Modulating ER Stress Research Articles

Integrative Analysis of Primary SF3B1 mt Ring Sideroblasts Provides Fundamental Insights into MDS-RS Pathogenesis and Dyserythropoiesis

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematopoietic stem cell (HSC) disorders characterized by aberrant HSC differentiation, cytopenia, and an increased risk of progression to leukemia. The low-risk subtype MDS with ring sideroblasts (MDS-RS) is specifically characterized by expanded and ineffective erythropoiesis, with more than 80% of patients displaying mutations in the core spliceosome component SF3B1 (SF3B1 mt).A hallmark of the MDS-RS bone marrow (BM) is the progressive accumulation of ring sideroblasts (RS), erythroblasts displaying perinuclear mitochondria loaded with aberrant ferritin-iron complexes. Whilst several in vitro and in vivo model systems exist for studying the impact of SF3B1 mt on erythropoiesis and RS development, primary SF3B1 mt erythroid biology remains largely unexplored due to the inability to purify live SF3B1 mt cells or fully replicate BM conditions in vitro.To address this issue, we developed an innovative two-step method to isolate live ring sideroblasts from SF3B1 mt MDS-RS BM aspiration material with extremely high purity (as determined through droplet digital PCR-based genotyping [Fig. 1A] and morphology-based detection through Prussian blue staining [Fig. 1B,C]). Unexpectedly, evaluation of matching peripheral blood samples showed that circulating ring sideroblasts are strikingly common in MDS-RS (Fig. 1D), with their abundance being significantly positively associated with clinically-determined BM RS frequencies and serum erythropoietin levels, as well as negatively associated with hemoglobin levels.Through high-throughput Chromium 3'-based single-cell RNA sequencing (scRNAseq) analysis of purified RS, we then showed that these cells comprise a heterogeneous population encompassing all stages of the erythroid differentiation continuum, from early progenitors to orthochromatic erythroblasts (Fig. 1E). The RS transcriptome was shown to be dynamically regulated towards the maintenance of cell survival during late terminal erythroid differentiation (exemplified through parkin 1 [PINK1] expression), with SF3B1 K700E erythroblasts employing multiple strategies to preserve homeostasis despite undergoing extreme oxidative stress. These observations were confirmed through a parallel whole-transcript RNAseq investigation comprising CD34 + and GPA +-enriched samples obtained from normal bone marrow (NBM) donors and SF3B1 K700E MDS-RS patients, as well as purified RS samples. This bulk RNAseq experiment validated the RS transcriptomic signature observed in scRNAseq (Fig. 1F) and allowed for a detailed investigation of RNA splicing. SF3B1 K700E-associated alternative splicing in CD34 + and RS was consistent with previous literature, but also highly context-dependent and with substantial changes in scope and magnitude throughout erythroid differentiation (Fig. 1G-I).Finally, we substantiated these RNAseq results through Tandem Mass Tag-based semi-quantitative proteomic analysis of purified RS and GPA-enriched cells from NBM donors and MDS-RS patients. We confirmed that ring sideroblast survival is heavily dependent on redox balance modulation and suppression of ER stress via an increased dependence on glutamine, mirroring the molecular mechanisms observed in malignancy. Additionally, our data strongly indicate that the RS population is a major modulator of the MDS-RS BM microenvironment due to expression of stress factors (with particular emphasis on GDF15, erythroferrone and IL-18). In conclusion, our integrative analysis of primary RS constitutes a unique platform for the study of MDS-RS, with special interest for the investigation of potential drivers of disease severity or treatment avenues. [Display omitted] DisclosuresKretzschmar: Vanadis Diagnostics, a PerkinElmer company.: Current Employment.

Can modulation of beta cell ER stress in the KINGS (Ins2+/G32S) mouse abolish sex differences in diabetic phenotype?

Can modulation of beta cell ER stress in the KINGS (<i>Ins2</i>^+/G32S) mouse abolish sex differences in diabetic phenotype?

