Chronic Endoplasmic Reticulum Stress Research Articles

4-Phenylbutyric acid mitigates ER stress-induced neurodegeneration in the spinal cords of a GM2 gangliosidosis mouse model.

Sandhoff disease (SD), a fatal and rare lysosomal storage disorder (LSD), is caused by a deficiency of the enzyme β-hexosaminidase B and leads to severe accumulation of GM2 gangliosides in lysosomes, primarily within the central nervous system (CNS). This accumulation results in severe neurological impairment, lower motor neuron disease, and death. Currently, there are no effective therapies available for SD. Here, we explored the role of endoplasmic reticulum (ER) stress in the spinal cord during disease progression in an established mouse model of SD and revealed the beneficial outcome of off-label treatment with the FDA-approved drug, 4-phenylbutyric acid (4-PBA). We analyzed the expression and localization of ER stress and cellular apoptosis markers, which revealed significant upregulation of these factors within motor neurons. Additionally, we observed a > 50% reduction in neuronal numbers throughout all spinal cord regions. Our studies also tested the impact of the chemical chaperone 4-PBA on ER stress in mice, and following administration, we observed significant improvements in motor neuromuscular function and life span throughout disease progression. 4-PBA treatment significantly reduced apoptosis in spinal cord neurons and increased the number of choline acetyltransferase (ChAT)-positive neurons, with little effect on astrogliosis or sensory interneurons. Overall, this study provides strong evidence for the role of chronic ER stress in the pathophysiology of SD and highlights 4-PBA as a promising therapeutic treatment for SD and potentially other related LSDs.

Sigma-1 receptor modulation by fluvoxamine ameliorates valproic acid-induced autistic behavior in rats: Involvement of chronic ER stress modulation, enhanced autophagy and M1/M2 microglia polarization

Autism spectrum disorder (ASD) is a neurodevelopmental disorder. While, fluvoxamine (FVX) is an antidepressant and widely prescribed to ASD patients, clinical results are inconclusive and the mechanism of FVX in the management of ASD is unclear. This study determined the potential therapeutic impact of FVX, a sigma-1 receptor (S1R) agonist, against the valproic acid (VPA)-induced model of autism. On gestational day 12.5, Wistar pregnant rats were given a single intraperitoneal (i.p.) injection of either VPA (600 mg/kg) or normal saline (10 mL/kg, vehicle-control). Starting on postnatal day (PND) 21 to PND 50, FVX (30 mg/kg, P·O. daily) and NE-100, (S1R) antagonist, (1 mg/kg, i.p. daily) were given to male pups. Behavior tests and histopathological changes were identified at the end of the experiment. In addition, the cerebellum biomarkers of endoplasmic reticulum (ER) stress and autophagy were assessed. Microglial cell polarization to M1 and M2 phenotypes was also assessed. FVX effectively mitigated the histopathological alterations in the cerebellum caused by VPA. FVX enhanced sociability and stereotypic behaviors in addition to its noteworthy impact on autophagy enhancement, ER stress deterioration, and controlling microglial cell polarization. The current investigation confirmed that the S1R agonist, FVX, can lessen behavioral and neurochemical alterations in the VPA-induced rat model of autism.

Interaction of PASTICCINO2 with Golgi anti-apoptotic proteins confers resistance to endoplasmic reticulum stress and is dependent on very-long-chain fatty acids.

The endoplasmic reticulum (ER) is crucial for maintaining cell homeostasis because it is the primary site for synthesizing secreted and transmembrane proteins and lipids. The unfolded protein response (UPR) is activated to restore the homeostasis of the ER when it is under stress; however, the relationship between lipids and the ER stress response in plants is not well understood. Arabidopsis GOLGI ANTI-APOPTOTIC PROTEINS (GAAPs) are involved in resisting ER stress, and in this study, we found that PASTICCINO2 (PAS2), which is involved in very-long-chain fatty acid (VLCFA) synthesis, interacts with GAAPs and INOSITOL REQUIRING ENZYME 1. The pas2 single-mutant and the gaap1 pas2 and gaap2 pas2 double-mutants exhibited increased seedling damage and an impaired UPR response under chronic ER stress. Site mutation combined with genetic analysis revealed that the role of PAS2 in resisting ER stress depended on its VLCFA synthesis domain. VLCFA contents were increased under ER stress, and this required GAAPs. Exogenous VLCFAs partially restored the defect in the activation of the UPR caused by mutation of PAS2 or GAAP under chronic ER stress. Our findings demonstrate that the association of PAS2 with GAAPs confers plant resistance to ER stress by regulating VLCFA synthesis and the UPR. This provides a basis for further studies on the connection between lipids and cell-fate decisions under stress.

