stress is associated with inhibition of neurogenesis, mice were given two intraperitoneal injections of eIf2α inhibitor salubrinal (1 mg/kg) at 24 h and 30 min prior to the oral administration of deltamethrin (3 mg/kg). Salubrinal prevented hippocampal ER stress, as indicated by decreased levels of C/EBP-homologous protein (CHOP) and transcription factor 4 (ATF4) and attenuated deltamethrin-induced reductions in BrdU-, Ki-67-, and DCX-positive cells in the dentate gyrus (DG) of the hippocampus. To further explore the relationship between ER stress and adult neurogenesis, we used caspase-12 knockout (KO) mice. The caspase-12 KO mice exhibited significant protection against deltamethrin-induced reduction of BrdU-, Ki-67-, and DCX-positive cells in the hippocampus. In addition, deltamethrin exposure led to a notable upregulation of CHOP and caspase-12 expression in a significant portion of BrdU- and Ki-67-positive cells in WT mice. Conversely, both salubrinal-treated mice and caspase-12 KO mice exhibited a considerably lower number of CHOP-positive cells in the hippocampus. Together, these findings suggest that exposure to the insecticide deltamethrin triggers ER stress-mediated suppression of adult hippocampal neurogenesis, which may subsequently contribute to learning and memory deficits in mice.

Salubrinal-mediated activation of eIF2α signaling improves oxidative stress-induced BMSCs senescence and senile osteoporosis.

Bone cells of various lineages become senescent in bone microenvironment. Senotherapies that clear the senescent bone cells improve bone microarchitecture of aged bones. However, the mechanisms underlie for the formation and maintenance of senescent bone cells are largely unknown. Here, we focus on the relationship between endoplasmic reticulum stress (ER stress)-activated unfolded protein response (UPR) signaling and cellular senescence of bone marrow mesenchymal stem cells (BMSCs). The PKR-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2 α(eIF2α) signaling branch was specifically activated and tightly regulated in senescent BMSCs induced by hydrogen peroxide (H2O2). However, blocking PERK-eIF2α signaling with AMG'44 could not reverse the cellular senescence phenotype of senescent BMSCs. Treated the senescent cells with salubrinal, an inhibitor for dephosphorylation of eIF2α, decreased SA-ß-Gal positive cells and the expression of markers for cellular senescence. Moreover, salubrinal enhanced the apoptosis of senescent BMSCs and upregulated expression of Chop and BIM. Furthermore, salubrinal treatment significantly improved the osteogenesis capacity of senescent BMSCs as reflected by the increase of Alp, Runx2 and Osteocalcin, the formation of Alp-positive staining cells and matrix mineralization. Salubrinal administration results in significant recovery in the bone microarchitecture of senile SAMP6 mice. Taken together, our data reveal an undefined role of PERK-eIF2α signaling in the maintenance of cellular senescent phenotype in BMSCs. The activation of eIF2α signaling with salubrinal is helpful for the clearance of senescent BMSCs and the improvement of bone integrity of aged mice.

Salubrinal protects against inflammatory response in macrophage and attenuates psoriasiform skin inflammation by antagonizing NF-κB signaling pathway.

Psoriasiform skin inflammation is the common chronic skin inflammatory disease with no effective clinical therapy. Salubrinal is a multifunctional molecule playing a protective role in several conditions. Recently, studies have reported that Salubrinal is a potential therapeutic agent for inflammatory diseases. However, the protective role of Salubrinal in psoriasis-like skin inflammation remains unknown. In this article, imiquimod (IMQ)-induced psoriasis models were established in wild-type mice to explore the role of Salubrinal in the development of psoriasis. As a result, the IMQ-induced mouse models exhibited typical skin inflammation, which was alleviated by the administration of Salubrinal. Furthermore, RAW264.7 macrophage was stimulated with Lipopolysaccharide(LPS) in the presence or absence of Salubrinal. LPS stimulation elevated the expression of various inflammatory biomarkers, while the administration of Salubrinal abolished the function of LPS in RAW264.7 macrophages. In addition, the activation of the nuclear factor-kappa B (NF-κB) signaling pathway in both the LPS-stimulated RAW264.7 macrophage and psoriasis mouse models was antagonized by the administration of Salubrinal. Collectively, Salubrinal might be considered as a promising therapeutic agent for psoriasis-like skin inflammation.