DDRE-05. STEAROYL COA DESATURASE IS ESSENTIAL FOR REGULATION OF ENDOPLASMIC RETICULUM HOMEOSTASIS AND TUMOR GROWTH IN GLIOBLASTOMA CANCER STEM CELLS

INTRODUCTIONEmerging evidence suggest that, in addition to glucose, fatty acids can also drive glioma growth. Increased lipid synthesis is one of the metabolic hallmarks of cancer, and indeed, unsaturated fatty acids (UFA) are particularly abundant in glioblastoma. However, the exact role of fatty acids in GBM tumors remains unclear. Blocking fatty acids synthesis can present a new therapy for GBM.METHODSThrough targeted inhibitors screening on glioma stem cells (GSCs), we found that they are highly susceptible to Stearoyl CoA Desaturase 1 (SCD1) inhibitors. SCD1 is a key enzyme responsible for the conversion of saturated fatty acids (SFA) to UFA. 1) Through cell-based assays and immunoblot analyses, we tried to understand the role of UFA, SFA and SCD1 in GSCs differentiation and proliferation. We investigated the mechanism between fatty acids and tumor growth through ER stress modulation linked with SCD1 expression. 2) As we found that GSCs are highly susceptible to SCD1 inhibition, we tested CAY, SCD1 inhibitor, in GSCs orthotopic mouse models and assess its effect on tumor growth and overall survival.RESULTSWe found that GSCs with extensive self-renewal capacity have an increased dependence on SCD1 activity. Through immunoblot analyses, we demonstrated that SCD1 inhibition exacerbates ER stress through accumulation of SFA and SCD1-mediated UFA synthesis mitigates ER stress. Survival analyses between SCD1 inhibitor-treated group and control group showed significant survival benefit in SCD1-inhibitor-treated group, in both mesenchymal (p=0.008, 35 days vs 18) and proneural (p=0.0002) type glioma cells (n=8/groups).CONCLUSIONSWe demonstrate that SCD1, the fatty acid desaturase, is essential for the maintenance of glioblastoma cancer stem cells. SCD1 is activated by ER stress and exerts a cytoprotective function by regulating ER homeostasis, thus favoring survival and tumor growth. Pharmacological targeting of SCD1 exhibits potent therapeutic efficacy in brain tumor mouse models.

Neuroprotective Effects of Thymoquinone by the Modulation of ER Stress and Apoptotic Pathway in In Vitro Model of Excitotoxicity.

Experimental evidence indicates that the activation of ionotropic glutamate receptors plays an important role in neurological disorders’ models such as epilepsy, cerebral ischemia and trauma. The glutamate receptor agonist kainic acid (KA) induces seizures and excitotoxic cell death in the CA3 region of the hippocampus. Thymoquinone (TQ) is the most important component of the essential oil obtained from black cumin (Nigella sativa L.) seeds. It has many pharmacological actions including antioxidant, anti-inflammatory, and anti-apoptotic effects. TQ was used in an in vitro experimental model of primary cultures where excitotoxicity was induced. Briefly, rat organotypic hippocampal slices were exposed to 5 µM KA for 24 h. Cell death in the CA3 subregions of slices was quantified by measuring propidium iodide fluorescence. The cross-talk between TQ, ER stress and apoptotic pathways was investigated by Western blot. In untreated slices TQ (10 µM) induced a significant increase on the PSD95 levels and it decreased the excitotoxic injury induced by KA. Additionally, TQ was able to ameliorate the KA-induced increase in unfolded proteins GRP78 and GRP94 expression. Finally, TQ was able to partially rescue the reduction of the KA-induced apoptotic pathway activation. Our results suggest that TQ modulates the processes leading to post-kainate neuronal death in the CA3 hippocampal area.

