Chronic Endoplasmic Reticulum Research Articles

Abstract 014: Brain Endoplasmic Reticulum Stress Mediates the Development of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD), characterized by an accumulation of liver lipids without excess alcohol consumption, is a major risk factor for the development of hypertension and Type-II diabetes. Liver endoplasmic reticulum (ER) stress and activation of the unfolded protein response have been implicated in the development of NAFLD. Brain ER stress has emerged as the basis of a number of chronic diseases, although the role of the central nervous system in mediating NAFLD remains unknown. We hypothesized that brain ER stress contributes to the development of NAFLD. First, male C57Bl/6 mice were instrumented with an intracerebroventricular (ICV) cannula for administration of the chemical ER stress inducer thapsigargin (TG; 1 μg/day; 3 days; n=5) or vehicle control (n=4). Daily short-term induction of ER stress in the brain with TG did not alter body weight (27±1 vs. 27±1 g; vehicle vs. TG; p>0.05), food intake or adiposity, but caused a ~40% increase in liver weight (1.04±0.03 vs. 1.39±0.10 g; vehicle vs. TG; p<0.05). ICV TG also elicited a significant increase in liver triglycerides (36±1 vs. 135±6 mmol; vehicle vs. TG; p<0.05), and real-time PCR analysis revealed upregulation of hepatic gluconeogenic and lipogenic markers in response to short-term brain ER stress (e.g. Pepck 2.45±0.24 fold vehicle; p<0.05). Second, male C57Bl/6 mice were fed high fat diet (HFD, 60% fat, n=8), a model that causes NAFLD and brain ER stress, or remained on normal chow (5% fat, n=6) for 20 weeks. ICV cannula were then implanted for delivery of the chemical ER chaperone TUDCA (5 μg/day) or vehicle control. Inhibition of brain ER stress with ICV TUDCA over 3 days rescued HFD-induced hepatomegaly (2.2±0.2 vs. 1.5±0.1 g; HFD vehicle vs. HFD TUDCA; p<0.05) and reduced obesity-induced liver triglyceride accumulation (365±44 vs. 127±36 mmol; HFD vehicle vs. HFD TUDCA; p<0.05), independent of changes in body weight, food intake or adiposity. These data demonstrate that induction of brain ER stress caused a NAFLD phenotype, whereas inhibition of chronic HFD-induced brain ER stress reduced hepatomegaly and hepatic steatosis. Collectively, these findings illustrate a novel role for ER stress in the brain as a contributor to NAFLD. HL63887, HL84207, AHA13POST14410020, K99HL166776

Dipeptidyl peptidase-4 inhibitor, vildagliptin, inhibits pancreatic beta cell apoptosis in association with its effects suppressing endoplasmic reticulum stress in db/db mice

AimsVildagliptin promotes beta cell survival by inhibiting cell apoptosis. It has been suggested that chronic ER (endoplasmic reticulum) stress triggers beta cell apoptosis. The objective of the study is to explore whether the pro-survival effect of vildagliptin is associated with attenuation of endoplasmic reticulum stress in islets of db/db mice. MethodsVildagliptin was orally administered to db/db mice for 6weeks, followed by evaluation of beta cell apoptosis by caspase3 activity and TUNEL staining method. Endoplasmic reticulum stress markers were determined with quantitative RT-PCR, immunohistochemistry and immunoblot analysis. ResultsAfter 6weeks of treatment, vildagliptin treatment increased plasma active GLP-1 levels (22.63±1.19 vs. 11.69±0.44, P<0.001), inhibited beta cell apoptosis as demonstrated by lower amounts of TUNEL staining nuclei (0.37±0.03 vs. 0.55±0.03, P<0.01) as well as decreased caspase3 activity (1.48±0.11 vs. 2.67±0.13, P<0.01) in islets of diabetic mice compared with untreated diabetic group. Further, vildagliptin treatment down-regulated several genes related to endoplasmic reticulum stress including TRIB3 (tribbles homolog 3) (15.9±0.4 vs. 33.3±1.7, ×10−3, P<0.001), ATF-4(activating transcription factor 4) (0.83±0.06 vs. 1.42±0.02, P<0.001) and CHOP(C/EBP homologous protein) (0.07±0.01 vs. 0.16±0.01, P<0.001). ConclusionsVildagliptin promoted beta cell survival in db/db mice in association with down-regulating markers of endoplasmic reticulum stress including TRIB3, ATF-4 as well as CHOP.

Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution.

Chronic endoplasmic reticulum (ER) stress results in toxicity that contributes to multiple human disorders. We report a stress resolution pathway initiated by the nuclear receptor LRH-1 that is independent of known unfolded protein response (UPR) pathways. Like mice lacking primary UPR components, hepatic Lrh-1-null mice cannot resolve ER stress, despite a functional UPR. In response to ER stress, LRH-1 induces expression of the kinase Plk3, which phosphorylates and activates the transcription factor ATF2. Plk3-null mice also cannot resolve ER stress, and restoring Plk3 expression in Lrh-1-null cells rescues ER stress resolution. Reduced or heightened ATF2 activity also sensitizes or desensitizes cells to ER stress, respectively. LRH-1 agonist treatment increases ER stress resistance and decreases cell death. We conclude that LRH-1 initiates a novel pathway of ER stress resolution that is independent of the UPR, yet equivalently required. Targeting LRH-1 may be beneficial in human disorders associated with chronic ER stress. DOI: http://dx.doi.org/10.7554/eLife.01694.001.

Translational and post‐translational regulation of XIAP by eIF2α and ATF4 promotes ER stress‐induced cell death during the unfolded protein response (151.5)

Chronic endoplasmic reticulum (ER) stress causes cell death and contributes to disease pathogenesis and progression. ER stress activates intracellular signaling pathways termed the Unfolded Protein Response (UPR). The mechanisms by which chronic ER stress and UPR promote cell death are poorly understood. The PERK arm of the UPR has been implicated in the induction of cell death through its upregulation of proapoptotic CHOP transcription factor. Chop‐/‐ cells are only partially resistant to ER stress‐induced apoptosis, and CHOP overexpression alone does not induce apoptosis. These findings strongly suggest that additional mechanisms are involved in regulating cell death downstream of PERK. Here, we find chronic ER stress dramatically suppresses XIAP protein levels. We find that PERK signaling is responsible for suppressing XIAP by eIF2a‐dependent translational attenuation and ATF4‐mediated proteasomal degradation. PERK’s down‐regulation of XIAP occurs independently of CHOP. Loss of XIAP leads to increased cell death, while XIAP overexpression significantly enhances resistance to ER stress‐induced cell death, even in the absence of CHOP. We propose a “two‐hit” model of ER stress‐induced apoptosis involving concomitant CHOP upregulation and XIAP down‐regulation both regulated by PERK.

Oncogenic Activation of MEK/ERK Primes Melanoma Cells for Adaptation to Endoplasmic Reticulum Stress

Cancer cells commonly undergo chronic endoplasmic reticulum (ER) stress, to which the cells have to adapt for survival and proliferation. We report here that in melanoma cells intrinsic activation of the ER stress response/unfolded protein response (UPR) is, at least in part, caused by increased outputs of protein synthesis driven by oncogenic activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) and promotes proliferation and protects against apoptosis induced by acute ER stress. Inhibition of oncogenic BRAF(V600E) or MEK-attenuated activation of inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) signaling of the UPR in melanoma cells. This was associated with decreased phosphorylation of eukaryotic initiation factor 4E (eIF4E) and nascent protein synthesis and was recapitulated by knockdown of eIF4E. In line with this, introduction of BRAF(V600E) into melanocytes led to increases in eIF4E phosphorylation and protein production and triggered activation of the UPR. Similar to knockdown of glucose-regulated protein 78 (GRP78), inhibition of XBP1 decelerated melanoma cell proliferation and enhanced apoptosis induced by the pharmacological ER stress inducers tunicamycin and thapasigargin. Collectively, these results reveal that potentiation of adaptation to chronic ER stress is another mechanism by which oncogenic activation of the MEK/ERK pathway promotes the pathogenesis of melanoma.

