TULP1 missense mutations cause variable retinal phenotypes and activation of the endoplasmic reticulum unfolded protein response pathway.

Domain interaction, a structural property of apolipoprotein E4 (apoE4), is predicted to contribute to the association of apoE4 with Alzheimer disease. Arg-61 apoE mice, a gene-targeted mouse model specific for domain interaction, have lower brain apoE levels and synaptic, functional, and cognitive deficits. We hypothesized that domain interaction elicits an endoplasmic reticulum (ER) stress in astrocytes and an unfolded protein response that targets Arg-61 apoE for degradation. Primary Arg-61 apoE astrocytes had less intracellular apoE than wild-type astrocytes, and unfolded protein response markers OASIS (old astrocyte specifically induced substance), ATF4, and XBP-1 and downstream effectors were up-regulated. ER stress appears to cause global astrocyte dysfunction as glucose uptake was decreased in Arg-61 apoE astrocytes, and astrocyte-conditioned medium promoted neurite outgrowth less efficiently than wild-type medium in Neuro-2a cell cultures. We showed age-dependent up-regulation of brain OASIS levels and processing in Arg-61 apoE mice. ER stress and astrocyte dysfunction represent a new paradigm underlying the association of apoE4 with neurodegeneration.

The endoplasmic reticulum (ER) is a dynamic and multifunctional organelle responsible for protein biosynthesis, folding, assembly and modifications. Loss of protein folding regulation, which leads to unfolded or misfolded proteins accumulation inside the ER lumen, drives ER stress (ERS) and unfolded protein response (UPR) activation. During aging, there is a decline in the ability of the cell to handle protein folding, accumulation and aggregation, and the function of UPR is compromised. There is a progressive failure of the chaperoning systems and a decline in many of its components, so that the UPR activation cannot rescue the ERS. Physical activity has been proposed as a powerful tool against aged-related diseases, which are linked to ERS. Interventional studies have demonstrated that regular exercise is able to decrease oxidative stress and inflammation and reverse mitochondrial and ER dysfunctions. Exercise-induced metabolic stress could activate the UPR since muscle contraction is directly involved in its activation, mediating exercise-induced adaptation responses. In fact, regular moderate-intensity exercise-induced ERS acts as a protective mechanism against current and future stressors. However, biological responses vary according to exercise intensity and therefore induce different degrees of ERS and UPR activation. This article reviews the effects of aging and exercise on ERS and UPR, also analyzing possible changes induced by different types of exercise in elderly subjects.

Endoplasmic reticulum (ER) stress promotes placental dysmorphogenesis and is associated with poor pregnancy outcomes. We show that unfolded protein response signalling pathways located in the ER drive differentiation of mouse trophoblast stem cells into trophoblast subtypes involved in development of the placental labyrinth zone and trophoblast invasion. In a mouse model of chronic ER stress (Eif2s1tm1RjK ), higher ER stress in homozygous blastocysts is accompanied by reduced trophectoderm cell number and developmental delay and also is associated with an increased incidence of early pregnancy loss. Administration of the chemical chaperone, tauroursodeoxycholic acid, to Eif2s1+/tm1RjK heterozygous females during pregnancy alleviated ER stress in the mutant placenta, restored normal trophoblast populations and reduced the frequency of early pregnancy loss. Our results suggest that alleviation of intrauterine ER stress could provide a potential therapeutic target to improve pregnancy outcome in women with pre-gestational metabolic or gynaecological conditions. Women with pre-gestational health conditions (e.g. obesity, diabetes) or gynaecological problems (e.g. endometriosis) are at increased risk of adverse pregnancy outcomes including miscarriage, pre-eclampsia and fetal growth restriction. Increasing evidence suggests that unfavourable intrauterine conditions leading to poor implantation and/or defective placentation are a possible causative factor. The endoplasmic reticulum (ER) unfolded protein response (UPRER ) signalling pathways are a convergence point of various physiological stress stimuli that can be triggered by an unfavourable intrauterine environment. Therefore, we explored the impact of ER stress on mouse trophoblast differentiation in vitro, mouse blastocyst formation and early placenta development in the Eif2s1tm1RjK mutant mouse model of chronic ER stress. Chemically-manipulated ER stress or activation of UPRER pathways in a mouse trophoblast stem cell line promoted lineage-specific differentiation. Co-treatment with specific UPRER pathway inhibitors rescued this effect. Although the inner cell mass was unaffected, the trophectoderm of homozygous Eif2s1tm1RjK blastocysts exhibited ER stress associated with a reduced cell number. Furthermore, one-third of Eif2s1tm1RjK homozygous blastocysts exhibited severe developmental defects. We have previously reported a reduced trophoblast population and premature trophoblast differentiation in Eif2s1tm1RjK homozygous placentas at mid-gestation. Here, we demonstrate that treatment of Eif2s1+/tm1RjK heterozygous pregnant females with the chemical chaperone tauroursodeoxycholic acid alleviated ER stress, restored the trophoblast population and reduced the frequency of embryonic lethality. Our data suggest that therapeutic targeting of ER stress may improve pregnancy outcome in women with pre-gestational metabolic or gynaecological conditions.