MSI-1436 improves EMS adipose derived progenitor stem cells in the course of adipogenic differentiation through modulation of ER stress, apoptosis, and oxidative stress

BackgroundProtein tyrosine phosphatase 1B (PTP1B) is one of the major negative regulators of leptin and insulin signaling, and has been strongly implicated in insulin resistance development in the course of obesity and metabolic syndrome conditions; however, its exact role in controlling adipose tissue biogenesis is still poorly understood.ObjectivesThis investigation aimed to elucidate whether selective inhibition of PTP1B using MSI-1436 compound may improve and restore the defective adipogenicity of ASCs isolated from EMS-affected horses.MethodsEquine ASC EMS cells were cultured under adipogenic conditions in the presence of PTP1B inhibitor and were subsequently tested for expression of the main adipogenic-related genes using RT-qPCR, changes in free fatty acid profiles by means of GC-MS technique, and for mitochondrial dynamics improvement through the analysis of mitochondrial transmembrane potential and oxidative stress.ResultsSelective inhibition of PTP1B in equine ASC EMS cells improved substantially adipogenic differentiation by promoting cellular proliferation and normalizing expression of C/EBPalpha, PPARγ, and Adipoq markers that are critical for proper adipogenesis. Levels of secreted adiponectin and PPARγ were also shown to be increased in MSI-1436-conditioned cells, while total leptin levels markedly dropped under the same conditions. Moreover, MSI-1436 treatment enabled the regulation of metabolic-related transcripts that are crosslink to adipogenesis, namely Akt1, Akt2, and SHBG. The obtained results demonstrated also an obvious reduction in intracellular accumulated ROS and NO, as well as mitigated ER stress through the downregulation of Chop, Perk, Atf6, Ire1, and Xbp1 transcripts upon PTP1B inhibition. Furthermore, general fluctuations in FFA composition of all differentiated groups have been highlighted, where palmitic acid, palmitoleic acid, stearic acid, and linolelaidic acid that are known to be associated with the development of metabolic disorders were found to be normalized upon PTP1B inhibition during adipogenic differentiation.ConclusionThe presented data provides the evidence that the use of PTP1B inhibitor may be successful in controlling and enhancing adipogenic differentiation of impaired equine ASCs affected by metabolic syndrome, and thus offers new insights for the management of obesity through the regulation of adipose tissue dynamics.Graphical abstract

Lithium chloride functions as Newcastle disease virus-induced ER-stress modulator and confers anti-viral effect

Newcastle disease is a severe clinical manifestation of avian species caused by Newcastle disease virus (NDV). Although several vaccination strategies are available to protect poultry against NDV infection, even then, outbreaks have been reported in the vaccinated birds. The lack of therapeutics against NDV makes the need for effective anti-viral drugs is of utmost importance. Lithium Chloride (LiCl) is a widely prescribed drug for the treatment of bipolar disorder, acute brain injuries, and chronic neurodegenerative diseases. Also, LiCl has been repurposed as an effective anti-viral drug for some viral infections. In the present work, we have investigated the efficacy of LiCl to inhibit NDV replication using in vitro, in ovo, and in vivo models. Our results collectively showed the modulation of NDV replication after the LiCl treatment. We also demonstrated that NDV induces endoplasmic reticulum stress (ER-stress), and a stress-inducible ER chaperone, glucose-regulating protein 78 (GRP78), was found to be over-expressed after NDV infection. Subsequently, the treatment of NDV infected cells with LiCl significantly reduced the transcript and protein levels of GRP78. Finally, we concluded that LiCl treatment protects the cells from ER-stress induced by the NDV infection.

New Insight into Triple-Negative Breast Cancer Therapy: The Potential Roles of Endoplasmic Reticulum Stress and Autophagy Mechanisms.

Breast cancer is accounted as the fifth leading cause of mortality among the other cancers. Notwithstanding, Triple Negative Breast Cancer (TNBC) is responsible for 15-20% of breast cancer mortality. Despite many investigations, it remains incurable in part due to insufficient understanding of its exact mechanisms. A literature search was performed in PubMed, SCOPUS and Web of Science databases using the keywords autophagy, Endoplasmic Reticulum (ER) stress, apoptosis, TNBC and the combinations of these keywords. It was found that autophagy plays a dual role in cancer, so that it may decrease the viability of tumor cells or act as a cytoprotective mechanism. It then appears that using compounds having modulatory effects on autophagy is of importance in terms of induction of autophagic cell death and diminishing the proliferation and metastasis of tumor cells. Also, ER stress can be modulated in order to stimulate apoptotic and autophagic cell death in tumor cells. Perturbation in the signaling pathways related to cell survival leads to the initiation and progression of cancer. Regarding the advancement in the cancer pathology, it seems that modulation of autophagy and ER stress are promising.