The beneficial effect of salubrinal on neuroinflammation and neuronal loss in intranigral LPS-induced hemi-Parkinson disease model in rats.

OBJECTIVE: Endoplasmic reticulum stress (ERS) and neuroinflammation are triggers for neurodegenerative disorders. Salubrinal is a selective inhibitor of protein phosphatase 1 (PP1) complex involving dephosphorylation of phosphorylated eukaryotic initiation factor-2α (eIF2α), the key crucial pathway in the ERS. Therefore, this study assessed the effects of inhibition of the ERS with salubrinal in the intranigral hemi-Parkinson disease (PD) model. MATERIALS AND METHODS: Animals were treated with salubrinal for one week after the PD model was created by intranigral lipopolysaccharide (LPS) administration. Apomorphine-induced rotation, rotarod, cylinder, and pole tests were performed to evaluate behavioral changes. Proinflammatory cytokines and the expression level of the dual specificity protein phosphatase 2 (DUSP2), PP1, and p-eIF2α were evaluated. Nigral expression of inducible nitric oxide synthase (iNOS), nuclear factor kappaB (Nf-κB), and cyclooxygenase (COX)-2 was determined. Finally, tyrosine hydroxylase and caspase-3/ caspase-9 expressions were assessed by immunohistochemistry. RESULTS: Salubrinal reduced the motor impairments and dopamine-related behavioral deficiencies caused by the LPS. Salubrinal attenuated the LPS-induced increased levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, and salubrinal rescued the loss of TH expression and dopamine levels and prevented the caspase-3/9 increase in the substantial nigra (SN). LPS potently increased iNOS, Nf-κB, and COX-2 expression, but this effect was reduced after salubrinal treatment. Additionally, salubrinal attenuated the LPS-induced PP1 and DUSP2 increase. CONCLUSION: Our results reveal that salubrinal is attenuating several inflammatory mediators and thereby decreased the inflammatory effects of LPS in the neurons of the SN. Together this results in increased cellular survival and maintained integrity of SN. Taken together our data show the beneficial effects of inhibition of ERS to restrict neuroinflammatory progression and neuronal loss in a PD model.

Salubrinal Alleviates Collagen-Induced Arthritis through Promoting P65 Degradation in Osteoclastogenesis.

Rheumatoid arthritis (RA) is a complex systemic autoimmune disorder that primarily involves joints, further affects the life quality of patients, and has increased mortality. The pathogenesis of RA involves multiple pathways, resulting in some patients showing resistance to the existing drugs. Salubrinal is a small molecule compound that has recently been shown to exert multiple beneficial effects on bone tissue. However, the effect of Salubrinal in RA has not been clearly confirmed. Hence, we induced collagen-induced arthritis (CIA) in DBA/1J mice and found that Salubrinal treatment decreased the clinical score of CIA mice, inhibiting joint damage and bone destruction. Furthermore, Salubrinal treatment downregulated osteoclast number in knee joint of CIA in mice, and suppressed bone marrow-derived osteoclast formation and function, downregulated osteoclast-related gene expression. Moreover, Salubrinal treatment inhibited RANKL-induced NF-κB signaling pathway, and promoted P65 degradation through the ubiquitin-proteasome system, further restrained RANKL-induced osteoclastogenesis. This study explains the mechanism by which Salubrinal ameliorates arthritis of CIA in mice, indicating that Salubrinal may be a potential drug for RA, and expands the potential uses of Salubrinal in the treatment of bone destruction-related diseases.