Adiponectin may inhibit chronic intermittent hypoxia-induced endoplasmic reticulum stress and cell apoptosis in genioglossus

Introduction Adiponectin may inhibit chronic intermittent hypoxia-induced endoplasmic reticulum stress and cell apoptosis in genioglossus Background Adiponectin is a potent protective molecule against injury caused by chronic intermittent hypoxia (CIH). However, the protective mechanism is not well elucidated. Cell apoptosis was remarkable in genioglossus of rats undergoing CIH. Enhanced endoplasmic reticulum (ER) stress has been recognized as the precursor of acceleration of cell apoptosis. Objective The present study aims to investigate the effects of CIH and adoponectin supplement on ER stress and cell apoptosis in genioglossus of rats. Materials and methods 30 male Wistar rats were randomly divided into three groups: normal control group (group A), CIH group (group B) and CIH+Ad group (group C) with 10 rats in each. Rats in group A were kept breathing normal air, while rats in both group B and group C received same CIH environment (CIH 8 hr/day for successive 5 weeks). However, rats in group C was given intravenous Ad supplement at the dosage of 100 ∣ μg/kg, twice a week for sucessive 5 weeks. At the end of experiment (day 35), the expression of glucose-regulated protein 78(GRP78), P38, c-Jun NH-terminal kinase (JNK) and C/EBP homologous protein (CHOP) proteins of genioglossus were tested with western blot assay. In addition, the cell apoptosis of genioglossus in each group was measured through TUNEL and Annexin V/PI staining. Results Compared with control group, in CIH group the P38 was significantly activated, the expression of GRP78 and CHOP proteins of genioglossus were significantly upregulated, and the rate of cell apoptosis was significantly elevated (all P 0.01). However, compared with CIH group, in group C the activation of P38 was partially inhibited, the expression of GRP78 and CHOP proteins was significantly weakened, and the rate of cell apoptosis was remarkably lower (all P 0.05). There was no significant difference of JNK expression of genioglossus among three groups (P > 0.05). Conclusion Conclusions CIH could induce ER stress and significantly elevate the rate of cell apoptosis through ativation of P38-CHOP pathway in genioglossus of rats submitted to CIH. Supplement of extrinsic adiponectin could attenuate excessive ER stress, inhibit activation of P38-CHOP pathway, and thus further reduce the rate of cell apoptosis induced by CIH in genioglossus. Acknowledgements Thanks to the support from the Department of pathophysiology, Nanjing Medical University for prividing this study with CIH instrument.

HCV Infection Selectively Impairs Type I but Not Type III IFN Signaling

A stable and persistent Hepatitis C virus (HCV) replication cell culture model was developed to examine clearance of viral replication during long-term treatment using interferon-α (IFN-α), IFN-λ, and ribavirin (RBV). Persistently HCV-infected cell culture exhibited an impaired antiviral response to IFN-α+RBV combination treatment, whereas IFN-λ treatment produced a strong and sustained antiviral response that cleared HCV replication. HCV replication in persistently infected cells induced chronic endoplasmic reticulum (ER) stress and an autophagy response that selectively down-regulated the functional IFN-α receptor-1 chain of type I, but not type II (IFN-γ) or type III (IFN-λ) IFN receptors. Down-regulation of IFN-α receptor-1 resulted in defective JAK-STAT signaling, impaired STAT phosphorylation, and impaired nuclear translocation of STAT. Furthermore, HCV replication impaired RBV uptake, because of reduced expression of the nucleoside transporters ENT1 and CNT1. Silencing ER stress and the autophagy response using chemical inhibitors or siRNA additively inhibited HCV replication and induced viral clearance by the IFN-α+RBV combination treatment. These results indicate that HCV induces ER stress and that the autophagy response selectively impairs type I (but not type III) IFN signaling, which explains why IFN-λ (but not IFN-α) produced a sustained antiviral response against HCV. The results also indicate that inhibition of ER stress and of the autophagy response overcomes IFN-α+RBV resistance mechanisms associated with HCV infection.

SOD1 as a Molecular Switch for Initiating the Homeostatic ER Stress Response under Zinc Deficiency

Zinc is an essential trace element, and impaired zinc homeostasis is implicated in the pathogenesis of various human diseases. However, the mechanisms cells use to respond to zinc deficiency are poorly understood. We previously reported that amyotrophic lateral sclerosis (ALS)-linked pathogenic mutants of SOD1 cause chronic endoplasmic reticulum (ER) stress through specific interactions with Derlin-1, which is a component of the ER-associated degradation machinery. Moreover, we recently demonstrated that this interaction is common to ALS-linked SOD1 mutants, and wild-type SOD1 (SOD1(WT)) comprises a masked Derlin-1 binding region (DBR). Here, we found that, under zinc-deficient conditions, SOD1(WT) adopts a mutant-like conformation that exposes the DBR and induces the homeostatic ER stress response, including the inhibition of protein synthesis and induction of a zinc transporter. We conclude that SOD1 has a function as a molecular switch that activates the ER stress response, which plays an important role in cellular homeostasis under zinc-deficient conditions.