Multiple cellular stress responses have been implicated in chronic diseases such as obesity, diabetes, cardiovascular, and inflammatory bowel diseases. Even though phenotypically different, chronic diseases share cellular stress signaling pathways, in particular endoplasmic reticulum (ER) unfolded protein response (UPR). The purpose of the ER UPR is to restore ER homeostasis after challenges of the ER function. Among the triggers of ER UPR are changes in the redox status, elevated protein synthesis, accumulation of unfolded or misfolded proteins, energy deficiency and glucose deprivation, cholesterol depletion, and microbial signals. Numerous mouse models have been used to characterize the contribution of ER UPR to several pathologies, and ER UPR-associated signaling has also been demonstrated to be relevant in humans. Additionally, recent evidence suggests that the ER UPR is interrelated with metabolic and inflammatory pathways, autophagy, apoptosis, and mitochondrial stress signaling. Furthermore, microbial as well as nutrient sensing is integrated into the ER-associated signaling network. The data discussed in the present review highlight the interaction of ER UPR with inflammatory pathways, metabolic processes and mitochondrial function, and their interrelation in the context of chronic diseases.

Endoplasmic reticulum stress in Alzheimer's disease: Molecular mechanisms and therapeutic prospects

Klotho is a transmembrane protein, which can be shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. We previously identified klotho as a breast and pancreatic tumor suppressor and as an inhibitor of the IGF-1 pathway. Recent data indicate klotho as a tumor suppressor in an array of malignancies, including colorectal cancer (CRC). We aimed to decipher the role of klotho as a tumor suppressor in CRC. Analysis of a public database (Oncomine) indicated reduced expression of klotho in CRC. Two models were employed to study the effect of klotho in vivo. Using the azoxymethane inflammation model, we discovered that klotho inhibited colon polyp formation. Using an orthotopic model consisting of direct injection of MC38 cells to the mice colons, we found that klotho inhibited colonic obstruction and tumor formation. Similarly, klotho inhibited colony formation and proliferation of CRC cell lines. Aberrant activation of the canonical Wnt pathway is implicated in CRC development and progression. β-catenin, the key effector of this pathway, functions with T-cell factor/lymphoid-enhancer-factor (TCF/LEF) to activate expression of Wnt target genes. While we did not see an effect of klotho on the IGF-1 pathway, it reduced Wnt3A and β-catenin levels as measured by Western blotting, and inhibited TFC/LEF transcriptional activation in luciferase assay. As transcriptional inhibition was abrogated by a constitutively active β-catenin, we suspected that klotho inhibited the pathway upstream of β-catenin. Indeed, co-immunoprecipitation analyses indicated direct interaction between klotho and Wnt3A. Yet, the inhibitory effect of klotho on CRC cell colony formation was only partially rescued by a constitutively active β-catenin. These data indicate on additional mechanisms involved in the TS activity of klotho. Thus, we conducted an RNA expression array and observed involvement of klotho in endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Further studies showed that klotho induced elevation in XBP1 RNA levels, GRP78 protein levels, and eIF2α phosphorylation, all indicative of UPR regulation. Importantly, pharmacologic inhibition of ER stress and UPR overcame the growth suppression effect of klotho in CRC cells. Our data indicate klotho as a potent tumor suppressor in CRC, and suggest its role at very early stages of tumor formation, already at polyp formation. Our data also indicate, for the first time, klotho as a regulator of ER stress and UPR in cancer. These findings may pave the way for the development of novel therapeutic strategies based on enhancement of UPR. Citation Format: Tal Etan, Tammi Rubinstein, Tami Rubinek, Shiri Shahmoon, Chen Varol, Ehud Zigmond, Rina Rosin-Arbesfeld, Iris Barshack, Ido Wolf. Upregulation of unfolded protein response (UPR): A novel activity of the tumor suppressor klotho in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4507. doi:10.1158/1538-7445.AM2017-4507