IL-18BP Therapy Ameliorates Reproductive and Metabolic Phenotypes in a PCOS Mouse Model by Relieving Inflammation, Fibrosis and Endoplasmic Reticulum Stress.

There is a chronic inflammation in PCOS patients, which is correlated with the pathogenesis of PCOS. IL-18 and IL-18BP are related with some inflammatory diseases, while less explored in PCOS. Whether IL-18BP could be a potential drug of PCOS remains unknown.IL-18 and testosterone levels were evaluated in serum of 10 non-PCOS control patients and 20 PCOS patients. Female C57/BL6 mice were gavaged with letrozole to induce PCOS mouse model and IL-18 level was evaluated in the serum of PCOS mouse model, and IL-18 is intraperitoneally injected in female mice, IL-18BP is intraperitoneally injected in the PCOS mice models. Then the body weights, estrous cycles, reproductive hormones and morphology of ovaries were analyzed. The level of ovarian chronic inflammation, fibrosis and endoplasmic reticulum (ER) stress are evaluated.IL-18 levels are increased in the serum of PCOS patients and PCOS mice models respectively. The serum DHEAS, iWAT weight and adipocyte size were increased in IL-18 group compared to the control group (P < 0.05). In the PCOS mouse model treated with IL-18BP, the body weight and serum LH/FSH ratio was decreased compared to the PCOS group (P < 0.05). The expression levels of inflammatory factors and fibrosis-related genes, the expression level of endoplasmic reticulum stress-related genes, and the ROS positive area of ovarian tissue was decreased (P < 0.05).IL-18 is involved in inducing PCOS phenotypes, while IL-18BP relieves PCOS phenotypes by alleviating ovarian chronic inflammation, fibrosis and ER stress in PCOS mice.

Endoplasmic reticulum stress-induced senescence in human lung fibroblasts.

Loss of proteostasis and cellular senescence have been previously established as characteristics of aging; however, their interaction in the context of lung aging and potential contributions to aging-associated lung remodeling remains understudied. In this study, we aimed to characterize endoplasmic reticulum (ER) stress response, cellular senescence, and their interaction in relation to extracellular matrix (ECM) production in lung fibroblasts from young (25-45 yr) and old (>60 yr) humans. Fibroblasts from young and old patients without significant preexisting lung disease were exposed to vehicle, MG132, etoposide, or salubrinal. Afterward, cells and cell lysates or supernatants were analyzed for ER stress, cellular senescence, and ECM changes using protein analysis, proliferation assay, and senescence-associated beta-galactosidase (SA-β-Gal) staining. At baseline, fibroblasts from aging individuals showed increased levels of ER stress (ATF6 and PERK), senescence (p21 and McL-1), and ECM marker (COL1A1) compared to those from young individuals. Upon ER stress induction and etoposide exposure, fibroblasts showed an increase in senescence (SA-β-Gal, p21, and Cav-1), ER stress (PERK), and ECM markers (COL1A1 and LUM) compared to vehicle. Additionally, IL-6 and IL-8 levels were increased in the supernatants of MG132- and etoposide-treated fibroblasts, respectively. Finally, the ER stress inhibitor salubrinal decreased the expression of p21 compared to vehicle and MG132 treatments; however, salubrinal inhibited COL1A1 but not p21 expression in MG132-treated fibroblasts. Our study suggests that ER stress response plays an important role in establishment and maintenance of a senescence phenotype in lung fibroblasts and therefore contributes to altered remodeling in the aging lung.NEW & NOTEWORTHY The current study establishes functional links between endoplasmic reticulum (ER) stress and cellular senescence per se in the specific context of aging human lung fibroblasts. Recognizing that the process of aging per se is complex, modulated by the myriad of lifelong and environmental exposures, it is striking to note that chronic ER stress may play a crucial role in the establishment and maintenance of cellular senescence in lung fibroblasts.