Abstract 37: Endoplasmic Reticulum Stress in the Brain Contributes to the Pathogenesis of Non-alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a major risk factor for the development of cardiovascular diseases (CVD), including hypertension. While most studies have focused on peripheral mechanisms mediating hepatic dysfunction, a role for the central nervous system in the pathogenesis of NAFLD is less appreciated. Here we tested the hypothesis that endoplasmic reticulum (ER) stress in the brain contributes to the development of NAFLD. First , adult male C57Bl/6 mice were instrumented with an intracerebroventricular (ICV) cannula for administration of the ER stress inducer thapsigargin (TG; 1 μg/day; 3 days; n=5), or vehicle control (n=4), and allowed 1 week recovery. Short-term induction of ER stress in the brain, using TG, did not alter body weight (26±1 vs. 27±1 g; vehicle vs. TG; p&lt;0.05), food intake or adiposity, but caused a ~20% increase in liver weight (1.04±0.5 vs. 1.25±0.07 g; vehicle vs. TG; p&lt;0.05). ICV TG also elicited an increase in liver lipid droplet accumulation (0.074±0.002 vs. 0.665±0.060 droplets/cm 2 ; vehicle vs. TG; p&lt;0.05) and up-regulated hepatic gluconeogenic and lipogenic markers (e.g. Pepck mRNA 1.56±0.13 fold vehicle; p&lt;0.05). Second , male C57Bl/6 mice either remained on normal chow (n=6) or were fed a high fat diet (HFD; n=8) for a period of 20 weeks, a condition that we and others have shown results in ER stress in the brain. Mice were then implanted with an ICV cannula for administration of the chemical ER chaperone TUDCA (5 μg/day), or vehicle control, and allowed 1 week of recovery. ICV TUDCA administration over 5 days rescued HFD-induced hepatomegaly (1.77±0.18 vs. 1.19±0.07 g; HFD vehicle vs. HFD TUDCA; p&lt;0.05) and reduced obesity-induced liver triglyceride accumulation (365±44 vs. 127±36 mM; HFD vehicle vs. HFD TUDCA; p&lt;0.05), such that HFD ICV TUDCA treated mice were not different from controls. These data demonstrate that induction of ER stress in the brain causes hepatic lipid accumulation, independent of changes in body weight, food intake and adiposity. Furthermore, inhibition of chronic brain ER stress reduces HFD-induced hepatomegaly and hepatic steatosis. Collectively these findings illustrate a novel role for brain ER stress as a contributor to a major risk factor for CVD, NAFLD. HL63887, HL84207, AHA13POST14410020

Pharmacological brake-release of mRNA translation enhances cognitive memory

Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the 'integrated stress response' (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders. DOI:http://dx.doi.org/10.7554/eLife.00498.001.

Association of cyclin D and estrogen receptor α36 with hepatocellular adenomas of female mice under chronic endoplasmic reticulum stress

Hepatocellular adenomas (HCAs) are benign tumors that can lead to medical complications. Chronic inflammation and mutations in β-catenin, hepatocyte nuclear factor 1α, or glycoprotein 130 are potential causes for human HCA. However, additional causes may exist due to heterogeneity of HCA. We investigated whether HCA are caused by endoplasmic reticulum (ER) stress. Mice with a liver-specific deletion of the major chaperone BiP (LGKO) were used. Liver tumor occurrence was examined in LGKO with or without feeding of a high-fat diet (HFD) and characterized with immunohistochemistry with molecular markers of proliferation/malignancy. Molecular changes were analyzed with immunoblotting. Spontaneous monoclonal liver tumors were observed in 34% of LGKO females with constitutive hepatic ER stress. Lack of portal tracks or central veins, dilated sinusoidal spaces, hemorrhagic areas, active proliferation, and lipid deposits were observed in the liver tumors. HFD feeding induced multiclonal liver tumors in 83% of the LGKO females versus 0 in wild-type females. Hepatocytes reactive to antiglypican 3 antibodies were detected in the HFD-induced, but not spontaneous, tumors. In the liver tumors, inhibition of cyclin D and increase of the 36 kD estrogen receptor variant (estrogen receptor α36), active transcription activator 4/6, glycogen synthase kinase 3β, and extracellular signal-regulated protein kinases 1 and 2 were detected, whereas no change of hepatocyte nuclear factor 1α, β-catenin, p-53, androgen receptor α, or estrogen receptor α was detected. HFD activated Janus kinase and signal transducers and activators of transcription 3. Our evidence supports a novel link of HCA with ER stress and altered expression of cyclin D and estrogen receptor α36. Additional stress such as HFD may promote malignant transformation of HCA through the Janus kinase-signal transducers and activators of transcription pathway.