Transient upregulation of translational efficiency in prodromal and early symptomatic Tg2576 mice contributes to Aß pathology.

TG2576 mice show highest levels of the full length mutant Swedish Human Amyloid Precursor Protein (APPKM670/671LN) during prodromal and early sympotomatic stages. Interestingly, this occurs in association with the unbalanced expression of two of its RNA Binding proteins (RBPs) opposite regulators, the Fragile-X Mental Retardation Protein (FMRP) and the heteronuclear Ribonucleoprotein C (hnRNP C). Whether an augmentation in overall translational efficiency also contributes to the elevation of APP levels at those early developmental stages is currently unknown. We investigated this possibility by performing a longitudinal polyribosome profiling analysis of APP mRNA and protein in total hippocampal extracts from Tg2576 mice. Results showed that protein polysomal signals were exclusively detected in pre-symptomatic (1 months) and early symptomatic (3 months) mutant mice. Differently, hAPP mRNA polysomal signals were detected at any age, but a peak of expression was found when mice were 3-month old. Consistent with an early but transient rise of translational efficiency, the phosphorylated form of the initial translation factor eIF2α (p-eIF2α) was reduced at pre-symptomatic and early symptomatic stages, whereas it was increased at the fully symptomatic stage. Pharmacological downregulation of overall translation in early symptomatic mutants was then found to reduce hippocampal levels of full length APP, Aßspecies, BACE1 and Caspase-3, to rescue predominant LTD at hippocampal synapses, to revert dendritic spine loss and memory alterations, and to reinstate memory-induced c-fosactivation. Altogether, our findings demonstrate that overall translation is upregulated in prodromal and early symptomatic Tg2576 mice, and that restoring proper translational control at the onset of AD-like symptoms blocks the emergence of the AD-like phenotype.

Impact of endoplasmic reticulum stress on oocyte aging mechanisms.

Endoplasmic reticulum (ER) stress is associated with several aging-related diseases; however, the mechanism underlying age-related deterioration of oocyte quality is unclear. Here, we used post-ovulatory, in vivo aged mouse oocytes as a model. Super-ovulated oocytes harvested from the oviduct at 14 h and 20 h post-hCG injection were designated as 'fresh' and 'aged', respectively. Embryo development following IVF was compared between fresh, aged and ER stress-induced oocytes. Expression of the ER stress marker GRP78 was examined at each stage. To evaluate the effect of salubrinal, an ER stress suppressor, on embryo development following IVF, expression levels of GRP78 and phospho-eukaryotic initiation factor 2 alpha were compared between aged and salubrinal-treated aged oocytes. Embryo transfer of salubrinal-treated aged oocytes was performed to examine the safety of salubrinal. Similar to aged oocytes, ER stress-induced oocytes showed lower fertilization rates and poor embryo development. Following IVF, expression of GRP78 decreased with embryo development. GRP78 expression was significantly higher in aged oocytes than in fresh oocytes. Salubrinal lowered GRP78 levels and improved embryo development. No adverse effect of salubrinal treatment was found on the birth weight of pups or on organogenesis in mice. The limitation of this study was that protein kinase-like ER kinase was the only ER stress pathway examined; the role of IRE1 and ATF6 pathways was not considered. Nevertheless, salubrinal can significantly improve embryo development in in vivo aged oocytes undergoing ER stress. Hence, regulation of ER stress might represent a promising therapeutic strategy to overcome poor oocyte quality.

Endoplasmic reticulum stress signaling in cancer and neurodegenerative disorders: Tools and strategies to understand its complexity.