Lithium-Induced Disruption of Intracellular Ca2+ Balance in the Collecting Duct Intercalated Cells Contributes to Metabolic Acidosis in Mice

INTRODUCTION. Lithium (Li+) is an effective mood stabilizer that continues to be widely used in modern psychiatric practice. One of the most common adverse effects of Li+ therapy is nephrogenic diabetes insipidus manifesting in reduced urinary concentrating ability due to impaired vasopressin (AVP) signaling in the collecting duct principal cells. While distal tubular acidosis has also been reported in patients receiving Li+, molecular determinants underlying the pathophysiological effects of Li+ on the collecting duct intercalated cells and renal acid-base transport remain to be fully elucidated. Activation of Gq-coupled vasopressin 1a receptors (V1aR) in A-intercalated cells reportedly induces intracellular Ca2+ ([Ca2+]i) mobilization and stimulates luminal H+ secretion, resulting in urine acidification. OBJECTIVE. The objective of this study was to test if Li+ impaired V1aR-induced [Ca2+]i response in the intercalated cells of murine collecting ducts. HYPOTHESIS. We hypothesized that Li+ markedly impaired V1aR-dependent [Ca2+]i signaling in A-intercalated cells causing the reduction of luminal proton secretion in the collecting duct. METHODS. We combined immunofluorescent imaging in freshly isolated collecting duct segments with metabolic cage studies in C57BL/6NJ (Jackson Labotatory) mice receiving a regular Teklad 2918 chow (control) or a Teklad 2918-based diet containing 0.3% lithium carbonate (Li+-treated) to evaluate how Li+ affects V1aR-mediated [Ca2+]i signal, H+ transport by intercalated cells, as well as urinary pH and ammonia excretion. RESULTS. We found that AVP elicited a transient [Ca2+]i response in the aquaporin-2 (AQP2) negative cells from collecting duct segments isolated from control mice. The response was mediated by Ca2+ release from the endoplasmic reticulum (ER), as it was inhibited by pretreatment of collecting duct segments with thapsigargin, a SERCA pump inhibitor. The amplitude of the AVP-induced calcium transient was 4 times lower in the AQP2-negative cells isolated from Li+-treated mice. Li+ intake remarkably facilitated store-operated calcium entry (SOCE) in the collecting duct cells, indicating chronic ER depletion and stress. At the systemic level Li+-treated mice exhibited urine alkalinization (by 0.8 pH units) and a 6-fold elevation in urinary ammonium excretion, when compared to controls. CONCLUSIONS. Intracellular Ca2+ signaling in the intercalated cells is an essential determinant of acid-base handling in the collecting duct. Our findings reveal that Li+ markedly alters [Ca2+]i signaling in the intercalated cells, causing urine alkalinization and elevated ammonium excretion, likely, to compensate for ensuing metabolic acidosis. NIDDK R01DK125464. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Gene Expression Profiling to Unfolded Proteins Response as a Risk Modulator of Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease. Despite new methods of diagnostics and treatment as well as extensive biological and immunosuppressive treatment, the etiology of RA is not fully understood. Moreover, the problem of diagnosis and treatment of RA patients is still current and affects a large group of patients. It is suggested that endoplasmic reticulum (ER)-related features may impair adaptation to chronic stress, inferring the risk of rheumatoid arthritis. The main goal in this study was evaluation of changes in mRNA translation to determine chronic ER stress conditions in rheumatoid arthritis patients. The study group consist of 86 individuals including a total of 56 rheumatoid arthritis patients and 30 healthy controls. The expression level of mRNA form blood samples of RA patients as well as controls of the unfolded protein response (UPR)-associated genes (p-eIF2, BCL-2, PERK, ATF4, and BAX) were investigated using real-time qPCR. GAPDH expression was used as a standard control. Considering the median, the expression levels of PERK, BCL-2, p-eIF2, ATF4, and BAX were found to be significantly increased in the blood of RA patients compared with the control group. The p-value for the PERK gene was 0.0000000036, the p-value for the BCL-2 gene was 0.000000014, the p-value for the p-eIF2 gene was 0.006948, the p-value for the ATF4 gene was 0.0000056, and the p-value for the BAX gene was 0.00019, respectively. Thus, it can be concluded that the targeting of the components of the PERK-dependent UPR signaling pathway via small-molecule PERK inhibitors may contribute to the development of novel, innovative treatment strategies against rheumatoid arthritis.