Endoplasmic reticulum (ER) stress is an imbalance between the protein-folding load and capacity of ER. It can be induced by various physiological conditions, activating the unfolded protein response (UPR) to re-establish homeostasis, promoting cell survival. Under severe or chronic stress, apoptosis is induced. Normal cells generally do not experience continuous ER stress induction. The stressful conditions experienced in the tumor microenvironment facilitate chronic ER stress and UPR activation, which plays a pivotal role in tumour survival. Exacerbation of pre-existing ER stress can trigger cancer cell death, with a minimal effect on normal cells. Current literature suggests that cannabinoid treatment may induce cancer cell death via ER stress; however, little is known about the mechanisms of induction. This study proposed that cannabidiol (CBD) mechanism occurred through the influx of Ca2+ via the TRPV1 receptor, and increasing reactive oxygen species (ROS) production affects protein folding and induces ER stress. ER stress was induced, and detection and quantification were completed using Thioflavin T staining and GRP78 by western blot analysis. The effect of cannabinoid treatment on ROS production and Ca2+ influx was measured. CBD was the most potent ER stress inducer, significantly increasing Ca2+ and ROS accumulation. Concomitant treatment with CBD and an antioxidant significantly increased cell viability and decreased ER stress induction in the MCF7 cell line. Concomitant treatment with a TRPV1 antagonist increased viability in this cell line. In conclusion, the data suggested that CBD induced ER stress via Ca2+ influx through the TRPV1 receptor, thereby elevating intracellular ROS levels and disrupting protein folding.

In human tauopathies, pathological aggregation of misfolded/unfolded proteins, particularly microtubule-associated protein tau (MAPT, tau) is considered to be an essential mechanism that triggers the induction of endoplasmic reticulum (ER) stress. Here, we assessed the molecular effects of natural antioxidant alpha-lipoic acid (ALA) in human tauR406W (hTau)-induced ER unfolded protein response (ERUPR) in fruit flies. In order to reduce hTau neurotoxicity during brain development, we used a transgenic model of tauopathy where the maximum toxicity was observed in adult flies. Then, the effects of ALA (0.001, 0.005, and 0.025% w/w of diet) in htau-induced ERUPR and behavioral dysfunctions in the ages 20 and 30 days were evaluated in Drosophila melanogaster. Data from expression (mRNA and protein) patterns of htau, analysis of eyes external morphology as well as larvae olfactory memory were confirmed by our tauopathy model. Moreover, the expression of ERUPR-related proteins involving Activating Transcription Factor 6 (ATF6), inositol regulating enzyme 1 (IRE1), and protein kinase RNA-like ER kinase (PERK) wase upregulated and locomotor function decreased in both ages of the model flies. Remarkably, the lower dose of ALA modified ERUPR and supported the reduction of behavioral deficits in youngest adults through the enhancement of GRP87/Bip, reduction of ATF6, downregulation of PERK-ATF4 pathway, and activation of the IRE1-XBP1 pathway. On the other hand, only a higher dose of ALA affected the ERUPR via moderation of PERK-ATF4 signaling in the oldest adults. As ALA also exerts higher protective effects on the locomotor function of younger adults when htauR406Wis expressed in all neurons (htau-elav) and mushroom body neurons (htau-ok), we proposed that ALA has age-dependent effects in this model. Taken together, based on our results, we conclude that aging potentially influences the ALA effective dose and mechanism of action on tau-induced ERUPR. Further molecular studies will warrant possible therapeutic applications of ALA in age-related tauopathies.

The replication of coronavirus, a family of important animal and human pathogens, is closely associated with the cellular membrane compartments, especially the endoplasmic reticulum (ER). Coronavirus infection of cultured cells was previously shown to cause ER stress and induce the unfolded protein response (UPR), a process that aims to restore the ER homeostasis by global translation shutdown and increasing the ER folding capacity. However, under prolonged ER stress, UPR can also induce apoptotic cell death. Accumulating evidence from recent studies has shown that induction of ER stress and UPR may constitute a major aspect of coronavirus–host interaction. Activation of the three branches of UPR modulates a wide variety of signaling pathways, such as mitogen-activated protein (MAP) kinase activation, autophagy, apoptosis, and innate immune response. ER stress and UPR activation may therefore contribute significantly to the viral replication and pathogenesis during coronavirus infection. In this review, we summarize the current knowledge on coronavirus-induced ER stress and UPR activation, with emphasis on their cross-talking to apoptotic signaling.