The endoplasmic reticulum (ER) comprises a network of tubules and vesicles that constitutes the largest organelle of the eukaryotic cell. Being the location where most proteins are synthesized and folded, it is crucial for the upkeep of cellular homeostasis. Disturbed ER homeostasis triggers the activation of a conserved molecular machinery, termed the unfolded protein response (UPR), that comprises three major signaling branches, initiated by the protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1) and the activating transcription factor 6 (ATF6). Given the impact of this intricate signaling network upon an extensive list of cellular processes, including protein turnover and autophagy, ER stress is involved in the onset and progression of multiple diseases, including cancer and neurodegenerative disorders. There is, for this reason, an increasing number of publications focused on characterizing and/or modulating ER stress, which have resulted in a wide array of techniques employed to study ER-related molecular events. This review aims to sum up the essentials on the current knowledge of the molecular biology of endoplasmic reticulum stress, while highlighting the available tools used in studies of this nature.

Effects of salubrinal on ER stress in an experimental model of polycystic ovary syndrome.

This study aimed to investigate the role of endoplasmic reticulum (ER) stress in polycystic ovary syndrome (PCOS) and the effect of salubrinal (SAL) on this role. Animals were divided into four groups as control, PCOS, PCOS+SAL and SAL. Weights and serum testosterone levels were increased in the PCOS group while serum LH and ATF4 expressions were decreased. Morphometrically, number of follicles with a diameter between 150 and 300 µm were declined and number of follicles larger than 300 µm as well as the percentage of cystic follicles (CFs) were increased. Immunoreactivities of GRP78 and p-eIF2α were decreased, whereas oxidative stress (OS) dependent PAR expression was increased. Ultrastructurally, the PCOS group had no ER enlargement which was observed in the control group, while there were mitochondrial damage in granulosa cells (GCs). Elevated OS levels did not induce but rather decreased ER stress in GCs, and SAL injection in the PCOS model was ineffective on searched parameters. Since ER stress plays roles in certain physiological processes, we suggest that inhibitors of ER stress may not be always useful for reproductive tissues.

Salubrinal, a novel inhibitor of eIF-2α dephosphorylation, promotes erythropoiesis at early stage targeted by ufmylation pathway.

Ufmylation was proved to play a crucial role in hematopoietic stem cell (HSC) survival and erythroid differentiation, ufmylation deficiency induces acute anemia and lethality of embryos and adults in mouse models. To screen some compounds to rescue phenotypes induced by gene deletion, in this study, we used DDRGK1F/F ; CreERT2 conditional knockout mice, DDRGK1F/F ; CreERT2 bone marrow (BM) and fetal liver cells (FL), Uba5, and DDRGK1 knockdown human CD34 cell in vivo and in vitro, we found salubrinal, a novel inhibitor of eIF-2α dephosphorylation, promoted erythropoiesis at early stage, and partly rescued the acute anemia induce by DDRGK1 deficiency through upregulation of ufmylation and erythroid transcription factors. In phenylhydrazine (PHZ)-induced hemolytic anemia mice, interestingly, salubrinal could significantly improve hemocrit and red blood cell (RBC) indices of the mice treated with PHZ via upregulation of ufmylation. Its novel function was verified to attenuate unfolded protein response (UPR) and cell death programs, and to keep endoplasmic reticulum (ER) homeostasis in HSCs. Taken together results, it suggested that salubrinal may be a promising antianemic agent targeted by ufmylation.

Post-ischemic salubrinal administration reduces necroptosis in a rat model of global cerebral ischemia.