Berberine inhibits free fatty acid and LPS-induced inflammation via modulating ER stress response in macrophages and hepatocytes.

Inflammation plays an essential role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Berberine (BBR), an isoquinoline alkaloid isolated from Chinese medicinal herbs, has been widely used to treat various diseases, including liver diseases for hundreds of years. The previous studies have shown that BBR inhibits high fat-diet-induced steatosis and inflammation in rodent models of NAFLD. However, the underlying molecular mechanisms remain unclear. This study is aimed to identify the potential mechanisms by which BBR inhibits free fatty acid (FFA) and LPS-induced inflammatory response in mouse macrophages and hepatocytes. Mouse RAW264.7 macrophages and primary mouse hepatocytes were treated with palmitic acid (PA) or LPS or both with or without BBR (0-10 μM) for different periods (0-24 h). The mRNA and protein levels of proinflammatory cytokines (TNF-α, IL-6, IL-1β, MCP-1) and ER stress genes (CHOP, ATF4, XBP-1) were detected by real-time RT-PCR, Western blot and ELISA, respectively. The results indicated that BBR significantly inhibited PA and LPS-induced activation of ER stress and expression of proinflammatory cytokines in macrophages and hepatocytes. PA/LPS-mediated activation of ERK1/2 was inhibited by BBR in a dose-dependent manner. In summary, BBR inhibits PA/LPS-induced inflammatory responses through modulating ER stress-mediated ERK1/2 activation in macrophages and hepatocytes.

Klotho regulation by albuminuria is dependent on ATF3 and endoplasmic reticulum stress.

Proteinuria is associated with renal function decline and cardiovascular mortality. This association may be attributed in part to alterations of Klotho expression induced by albuminuria, yet the underlying mechanisms are unclear. The presence of albumin decreased Klotho expression in the POD-ATTAC mouse model of proteinuric kidney disease as well as in kidney epithelial cell lines. This downregulation was related to both decreased Klotho transcription and diminished protein half-life, whereas cleavage by ADAM proteases was not modified. The regulation was albumin specific since it was neither observed in the analbuminemic Col4α3-/- Alport mice nor induced by exposure of kidney epithelial cells to purified immunoglobulins. Albumin induced features of ER stress in renal tubular cells with ATF3/ATF4 activation. ATF3 and ATF4 induction downregulated Klotho through altered transcription mediated by their binding on the Klotho promoter. Inhibiting ER stress with 4-PBA decreased the effect of albumin on Klotho protein levels without altering mRNA levels, thus mainly abrogating the increased protein degradation. Taken together, albuminuria decreases Klotho expression through increased protein degradation and decreased transcription mediated by ER stress induction. This implies that modulating ER stress may improve proteinuria-induced alterations of Klotho expression, and hence renal and extrarenal complications associated with Klotho loss.

ER stress and UPR activation in glioblastoma: identification of a noncanonical PERK mechanism regulating GBM stem cells through SOX2 modulation

Patients with aggressive brain tumors, named glioblastoma multiforme (GBM), have a poor prognoses. Here we explored if the ER stress/unfolded protein response (UPR) is involved in the pathophysiology of GBM and may provide novel therapeutic targets. Immunohistochemical analyses of a tissue microarray containing primary GBM specimens showed strong variability in expression of the UPR markers GRP78/BiP, XBP1, and ATF4. Interestingly, high ATF4 expression was associated with poor overall survival suggesting involvement of PERK signaling in GBM progression. In vitro experiments using patient-derived neurospheres, enriched for GBM stem cells (GSCs), showed high sensitivity for the ER stressor thapsigargin (Tg) mainly via PERK signaling. In contrast, neurospheres-derived differentiated GBM cells were less sensitive likely due to lower UPR activity as indicated by comparative transcriptional profiling. Tg and Tunicamycin strongly reduced neurosphere forming ability of GSCs that was linked with potent PERK-dependent downregulation of SOX2 protein. Interestingly, SOX2 downregulation occurred directly via PERK, not requiring downstream activation of the PERK-UPR pathway. Moreover, PERK inactivation resulted in aberrant serum-induced differentiation of GBM neurospheres accompanied by persistent SOX2 expression, delayed upregulation of GFAP and reduced cell adherence. In conclusion, we provide evidence that PERK signaling contributes to the prognoses of primary GBM patients and identified PERK as a novel regulator of SOX2 expression and GSC differentiation. The role of PERK appeared to be pleiotropic involving UPR-dependent, as well as novel identified noncanonical mechanisms regulating SOX2. ER stress and PERK modulation appear to provide promising therapeutic targets for therapy in GBM.