Proteasome Dysfunction Mediates Obesity-Induced Endoplasmic Reticulum Stress and Insulin Resistance in the Liver

Chronic endoplasmic reticulum (ER) stress is a major contributor to obesity-induced insulin resistance in the liver. However, the molecular link between obesity and ER stress remains to be identified. Proteasomes are important multicatalytic enzyme complexes that degrade misfolded and oxidized proteins. Here, we report that both mouse models of obesity and diabetes and proteasome activator (PA)28-null mice showed 30–40% reduction in proteasome activity and accumulation of polyubiquitinated proteins in the liver. PA28-null mice also showed hepatic steatosis, decreased hepatic insulin signaling, and increased hepatic glucose production. The link between proteasome dysfunction and hepatic insulin resistance involves ER stress leading to hyperactivation of c-Jun NH2-terminal kinase in the liver. Administration of a chemical chaperone, phenylbutyric acid (PBA), partially rescued the phenotypes of PA28-null mice. To confirm part of the results obtained from in vivo experiments, we pretreated rat hepatoma-derived H4IIEC3 cells with bortezomib, a selective inhibitor of the 26S proteasome. Bortezomib causes ER stress and insulin resistance in vitro—responses that are partly blocked by PBA. Taken together, our data suggest that proteasome dysfunction mediates obesity-induced ER stress, leading to insulin resistance in the liver.

Antidiabetic Potential of the Heme Oxygenase-1 Inducer Curcumin Analogues

Although there is a therapeutic treatment to combat diabetes, the identification of agents that may deal with its more serious aspects is an important medical field for research. Diabetes, which contributes to the risk of cardiovascular disease, is associated with a low-grade chronic inflammation (inflammatory stress), oxidative stress, and endoplasmic reticulum (ER) stress. Because the integration of these stresses is critical to the pathogenesis of diabetes, agents and cellular molecules that can modulate these stress responses are emerging as potential targets for intervention and treatment of diabetic diseases. It has been recognized that heme oxygenase-1 (HO-1) plays an important role in cellular protection. Because HO-1 can reduce oxidative stress, inflammatory stress, and ER stress, in part by exerting antioxidant, anti-inflammatory, and antiapoptotic effects, HO-1 has been suggested to play important roles in pathogenesis of diabetes. In the present review, we will explore our current understanding of the protective mechanisms of HO-1 in diabetes and present some emerging therapeutic options for HO-1 expression in treating diabetic diseases, together with the therapeutic potential of curcumin analogues that have their ability to induce HO-1 expression.

Diabetes and beta cell function: from mechanisms to evaluation and clinical implications

Diabetes is a complex, heterogeneous condition that has beta cell dysfunction at its core. Many factors (e.g. hyperglycemia/glucotoxicity, lipotoxicity, autoimmunity, inflammation, adipokines, islet amyloid, incretins and insulin resistance) influence the function of pancreatic beta cells. Chronic hyperglycaemia may result in detrimental effects on insulin synthesis/secretion, cell survival and insulin sensitivity through multiple mechanisms: gradual loss of insulin gene expression and other beta-cell specific genes; chronic endoplasmic reticulum stress and oxidative stress; changes in mitochondrial number, morphology and function; disruption in calcium homeostasis. In the presence of hyperglycaemia, prolonged exposure to increased free fatty acids result in accumulation of toxic metabolites in the cells (“lipotoxicity”), finally causing decreased insulin gene expression and impairment of insulin secretion. The rest of the factors/mechanisms which impact on the course of the disease are also discusses in detail.The correct assessment of beta cell function requires a concomitant quantification of insulin secretion and insulin sensitivity, because the two variables are closely interrelated. In order to better understand the fundamental pathogenetic mechanisms that contribute to disease development in a certain individual with diabetes, additional markers could be used, apart from those that evaluate beta cell function.The aim of the paper was to overview the relevant mechanisms/factors that influence beta cell function and to discuss the available methods of its assessment. In addition, clinical considerations are made regarding the therapeutical options that have potential protective effects on beta cell function/mass by targeting various underlying factors and mechanisms with a role in disease progression.