Ischemic stroke is one of the most important causes of death and disability worldwide. Subroutines underlying cell death after stroke are largely unknown despite their importance in the design of novel therapies for this pathology. Necroptosis, a recently described form of regulated cell death, has been related with inflammation and, in some models, with endoplasmic reticulum (ER) stress. We hypothesize that alleviation of ER stress following a salubrinal treatment will reduce the ischemic-dependent necroptosis. To probe the hypothesis, we measured, at 48 and 72 h after transient global cerebral ischemia in rat, in cerebral cortex and cornu ammonis 1, the main hallmarks of necroptosis: mRNA levels and phosphorylation of mixed lineage kinase domain like pseudokinase as well as receptor interacting serine/threonine protein kinase 3, along the years 2017-2018. Selective neuronal loss after 7 days of the ischemic insult, and other markers related with the inflammatory response were also measured. This study shows that necroptosis in cerebral cortex can be detected after 72 h of the insult and seems to be elicited before 48 h of reperfusion. The type of necroptosis here observed seems to be tumor necrosis factor receptor 1 independent. Necroptotic response is less evident in the cornu ammonis 1 hippocampal area than in cerebral cortex. The treatment with salubrinal administered 1 and 24 h after the ischemia, decreased the necroptotic marker levels and reduced the areas of selective neuronal loss, supporting the presence of ischemic-dependent necroptosis, and the notion that ER stress is involved in the necroptotic response. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Salubrinal offers neuroprotection through suppressing endoplasmic reticulum stress, autophagy and apoptosis in a mouse traumatic brain injury model.

Traumatic brain injury (TBI) is a complex injury that can cause severe disabilities and even death. TBI can induce secondary injury cascades, including but not limited to endoplasmic reticulum (ER) stress, apoptosis and autophagy. Although the investigators has previously shown that salubrinal, the selective phosphatase inhibitor of p-eIF2α, ameliorated neurologic deficits in murine TBI model, the neuroprotective mechanisms of salubrinal need further research to warrant the preclinical value. This study was undertaken to characterize the effects of salubrinal on cell death and neurological outcomes following TBI in mice and the potential mechanisms. In the current study, ER stress-related proteins including p-eIF2α, GRP78 and CHOP showed peak expressions both in the cortex and hippocampus from day 2 to day 3 after TBI, indicating ER stress was activated in our TBI model. Immunofluorescence staining showed that CHOP co-located NeuN-positive neuron, GFAP-positive astrocyte, Iba-1-positive microglia, CD31-positive vascular endothelial cell and PDGFR-ß-positive pericyte in the cortex on day 2 after TBI, and these cells mentioned above constitute the neurovascular unit (NVU). We also found TBI-induced plasmalemma permeability, motor dysfunction, spatial learning and memory deficits and brain lesion volume were alleviated by continuous intraperitoneal administration of salubrinal post TBI. To investigate the underlying mechanisms further, we determined that salubrinal suppressed the expression of ER stress, autophagy and apoptosis related proteins on day 2 after TBI. In addition, salubrinal administration decreased the number of CHOP+/TUNEL+ and CHOP+/LC3+ cells on day 2 after TBI, detected by immunofluorescence. In conclusion, these data imply that salubrinal treatment improves morphological and functional outcomes caused by TBI in mice and these neuroprotective effects may be associated with inhibiting apoptosis, at least in part by suppressing ER stress-autophagy pathway.

Inhibition of the dephosphorylation of eukaryotic initiation factor 2α ameliorates murine experimental pancreatitis.

BACKGROUND: Endoplasmic reticulum (ER) stress in the pancreas is closely associated with the development of acute pancreatitis. However, the role of the protein kinase RNA-like ER kinase (PERK) in this disease is not fully understood. We investigated whether an inhibitor of the dephosphorylation of eukaryotic initiation factor 2α, salubrinal, could improve murine experimental pancreatitis through the amelioration of ER stress. METHODS: Acute pancreatitis was induced by the intraperitoneal administration of cerulein (50 µg/kg) six times at 1-h intervals followed by lipopolysaccharide (10 mg/kg). Salubrinal was administered intraperitoneally immediately after lipopolysaccharide injection and 3 h later. Mice were sacrificed 24 h after the first injection of cerulein, and serum amylase and proinflammatory cytokines were measured. The severity of pancreatitis was evaluated histologically using a scoring system. The expression levels of ER stress-related proteins were evaluated by Western blotting. RESULTS: The administration of salubrinal significantly attenuated the increase in serum amylase levels and improved histologically assessed pancreatitis. The serum levels of proinflammatory cytokines were significantly suppressed in salubrinal-treated mice, as was the expression of glucose-regulated protein 78, CCAAT/enhancer-binding protein homologous protein, and cleaved caspase-3. CONCLUSIONS: The amelioration of ER stress through augmentation of the PERK-signaling pathway may be a therapeutic target for the treatment of acute pancreatitis.