Resveratrol protects against cadmium chloride‐induced hippocampal neurotoxicity by inhibiting ER stress and GAAD 153 and activating sirtuin 1/AMPK/Akt

This study investigated whether the apoptotic effect induced by cadmium chloride (CdCl2 ) in rat's hippocampi and neuroprotection afforded by resveratrol (RES) are mediated by modulation of ER stress and involve sirtuin 1 (SIRT1)/AMPK/Akt axis. Adult male Wistar rats were divided into four groups (n = 24/group) as control, control + RES (300 mg/kg), CdCl2 (5 mg/kg), and CdCl2 + RES. All treatments were conducted orally for 45 days. Also, cultured hippocampal cells were treated with CdCl2 in the presence or absence of RES and with or without preincubation with SIRT1, AMPK, or PI3K inhibitors. CdCl2 impaired retention and spatial memories of rats and reduced levels and activities of SIRT1 and inhibited AMPK/Akt axis in their hippocamapi where SIRT1 was the upstream regulator. It also enahnced hippocampal levels of reactive oxygen species (ROS) and expression of caspase-12 and caspase-3, depleted glutathione (GSH) levels, and activated GRP78, activating transcription factor-6, GAAD 153, X-box binding protein-1 arms of ER stress. On the contrary, RES coadminsitration completley abolished all these events. Interstingly and in control rats, RES not only increased levels of GSH, but also enhenced protein levels of B-cell lymphoma 2 (Bcl-2) and dwonregulated GAAD 153. In both control and CdCl2 -treated rats, pharmacological inhibtion of SIRT1, AMPK, and Akt compleltely abolished all effects afforded by RES. In conclusion, CdCl2 -induced hippocampal apopotis is associated with reduction of SIRT1/AMPK/Akt activity levels, ROS generation, downregulation of Bcl-2, and activities, activation of ER stress, and GAAD 153, whereas RES is able to reverse these effects through activation of SIRT1/AMPK/Akt.

Proteomics-based functional studies reveal that galectin-3 plays a protective role in the pathogenesis of intestinal Beh\xe7et\u2019s disease

The pathogenesis of intestinal Behçet’s disease (BD) remains poorly understood. Therefore, we aimed to discover and validate biomarkers using proteomics analysis and subsequent functional studies. After two-dimensional electrophoresis, candidate proteins were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS). We validated these results by evaluating the protein levels and their functions in vitro using HT-29 colorectal cancer cells, colon tissues from patients and mice, and murine bone marrow derived macrophages (BMDMs). Of the 30 proteins differentially expressed in intestinal BD tissues, we identified seven using MALDI-TOF/TOF MS. Focusing on galectin-3, we found that TGF-B and IL-10 expression was significantly lower in shLGALS3-transfected cells. Expression of GRP78 and XBP1s and apoptosis rates were all higher in shLGALS3-transfected cells upon the induction of endoplasmic reticulum stress. In response to lipopolysaccharide stimulation, microtubule-associated protein 1 light chain 3B accumulated and lysosomes decreased in these cells. Finally, Salmonella typhimurium infection induced caspase-1 activation and increased IL-1β production, which facilitated activation of the NLRC4 inflammasome, in Lgals3−/− murine BMDMs compared to wild type BMDMs. Our data suggest that galectin-3 may play a protective role in the pathogenesis of intestinal BD via modulation of ER stress, autophagy, and inflammasome activation.