IRE1 signaling increases PERK expression during chronic ER stress

The Unfolded Protein Response (UPR) is an essential cellular process activated by the accumulation of unfolded proteins within the Endoplasmic Reticulum (ER), a condition referred to as ER stress. Three ER anchored receptors, IRE1, PERK and ATF6 act as ER stress sensors monitoring the health of the ER. Upon detection of ER stress, IRE1, PERK and ATF6 initiate downstream signaling pathways collectively referred to as the UPR. The overarching aim of the UPR is to restore ER homeostasis by reducing ER stress, however if that is not possible, the UPR transitions from a pro-survival to a pro-death response. While our understanding of the key signaling pathways central to the UPR is well defined, the same is not true of the subtle signaling events that help fine tune the UPR, supporting its ability to adapt to varying amplitudes or durations of ER stress. In this study, we demonstrate cross talk between the IRE1 and PERK branches of the UPR, wherein IRE1 via XBP1s signaling helps to sustain PERK expression during prolonged ER stress. Our findings suggest cross talk between UPR branches aids adaptiveness thereby helping to support the plasticity of UPR signaling responses.

ER stress signaling at the interphase between MASH and HCC

HCC is the most frequent primary liver cancer with an extremely poor prognosis and often develops on preset of chronic liver diseases. Major risk factors for HCC include metabolic dysfunction–associated steatohepatitis, a complex multifactorial condition associated with abnormal endoplasmic reticulum (ER) proteostasis. To cope with ER stress, the unfolded protein response engages adaptive reactions to restore the secretory capacity of the cell. Recent advances revealed that ER stress signaling plays a critical role in HCC progression. Here, we propose that chronic ER stress is a common transversal factor contributing to the transition from liver disease (risk factor) to HCC. Interventional strategies to target the unfolded protein response in HCC, such as cancer therapy, are also discussed.

Abstract LB191: A genome-wide CRISPR-based functional screen uncovers the KLF4-Survivin/BIRC5 signaling pathways as a key regulator of cell fate following endoplasmic reticulum stress: Implications for tumor growth and therapeutic targeting

Abstract The growth and spread of tumors involve intricate interactions with the surrounding tissue microenvironment. As a tumor develops, it faces various stresses from its microenvironment, including hypoxia, nutrient deficiency, and acidosis. In response to these challenges, cancer cells can hijack existing cytoprotective mechanisms, such as the Unfolded Protein Response (UPR). The UPR activates signaling pathways at the translational and later transcriptional levels to alleviate cellular stress and prevent cell death. However, under unresolved or chronic endoplasmic reticulum (ER) stress, the UPR may paradoxically promote cell death. The precise mechanisms through which the UPR influences cellular fate during ER stress remain poorly understood. To address this knowledge gap, we employed a functional CRISPR-based genetic knockout screen to identify novel regulators of the UPR. Using a lentiviral genome-wide CRISPR-Cas9 knockout library, we identified over-represented sgRNAs (associated with pro-apoptotic genes) and underrepresented sgRNAs (associated with pro-survival genes) following ER stress induced by thapsigargin and tunicamycin. One of the top candidates identified was Survivin/BIRC5, a protein with anti-apoptotic and cell cycle regulatory activities and which is overexpressed in tumor cells compared to healthy tissues. Functional studies revealed that genetic or pharmacological inhibition of Survivin expression enhanced ER stress-induced apoptosis, but not apoptosis in response to other forms of stress. These findings were validated in multiple cancer cell lines, ruling out off-target effects. Mechanistically, ER stress led to PERK-dependent downregulation of Survivin, and overexpression of Survivin blocked Thapsigargin-induced apoptosis. In vivo xenograft models showed that combining Survivin ablation with PERK inhibition significantly delayed tumor growth and improved overall survival. Through a combination of bioinformatics analysis and loss-of-function studies, we identified the transcription factor KLF4 as a mediator of PERK-dependent effects on Survivin repression. Notably, CRISPR-mediated KLF4 knockout successfully rescued Survivin levels post-ER stress. Additionally, an analysis of TCGA data across various tumor types provided supporting evidence of a negative correlation between KLF4 and Survivin expression. In summary, this study unveils a novel regulatory axis, PERK-KLF4-Survivin, as a key determinant of cell fate in the cellular responses to ER stress. Targeting Survivin in conjunction with PERK inhibition showed promising antitumor effects in vivo. Citation Format: Nektaria M. Leli, Hengxi Liu, Souvik Dey, Lauren Brady, Carlo Salas Salinas, Victoria Wu, Spiros Tastsoglou, Giorgos Skoufos, Ioannis Verginadis, Ilias Karagounis, Artemis G. Hatzigeorgiou, Constantinos Koumenis. A genome-wide CRISPR-based functional screen uncovers the KLF4-Survivin/BIRC5 signaling pathways as a key regulator of cell fate following endoplasmic reticulum stress: Implications for tumor growth and therapeutic targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB191.

Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOCY437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

Chronic endoplasmic reticulum stress in myotonic dystrophy type 2 promotes autoimmunity via mitochondrial DNA release

Myotonic dystrophy type 2 (DM2) is a tetranucleotide CCTG repeat expansion disease associated with an increased prevalence of autoimmunity. Here, we identified an elevated type I interferon (IFN) signature in peripheral blood mononuclear cells and primary fibroblasts of DM2 patients as a trigger of chronic immune stimulation. Although RNA-repeat accumulation was prevalent in the cytosol of DM2-patient fibroblasts, type-I IFN release did not depend on innate RNA immune sensors but rather the DNA sensor cGAS and the prevalence of mitochondrial DNA (mtDNA) in the cytoplasm. Sublethal mtDNA release was promoted by a chronic activation of the ATF6 branch of the unfolded protein response (UPR) in reaction to RNA-repeat accumulation and non-AUG translated tetrapeptide expansion proteins. ATF6-dependent mtDNA release and resulting cGAS/STING activation could also be recapitulated in human THP-1 monocytes exposed to chronic endoplasmic reticulum (ER) stress. Altogether, our study demonstrates a novel mechanism by which large repeat expansions cause chronic endoplasmic reticulum stress and associated mtDNA leakage. This mtDNA is, in turn, sensed by the cGAS/STING pathway and induces a type-I IFN response predisposing to autoimmunity. Elucidating this pathway reveals new potential therapeutic targets for autoimmune disorders associated with repeat expansion diseases.

Stress and the domestic cat: have humans accidentally created an animal mimic of neurodegeneration?

Many neurodegenerative diseases (NDD) appear to share commonality of origin, chronic ER stress. The endoplasmic reticulum (ER) is a dynamic organelle, functioning as a major site of protein synthesis and protein posttranslational modifications, required for proper folding. ER stress can occur because of external stimuli, such as oxidative stress or neuroinflammatory cytokines, creating the ER luminal environment permissive for the accumulation of aggregated and misfolded proteins. Unresolvable ER stress upregulates a highly conserved pathway, the unfolded protein response (UPR). Maladaptive chronic activation of UPR components leads to apoptotic neuronal death. In addition to other factors, physiological responses to stressors are emerging as a significant risk factor in the etiology and pathogenesis of NDD. Owned cats share a common environment with people, being exposed to many of the same stressors as people and additional pressures due to their "quasi" domesticated status. Feline Cognitive Dysfunction Syndrome (fCDS) presents many of the same disease hallmarks as human NDD. The prevalence of fCDS is rapidly increasing as more people welcome cats as companions. Barely recognized 20 years ago, veterinarians and scientists are in infancy stages in understanding what is a very complex disease. This review will describe how cats may represent an unexplored animal mimetic phenotype for human NDD with stressors as potential triggering mechanisms. We will consider how multiple variations of stressful events over the short-life span of a cat could affect neuronal loss or glial dysfunction and ultimately tip the balance towards dementia.