Hispidulin induces ER stress-mediated apoptosis in human hepatocellular carcinoma cells in vitro and in vivo by activating AMPK signaling pathway.

Hispidulin (4',5,7-trihydroxy-6-methoxyflavone) is a phenolic flavonoid isolated from the medicinal plant S. involucrata, which exhibits anti-neoplastic activity against several types of cancer. However, the mechanism underlying its anti-cancer activity against hepatocellular carcinoma (HCC) has not been fully elucidated. In this study, we investigated whether and how hispidulin-induced apoptosis of human HCC cells in vitro and in vivo. We showed that hispidulin (10, 20 µmol/L) dose-dependently inhibited cell growth and promoted apoptosis through mitochondrial apoptosis pathway in human HCC SMMC7721 cells and Huh7 cells. More importantly, we revealed that its pro-apoptotic effects depended on endoplasmic reticulum stress (ERS) and unfolded protein response (UPR), as pretreatment with salubrinal, a selective ERS inhibitor, or shRNA targeting a UPR protein CHOP effectively abrogated hispidulin-induced cell apoptosis. Furthermore, we showed that hispidulin-induced apoptosis was mediated by activation of AMPK/mTOR signaling pathway as pretreatment with Compound C, an AMPK inhibitor, or AMPK-targeting siRNA reversed the pro-apoptotic effect of hispidulin. In HCC xenograft nude mice, administration of hispidulin (25, 50 mg/kg every day, ip, for 27 days) dose-dependently suppressed the tumor growth, accompanied by inducing ERS and apoptosis in tumor tissue. Taken together, our results demonstrate that hispidulin induces ERS-mediated apoptosis in HCC cells via activating the AMPK/mTOR pathway. This study provides new insights into the anti-tumor activity of hispidulin in HCC.

Differential Contribution of Calcium-Activated Proteases and ER-Stress in Three Mouse Models of Retinitis Pigmentosa Expressing P23H Mutant RHO.

Autosomal dominant retinitis pigmentosa (adRP) is mainly caused by mutations responsible for rhodopsin (RHO) misfolding. Although it was previously proved that unfolded RHO is retained into the endoplasmatic reticulum (ER) eliciting ER-stress, consequent mechanisms underlying photoreceptor degeneration need to be further clarified. Several animal models of RHO mutants have been developed for this purpose and for development of neuroprotective treatments. Here, we compared two of the most used models of adRP, the P23H mutant RHO transgenic and knock-in mouse models, in order to define which are their limits and potentials. Although they were largely used, the differences on the activation of the cell death pathways occurring in these two models still remain to be fully characterized. We present data proving that activation of calpains is a mechanism of cell death shared by both models and that molecules targeting calpains are neuroprotective. Conversely, the role of ER-stress contribution to cell death appears to be divergent and remains controversial.

Activated Integrated Stress Response Induced by Salubrinal Promotes Cisplatin Resistance in Human Gastric Cancer Cells via Enhanced xCT Expression and Glutathione Biosynthesis.