Berberine ameliorates oxidative stress-induced apoptosis by modulating ER stress and autophagy in human nucleus pulposus cells

AimNucleus pulposus (NP) cell apoptosis induced by oxidative stress is known to be closely involved in the pathogenesis of intervertebral disc (IVD) degeneration. Berberine, a small molecule derived from Rhizoma coptidis, has been found to exert antioxidative activity and preserve cell viability. The present study aims to investigate whether berberine can prevent NP cell apoptosis under oxidative damage and the potential underlying mechanisms. Methods and materialsThe effects of berberine on IVD degeneration were investigated both in vitro and in vivo. Key findingsOur results showed that berberine significantly mitigated oxidative stress-decreased cell viability as well as apoptosis in human NP cells. Berberine treatment could attenuate oxidative stress-induced ER stress and autophagy in a concentration-dependent manner. With 4-PBA (ER stress specific inhibitor) and 3-MA (autophagy specific inhibitor) administration, we demonstrated that berberine inhibited oxidative stress-induced apoptosis by modulating the ER stress and autophagy pathway. We also found that the IRE1/JNK pathway was involved in the induction of ER stress-dependent autophagy. With Ca2+ chelator BAPTA-AM utilization, we revealed that oxidative stress-mediated ER stress and autophagy repressed by berberine could be restored by inducing intracellular Ca2+ dysregulation. Furthermore, in vivo study provided evidence that berberine treatment could retard the process of puncture-induced IVD degeneration in a rat model. SignificanceOur results indicate that berberine could prevent oxidative stress-induced apoptosis by modulating ER stress and autophagy, thus offering a novel potential pharmacological treatment strategy for IVD degeneration.

Administration of Tonsil-Derived Mesenchymal Stem Cells Improves Glucose Tolerance in High Fat Diet-Induced Diabetic Mice via Insulin-Like Growth Factor-Binding Protein 5-Mediated Endoplasmic Reticulum Stress Modulation.

Type 2 diabetes mellitus (T2DM) is a prevalent chronic metabolic disorder accompanied by high blood glucose, insulin resistance, and relative insulin deficiency. Endoplasmic reticulum (ER) stress induced by high glucose and free fatty acids has been suggested as one of the main causes of β-cell dysfunction and death in T2DM. Stem cell-derived insulin-secreting cells were recently suggested as a novel therapy for diabetes. In the present study, we demonstrate the therapeutic potential of tonsil-derived mesenchymal stem cells (TMSCs) to treat high-fat diet (HFD)-induced T2DM. To explore whether TMSC administration can alleviate T2DM, TMSCs were intraperitoneally injected in HFD-induced T2DM mice once every 2 weeks. TMSC injection markedly improved glucose tolerance and glucose-stimulated insulin secretion and prevented HFD-induced pancreatic β-cell hypertrophy and cell death. In addition, TMSC injection relieved the ER-stress response and preserved gene expression related to glucose sensing and insulin secretion. Moreover, administration of TMSC-derived conditioned medium induced similar therapeutic outcomes, suggesting paracrine effects. Finally, proteomic analysis revealed high secretion of insulin-like growth factor-binding protein 5 by TMSCs, and its expression was critical for the protective effects of TMSCs against HFD-induced glucose intolerance and ER-stress response in pancreatic islets. TMSC administration can alleviate HFD-induced-T2DM via preserving pancreatic islets and their function. These results provide novel evidence of TMSCs as an ER-stress modulator that may be a novel, alternative cell therapy for T2DM.