Incretin Mimetics Restore the ER-Mitochondrial Axis and Switch Cell Fate Towards Survival in LUHMES Dopaminergic-Like Neurons: Implications for Novel Therapeutic Strategies in Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder that afflicts more than 10 million people worldwide. Available therapeutic interventions do not stop disease progression. The etiopathogenesis of PD includes unbalanced calcium dynamics and chronic dysfunction of the axis of the endoplasmic reticulum (ER) and mitochondria that all can gradually favor protein aggregation and dopaminergic degeneration. In Lund Human Mesencephalic (LUHMES) dopaminergic-like neurons, we tested novel incretin mimetics under conditions of persistent, calcium-dependent ER stress. We assessed the pharmacological effects of Liraglutide-a glucagon-like peptide-1 (GLP-1) analog-and the dual incretin GLP-1/GIP agonist DA3-CH in the unfolded protein response (UPR), cell bioenergetics, mitochondrial biogenesis, macroautophagy, and intracellular signaling for cell fate in terminally differentiated LUHMES cells. Cells were co-stressed with the sarcoplasmic reticulum calcium ATPase (SERCA) inhibitor, thapsigargin. We report that Liraglutide and DA3-CH analogs rescue the arrested oxidative phosphorylation and glycolysis. They mitigate the suppressed mitochondrial biogenesis and hyper-polarization of the mitochondrial membrane, all to re-establish normalcy of mitochondrial function under conditions of chronic ER stress. These effects correlate with a resolution of the UPR and the deficiency of components for autophagosome formation to ultimately halt the excessive synaptic and neuronal death. Notably, the dual incretin displayed a superior anti-apoptotic effect, when compared to Liraglutide. The results confirm the protective effects of incretin signaling in ER and mitochondrial stress for neuronal degeneration management and further explain the incretin-derived effects observed in PD patients.

Adaptive responses of neuronal cells to chronic endoplasmic reticulum (ER) stress

Accumulation of misfolded proteins or perturbation of calcium homeostasis leads to endoplasmic reticulum (ER) stress and is linked to the pathogenesis of neurodegenerative diseases. Hence, understanding the ability of neuronal cells to cope with chronic ER stress is of fundamental interest. Interestingly, several brain areas uphold functions that enable them to resist challenges associated with neurodegeneration. Here, we established novel clonal mouse hippocampal (HT22) cell lines that are resistant to prolonged (chronic) ER stress induced by thapsigargin (TgR) or tunicamycin (TmR) as in vitro models to study the adaption to ER stress. Morphologically, we observed a significant increase in vesicular und autophagosomal structures in both resistant lines and ‘giant lysosomes’, especially striking in TgR cells. While autophagic activity increased under ER stress, lysosomal function appeared slightly impaired; in both cell lines, we observed enhanced ER-phagy. However, proteomic analyses revealed that various protein clusters and signaling pathways were differentially regulated in TgR versus TmR cells in response to chronic ER stress. Additionally, bioenergetic analyses in both resistant cell lines showed a shift toward aerobic glycolysis (‘Warburg effect’) and a defective complex I of the oxidative phosphorylation (OXPHOS) machinery. Furthermore, ER stress-resistant cells differentially activated the unfolded protein response (UPR) comprising IRE1α and ATF6 pathways. These findings display the wide portfolio of adaptive responses of neuronal cells to chronic ER stress. ER stress-resistant neuronal cells could be the basis to uncover molecular modulators of adaptation, resistance, and neuroprotection as potential pharmacological targets for preventing neurodegeneration.

Large HBV Surface Protein-Induced Unfolded Protein Response Dynamically Regulates p27 Degradation in Hepatocellular Carcinoma Progression.