Unglycosylated clusterin variant accumulates in the endoplasmic reticulum and induces cytotoxicity

Clusterin is a stress-responsive and highly glycosylated secretory protein that plays cytoprotective role in most body fluids. In addition to extracellular clusterin, several intracellular clusterin variants that are rather cytotoxic have been recently uncovered under diverse pathological conditions. Although these variants revealed heterogeneity in their glycan modification, its significance in many diseases remains to be validated. Here, we found that clusterin is differentially metabolized by two well-characterized ER stress inducers. Thapsigargin induced retrotranslocation and rapid degradation of clusterin from the endoplasmic reticulum, whereas tunicamycin failed to degrade but rather retained clusterin in the endoplasmic reticulum. Important sorting determinant for these processes proved to be N-glycan moieties that are required for the prevention of terminal misfolding and aggregation of clusterin in the endoplasmic reticulum. This study provides a mechanistic insight into the generation of noble cytotoxic variant of intracellular clusterin and an idea about molecular pathogenesis of diseases associated with chronic endoplasmic reticulum stress, such as neurodegeneration.

A novel monoclonal antibody reveals a conformational alteration shared by amyotrophic lateral sclerosis‐linked SOD1 mutants

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by the selective loss of upper and lower motoneurons. Although >100 different Cu, Zn superoxide dismutase (SOD1) mutations have been identified in ALS patients, it remains controversial whether all of them are disease-causative mutations. Therefore, it is necessary to develop molecular mechanism-based diagnosis and treatment of ALS caused by SOD1 mutations. We previously reported that 3 pathogenic mutations of SOD1 cause chronic endoplasmic reticulum (ER) stress by inducing the binding of SOD1 to Derlin-1, a component of the ER homeostatic machinery. Here, we systematically analyzed 132 SOD1 mutants and found that most have a constitutively exposed Derlin-1-binding region (DBR) that is occluded in the wild-type protein. To develop the novel molecular mechanism-based antibody that can specifically recognize the aberrant structure of toxic SOD1 mutants, we generated the monoclonal antibody against the DBR. MS785, a monoclonal antibody generated against the DBR, distinguished most ALS-causative SOD1 mutants from both wild-type and nontoxic mutants. Moreover, MS785 recognized endogenous SOD1 in B lymphocytes derived from 14 ALS patients carrying SOD1 mutations but not from 11 healthy controls. This is the first study to address the common property of all ALS-causative SOD1 mutants. MS785 is the first molecular mechanism-based antibody that was shown to be able to distinguish ALS-linked toxic SOD1 mutants from both wild-type and nontoxic mutants. MS785 may thus become an innovative tool for the diagnosis of ALS.

Blockade of XBP1 splicing by inhibition of IRE1α is a promising therapeutic option in multiple myeloma

AbstractMultiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositol-requiring enzyme 1α (IRE1α) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM using a small-molecule IRE1α endoribonuclease domain inhibitor MKC-3946. MKC-3946 triggered modest growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Importantly, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG, even in the presence of bone marrow stromal cells or exogenous IL-6. Both bortezomib and 17-AAG induced ER stress, evidenced by induction of XBP1s, which was blocked by MKC-3946. Apoptosis induced by these agents was enhanced by MKC-3946, associated with increased CHOP. Finally, MKC-3946 inhibited XBP1 splicing in a model of ER stress in vivo, associated with significant growth inhibition of MM cells. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential therapeutic option in MM.

A BAX/BAK and cyclophilin D-independent intrinsic apoptosis pathway.

Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-XL overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.