Endoplasmic reticulum stress in melanoma pathogenesis and resistance

High rates of cell proliferation and protein synthesis in pancreatic cancer are among many factors leading to endoplasmic reticulum (ER) stress. To restore cellular homeostasis, the unfolded protein response (UPR) activates as an adaptive mechanism through either the IRE1α, PERK, or ATF6 pathways to reduce the translational load and process unfolded proteins, thus enabling tumor cells to proliferate. Under severe and prolonged ER stress, however, the UPR may promote adaptation, senescence, or apoptosis under these same pathways if homeostasis is not restored. In this review, we present evidence that high levels of ER stress and UPR activation are present in pancreatic cancer. We detail the mechanisms by which compounds activate one or many of the three arms of the UPR and effectuate downstream apoptosis and examine available data on the pre-clinical and clinical-phase ER stress inducers with the potential for anti-tumor efficacy in pancreatic cancer. Finally, we hypothesize a potential new approach to targeting pancreatic cancer by increasing levels of ER stress and UPR activation to incite apoptotic cell death.

Endoplasmic reticulum stress and unfolded protein response are involved in paediatric inflammatory bowel disease.

The structural protein Core of hepatitis C virus (HCV), a cytosolic protein, induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in hepatocytes, and is responsible for the pathogenesis of persistent HCV infection. Using yeast as a model system, we evaluated mechanisms underlying Core-induced interference of ER homeostasis and UPR, and found that UPR is induced by the immature Core (aa 1-191, Core191) but not by the mature Core (aa 1-177, Core177). Interestingly, Core191 inhibits both ERAD-L, a degradation system responsible for misfolded/unfolded proteins in the ER lumen, and ERAD-M, a degradation system responsible for proteins carrying a misfolded/unfolded region in the ER membrane. In contrast, Core177 inhibits ERAD-M but not ERAD-L. In addition, requirement of an unfolded protein sensor in the ER lumen suggested that inhibition of ERAD-L is probably responsible for Core191-dependent UPR activation. These results implicate inadequate maturation of Core as a trigger for induction of ER stress and UPR.

Background Differentiated intestinal organoids (ORG), which retain patient- and disease-specific features, offer a powerful model to study inflammatory bowel disease (IBD) mechanisms. While ORG preserve IBD characteristics, acute inflammation fades in culture. To address this, we developed a series of inflammatory mixes to reintroduce disease phenotypes. Given that IBD pathways also involve endoplasmic reticulum (ER) stress—a factor in pathogenesis and a potential therapeutic target—this study aimed to optimize two specific mixes: one to induce a purely inflammatory (INF) response and another to simulate ER stress-driven inflammation in differentiated intestinal organoids. Methods Biobanked intestinal ORG cultures, derived from non-inflamed endoscopy biopsies from UC patients (n=9), were expanded, mechanically split, and exposed to human expansion media (HM) for two days1. Next, ORG cultures were exposed to differentiation media (DM) for four days. From day 6 onwards, ORG were maintained in DM only, or supplemented with an INF mix (50 ng/ml TNF-α, 20 ng/ml IL-1β, 1 µg/ml Flagellin) or an ER stress mix (10 ng/ml TNFα, 20 ng/ml IL-1β, 1 µg/ml Flagellin, Tunicamycin (TM) 25ng/ml) for 48 hours (Figure1A). As TM can induce both inflammatory and ER stress pathways, a concentration range of TM (25 ng/ml – 1 µg/ml) and TNF-α (10 ng/ml – 100 ng/ml) has been evaluated by cell morphology to determine the threshold without comprising cell viability (Figure1B). Results Principal component analysis (PCA) demonstrated distinct clustering by treatment condition, with clear separation observed across all groups (Figure2A-B).PCA plots showed consistency with the directionality of key genes driving the PC. For instance, the upregulation of PLAU2 in the ER and INF-mix groups contributes to the separation along PC1, PSAT13, drives the separation along PC2, aligning with the ER mix group, and CCL204 is instead driving PC3 (Figure2C). In organoids treated with the ER stress mix, multiple ER stress markers showed strong upregulation compared to those exposed to the INF mix, with a more pronounced effect in terms of significance and fold change (Figure2D). Pathway analysis of the contrast of the NO-mix versus the ER mix further confirmed the activation of ER stress-related pathways (Figure2E). Conclusion We developed two mixes to model INF and ER stress in differentiated intestinal ORG. PCA analysis confirmed distinct clustering by condition, with key genes driving group separations. In addition, a stronger upregulation of ER stress markers and pathways was observed in the ORG treated with the ER-mix. This newly designed mixes will facilitate preclinical compound evaluation in a disease representative IBD model.

Undesirable ER stress induced by bavachin contributed to follicular atresia in zebrafish ovary