4-PBA prevents diabetic muscle atrophy in rats by modulating ER stress response and ubiquitin-proteasome system

PurposeSkeletal muscle is severely affected in diabetes leading to muscle atrophy. Previously we reported the role of ER stress in muscle atrophy due to hyperglycemia. Hence, in the present study, we investigated the effect of a classical ER stress inhibitor, 4-phenylbutyric acid (PBA), on muscle atrophy in diabetic rats. MethodsDiabetes was induced in male rats by streptozotocin, and PBA was administered (40 mg/kg/day; intraperitoneal) after two months of diabetes for two more months. Gastrocnemius muscle is collected after four months of experimental period. The cross-sectional area of myocytes was measured on Hematoxylin and Eosin stained muscle sections. Protein levels of ER stress markers, ubiquitin-proteasome system (UPS) components, and apoptosis were analysed by immunoblot. Proteasomal activity and apoptotic cells were measured. ResultsER stress markers (GRP78, ATF6, ATF4 and CHOP) that are elevated in diabetes are decreased with PBA treatment. PBA also averted diabetes-induced alterations in UPS (higher levels of E1, atrogin-1, UCHL1 and UCHL5, accumulation of ubiquitinated proteins and increased proteasomal activity). Apoptosis mediators-p53, BAX, and cleaved caspase-3 protein levels, and TUNEL positive cells were decreased in PBA treated diabetic rats. PBA notably improved the muscle-cross sectional area. ConclusionsResults highlighted the therapeutic potential of PBA in diabetes muscle wastage.

Protein Misfolding and ER Stress in Huntington's Disease.

Increasing evidence in recent years indicates that protein misfolding and aggregation, leading to ER stress, are central factors of pathogenicity in neurodegenerative diseases. This is particularly true in Huntington's disease (HD), where in contrast with other disorders, the cause is monogenic. Mutant huntingtin interferes with many cellular processes, but the fact that modulation of ER stress and of the unfolded response pathways reduces the toxicity, places these mechanisms at the core and gives hope for potential therapeutic approaches. There is currently no effective treatment for HD and it has a fatal outcome a few years after the start of symptoms of cognitive and motor impairment. Here we will discuss recent findings that shed light on the mechanisms of protein misfolding and aggregation that give origin to ER stress in neurodegenerative diseases, focusing on Huntington's disease, on the cellular response and on how to use this knowledge for possible therapeutic strategies.

Berberine inhibits free fatty acid and LPS‐induced inflammation via modulating ER stress response in macrophages

Inflammation plays a pivotal role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Berberine (BBR), an isoquinoline alkaloid isolated from many medicinal herbs, has been used to treat various diseases including liver diseases for hundreds of years. Our previous studies have shown that BBR inhibits high fat‐diet‐induced steatosis and inflammation in rat model. However, the underlying molecular mechanisms remain to be identified. This study was to identify the potential mechanism by which BBR inhibits free fatty acid (FFA) and LPS‐induced inflammatory response in mouse macrophages.MethodsMouse RAW2674.1 macrophages were treated with palmitic acid (PA) or LPS or PA+ LPS with or without different concentrations of BBR (0–10 μM) for different periods (0–24h). The mRNA and protein levels of IL‐1β, IL‐6, TNF‐α, MCP‐1, COX‐2, and ER stress genes (CHOP, ATF4, XBP‐1) were detected by real time RT‐PCR, Western blot and ELISA, respectively.ResultsBBR significantly inhibited PA‐ and LPS‐induced activation of ER stress and expression of proinflammatory cytokines (IL‐1β, IL‐6) and COX‐2. PA/LPS‐mediated activation of ERK1/2 was inhibited by BBR in a dose‐dependent manner.ConclusionBBR inhibits PA/LPS‐induced inflammatory responses through modulating ER stress‐mediated ERK1/2 activation in macrophages.Support or Funding InformationThis work was supported by VA Merit Award I01BX004033 and 1I01BX001390, VA Career Scientist Award IK6BX004477 and National Institutes of Health Grant R01 DK104893 and R01DK‐057543This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of PERK-eIF2α-ATF4 pathway contributes to diabetic hepatotoxicity: Attenuation of ER stress by Morin