The integrated stress response (ISR) pathway is essential for adaption of various stresses and is related to mitochondrion-to-nucleus communication. Mitochondrial dysfunction-induced reactive oxygen species (ROS) was demonstrated to activate general control nonderepressible 2 (GCN2)⁻eukaryotic translation initiation factor 2α (eIF2α)⁻activating transcription factor-4 (ATF4) pathway-mediated cisplatin resistance of human gastric cancer cells. However, whether or how ISR activation per se could enhance chemoresistance remains unclear. In this study, we used eIF2α phosphatase inhibitor salubrinal to activate the ISR pathway and found that salubrinal reduced susceptibility to cisplatin. Moreover, salubrinal up-regulated ATF4-modulated gene expression, and knockdown of ATF4 attenuated salubrinal-induced drug resistance, suggesting that ATF4-modulated genes contribute to the process. The ATF4-modulated genes, xCT (a cystine/glutamate anti-transporter), tribbles-related protein 3 (TRB3), heme oxygenase 1 (HO-1), and phosphoenolpyruvate carboxykinase 2 (PCK2), were associated with a poorer prognosis for gastric cancer patients. By silencing individual genes, we found that xCT, but not TRB3, HO-1, or PCK2, is responsible for salubrinal-induced cisplatin resistance. In addition, salubrinal increased intracellular glutathione (GSH) and decreased cisplatin-induced lipid peroxidation. Salubrinal-induced cisplatin resistance was attenuated by inhibition of xCT and GSH biosynthesis. In conclusion, our results suggest that ISR activation by salubrinal up-regulates ATF4-modulated gene expression, increases GSH synthesis, and decreases cisplatin-induced oxidative damage, which contribute to cisplatin resistance in gastric cancer cells.

Salubrinal protects human skin fibroblasts against UVB-induced cell death by blocking endoplasmic reticulum (ER) stress and regulating calcium homeostasis.

The role of UVB in skin photo damages has been widely reported. Overexposure to UVB will induce severe DNA damages in epidermal cells and cause most cytotoxic symptoms. In the present study, we tested the potential activity of salubrinal, a selective inhibitor of Eukaryotic Initiation Factor 2 (eIF2) -alpha phosphatase, against UV-induced skin cell damages. We first exposed human fibroblasts to UVB radiation and evaluated the cytosolic Ca2+ level as well as the induction of ER stress. We found that UVB radiation induced the depletion of ER Ca2+ and increased the expression of ER stress marker including phosphorylated PERK, CHOP, and phosphorylated IRE1α. We then determined the effects of salubrinal in skin cell death induced by UVB radiation. We observed that cells pre-treated with salubrinal had a higher survival rate compared to cells treated with UVB alone. Pre-treatment with salubrinal successfully re-established the ER function and Ca2+ homeostasis. Our results suggest that salubrinal can be a potential therapeutic agents used in preventing photoaging and photo damages.

Reversal of aberrant PI3K/Akt signaling by Salubrinal in a GalT-deficient mouse model.

Classic Galactosemia is an autosomal recessive disorder caused by deleterious mutations in the GALT gene, which encodes galactose-1 phosphate uridylyltransferase enzyme (GALT: EC 2.7.7.12). Recent studies of primary skin fibroblasts isolated from the GalT-deficient mice demonstrated a slower growth rate, a higher level of endoplasmic reticulum (ER) stress, and down-regulation of the Phosphoinositide 3 kinase/Protein kinase B (PI3K/Akt) signaling pathway. In this study, we compared the expression levels of the PI3K/Akt signaling pathway in normal and GalT-deficient mouse tissues. In mutant mouse ovaries, phospho-Akt [pAkt (Ser473)] and pGsk3ß were reduced by 62.5% and 93.5%, respectively (p<0.05 versus normal controls). In mutant cerebella, pAkt (Ser473) and pGsk3ß were reduced by 62%, 50%, respectively (p<0.05). To assess the role of ER stress in the down-regulation of PI3K/Akt signaling, we examined if administration of Salubrinal, a chemical compound that alleviates ER stress, to GalT-deficient fibroblasts and animals could normalize the pathway. Our results demonstrated that Salubrinal effectively reversed the down-regulated PI3K/Akt signaling pathway in the mutant cells and animals to levels close to those of their normal counterparts. Moreover, we revealed that Salubrinal can significantly slow down the loss of Purkinje cells in the cerebella, as well as the premature loss of primordial ovarian follicles in young mutant mice. These results open the door for a new therapeutic approach for the patients with Classic Galactosemia.