Integrating role of T antigen, Rb2/p130, CTCF and BORIS in mediating non-canonical endoplasmic reticulum-dependent death pathways triggered by chronic ER stress in mouse medulloblastoma

Distinct molecular pathways could be constitutively active in mouse T-Antigen positive and T-Antigen negative medulloblastoma cell lines, contributing to their phenotypic differences as well as to cellular responses, cell cycle progression, cell death and survival. The diversity of these responses may be due, at least in part, to distinct activities of Rb2/p130, CTCF and BORIS proteins in response to an altered network of signaling evoked by the T-Ag presence. Here, we provided evidence supporting a role for the T-Antigen in causing chronic endoplasmic reticulum (ER) stress and aberrant Caspase-12 expression and activation, subsequently driving to both massive cell death, and perhaps selection of cells with a higher malignant phenotype. Furthermore, we observed that the endoplasmic stress, either chronically caused by T-Ag or transiently induced by glucose deprivation, is accompanied by the formation of complexes between the retinoblastoma related protein Rb2/p130 and the chromatin insulator CCCTC-binding factor CTCF, or the CTCF-paralogue BORIS. Our study represents the first evidence supporting a role of the T-Antigen in inducing/maintaining chronic ER-stress, as well as, indicating a role of Rb2/p130, CTCF and BORIS as potential mediators of non-canonical ER-dependent death pathway in mouse medulloblastoma.

Blockade of XBP1 Splicing by Inhibition of IRE1α Is a Promising Therapeutic Option in Multiple Myeloma

Abstract 133Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response (UPR). Therefore blockade of UPR could provide a novel therapeutic option in MM. Upon UPR, inositol-requiring enzyme 1α (IRE1α) is activated by auto-phosphorylation, resulting in activation of its endoribonuclease domain to cleave XBP1 mRNA from XBP1 unspliced form (XBP1u: inactive) to generate the XBP1 spliced form (XBP1s: active). XBP1s protein in turn regulates genes responsible for protein folding and degradation, playing a pro-survival signaling role in the UPR. In this study, we specifically examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM. We first examined the biologic significance of IRE1α by knockdown using lentiviral shRNA and observed significant growth inhibition in IRE1α knockdown cells. We next examined the impact of inhibition of XBP1 splicing using a novel small molecule IRE1α endoribonuclease domain inhibitor MKC-3946 (MannKind, Valencia CA). MKC-3946 blocked not only the basal level, but also inducible (by tunicamycin) XBP1s, evidenced by RT-PCR analysis in RPMI8226 cells, without affecting phosphorylation of IRE1α. Importantly, MKC-3946 also inhibited XBP1s in primary tumor cells from MM patients. We also confirmed functional inhibition of XBP1s, with target genes including SEC61A1, p58IPK, and ERdj4 downregulated by MKC-3946 treatment. Importantly, MKC-3946 triggered growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Furthermore, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG in RPMI8226 and INA6 cells, as well as primary tumor cells from MM patients. Both bortezomib and 17-AAG induced ER stress with XBP1s, which was markedly blocked by MKC-3946. Moreover, apoptosis induced by bortezomib or 17-AAG was enhanced by MKC-3946, associated with increased CHOP mRNA and protein, a proapoptotic factor triggered by ER stress. We next demonstrated that XBP1s was induced by bortezomib in INA6 cells co-cultured with bone marrow (BM) stromal cells, which was inhibited by MKC-3946, associated with enhanced cytotoxicity induced by the combination. Finally, MKC-3946 inhibited XBP1s in a model of in vivo ER stress induced by tunicamycin. To evaluate the anti-MM effect of MKC-3946, we used the subcutaneous RPMI8226 xenograft model in mice. MKC-3946 significantly reduced MM tumor growth in the treatment versus control group, associated with prolonged overall survival. We also confirmed that MKC-3946 treatment significantly inhibited XBP1s in excised tumors, assessed by RT-PCR. In order to examine the activity of MKC-3946 on MM cell growth in the context of the human BM microenvironment in vivo, we used the SCID-hu model, in which INA6 cells are directly injected into a human bone chip implanted subcutaneously in SCID-mice. MKC-3946 treatment significantly inhibited tumor growth compared with vehicle control. Moreover, XBP1s in excised tumor cells was inhibited, evidenced by RT-PCR. In conclusion, these data demonstrate that blockade of XBP1s by MKC-3946 triggers MM cell growth inhibition in vivo and prolongs host survival. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential novel therapeutic option in MM. Disclosures:Tam:MannKind Corporation: Employment, Equity Ownership. Zeng:MannKind Corporation: Employment, Equity Ownership. Patterson:MannKind Corporation: Employment, Equity Ownership. Richardson:Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Millennium: Membership on an entity’s Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Anderson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Onyx: Membership on an entity’s Board of Directors or advisory committees; MannKind: Membership on an entity’s Board of Directors or advisory committees.