Hyperglycemia associated ER stress has been found as a critical contributor in the pathogenesis of type 2 diabetes mellitus. However, reports regarding molecular mechanisms involved are limited. This study was aimed to identify the role of ER stress in regulating hepatic glucose metabolism and its link with oxidative stress. Further, this study explores the novel role of Morin, a flavonol, in modulating ER stress in STZ/nicotinamide induced type 2 diabetic male Wistar rats. Results demonstrate that hyperglycemia induced ER stress in rats and significantly lowered the expression of glucose transporter proteins resulting in impaired glucose metabolism during diabetes. Morin was found to downregulate PERK-eIF2α-ATF4 pathway by interacting with PERK protein as confirmed through pull-down assay. Additionally, Morin maintained the reducing environment in ER and enhanced PDI activity compared to diabetic rats. Morin prevented cell death by suppressing the expression of PERK dependent pro-apoptotic proteins including ATF4 and CHOP. Findings from this study affirm the role of ER stress in hyperglycemia induced gluco-metabolic aberrations and liver injury as confirmed by ISRIB, a standard chemical ER stress inhibitor. Notably, Morin promoted deactivation of UPR sensors and upregulated PDI activity endorsing its anti-ER stress potential which may allow the development of new therapeutic avenues to target hyperglycemic hepatotoxicity.

Gamma-Tocotrienol Attenuates the Hepatic Inflammation and Fibrosis by Suppressing Endoplasmic Reticulum Stress in Mice.

Gamma-tocotrienol (γT3), an unsaturated isoform of vitamin E, is implicated in the hepatoprotective effects. The aim is to determine the effectiveness of γT3 on nonalcoholic fatty liver disease (NAFLD). C57BL/6 male mice are fed a diet containing high fat (45%) and cholesterol (0.2%) along with sucrose drink (HFCS) or HFCS diet supplemented with 0.1% γT3 (HFCS+γT3). The inclusion of γT3 robustly decreases the HFCS diet-induced de novo lipogenesis (DNL), ER stress, and inflammation leading to reduced hepatic steatosis and fibrosis. Next, mice are fed a methionine- and choline-deficient (MCD) diet or MCD diet with γT3 (MCD+γT3). The γT3 supplementation significantly reduces the MCD diet-induced hepatic ER stress and fibrosis despite the minimal impact on steatosis. To further investigate the role of ER stress, the mice with genetic ablation of CHOP are fed an MCD or MCD+γT3 diet. CHOP deletion abolishes the γT3-mediated suppression of hepatic fibrosis, suggesting that modulation of ER stress is a prerequisite to inhibit hepatic inflammation and fibrosis. γT3 supplementation is effective in attenuating NAFLD and fibrosis through a synergistic mechanism of decreased DNL and hepatic ER stress. This work strongly supports the translational potential of γT3 supplementation against NAFLD.

Protein tyrosine phosphatase 1B regulates endothelial endoplasmic reticulum stress; role in endothelial dysfunction

Protein tyrosine phosphatase 1B (PTP1B) impairs nitric oxide (NO) production and induces endothelial dysfunction in various diseases, including diabetes, septic shock and heart failure. In non-cardiovascular tissues, PTP1B modulates endoplasmic reticulum stress (ERS) however this role has never been assessed in endothelial cells. We evaluated the link between PTP1B, ERS and endothelial dysfunction in mice.Induction of ERS (Tunicamycin) in vivo in mice or ex vivo in mouse arteries led to severe arterial endothelial dysfunction (i.e. reduced flow-dependent, NO mediated dilatation in isolated small mesenteric arteries), and this was prevented by the PTP1B inhibitor trodusquemine and absent in PTP1B−/− mice. Trodusquemine also prevented the Tunicamycin -induced increased arterial levels of the molecular ERS actors 78 kDa glucose-regulated protein (GRP78) and Activating Transcription Factor 6 (ATF6α). Tunicamycin strongly increased the interactions of PTP1B with GRP78 and the activated forms of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and IRE1α (proximity Ligation Assay).Thus, PTP1B plays a central role in the regulation of ERS in the endothelium, and the endothelial protective effect of PTP1B inhibition appears likely due at least in part to reduction of endothelial ERS, notably by promoting PERK protective pathway. Modulation of ER stress via PTP1B inhibitors may be a promising approach to protect the endothelium in cardiovascular diseases.