Up to 50% of hepatocellular carcinoma (HCC) is caused by hepatitis B virus (HBV) infection, and the surface protein of HBV is essential for the progression of HBV-related HCC. The expression of large HBV surface antigen (LHB) is presented in HBV-associated HCC tissues and is significantly associated with the development of HCC. Gene set enrichment analysis revealed that LHB overexpression regulates the cell cycle process. Excess LHB in HCC cells induced chronic endoplasmic reticulum (ER) stress and was significantly correlated with tumor growth in vivo. Cell cycle analysis showed that cell cycle progression from G1 to S phase was greatly enhanced in vitro. We identified intensive crosstalk between ER stress and cell cycle progression in HCC. As an important regulator of the G1/S checkpoint, p27 was transcriptionally upregulated by transcription factors ATF4 and XBP1s, downstream of the unfolded protein response pathway. Moreover, LHB-induced ER stress promoted internal ribosome-entry-site-mediated selective translation of p27, and E3 ubiquitin ligase HRD1-mediated p27 ubiquitination and degradation. Ultimately, the decrease in p27 protein levels reduced G1/S arrest and promoted the progress of HCC by regulating the cell cycle.

MTORC1 inhibition impairs activation of the unfolded protein response and induces cell death during ER stress in cardiomyocytes.

The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of protein synthesis that senses and responds to a variety of stimuli to coordinate cellular metabolism with environmental conditions. To ensure that protein synthesis is inhibited during unfavorable conditions, translation is directly coupled to the sensing of cellular protein homeostasis. Thus, translation is attenuated during endoplasmic reticulum (ER) stress by direct inhibition of the mTORC1 pathway. However, residual mTORC1 activity is maintained during prolonged ER stress which is thought to be involved in translational reprogramming and adaption to ER stress. By analyzing the dynamics of mTORC1 regulation during ER stress, we unexpectedly found that mTORC1 is transiently activated in cardiomyocytes within minutes at the onset of ER stress before being inhibited during chronic ER stress. This dynamic regulation of mTORC1 appears to be mediated, at least in part, by ATF6, as its activation was sufficient to induce the biphasic control of mTORC1. We further showed that protein synthesis remains dependent on mTORC1 throughout the ER stress response and that mTORC1 activity is essential for posttranscriptional induction of several unfolded protein response elements. Pharmacological inhibition of mTORC1 increased cell death during ER stress, indicating that the mTORC1 pathway serves adaptive functions during ER stress in cardiomyocytes potentially by controlling the expression of the protective unfolded protein response.

MicroRNA Biogenesis in Cell Senescence Induced by Chronic Endoplasmic Reticulum Stress

MicroRNA Biogenesis in Cell Senescence Induced by Chronic Endoplasmic Reticulum Stress

Identification of potential phytochemicals and their inhibitory effect on the PERK receptor mediated UPR pathway for neuronal disease regulation: an in silico insight

The endoplasmic reticulum (ER) has been considered as the key site of protein biosynthesis and maturation in the eukaryotic cell. In recent years, the sequence at the N-terminal region of translated protein has shown a particular emphasis as a signal responsible for site-specific translocation mediated by post-translational modification. Once the native conformation is not achieved, the degradation pathway is activated, and therefore the restoration of the homeostasis of ER function in UPR pathway is initiated. One of the transmembrane proteins, PKR-like ER kinase (PERK) plays a key role in the activation of UPR through the inhibition of the translation process, thus preventing the cells from apoptosis due to chronic ER stress. Dysregulation of the neuronal proteostasis often results in neuronal dysfunction and its crucially associated neurodegenerative diseases or its manifestation of neuropathic pain. The correlation between ER stress and its associated signaling cascade, namely UPR, is well established in context of neuropathological modifications. This furthermore suggests that the proteins of the signaling cascade such as PERK can serve as a potential target during the onset of neuronal damage. The aim of this study was to identify the potential phytocompounds by evaluating the physicochemical properties, Lipinski screening, ADMET and toxicity properties of the selected phytocompounds by using SwissADME, MolInspiration and pKCSM webservers, which could establish a comparatively better affinity and binding energy than the control drug as GSK2606414 in set up the treatment of the neuronal diseases through molecular docking via PyRx and validating their structural stability through simulation using the Sybyl software for over100ns. Communicated by Ramaswamy H. Sarma

MicroRNA Biogenesis during Cellular Senesence Induced by Chronic Stress of the Endoplasmic Reticulum

MicroRNAs are small non-coding regulatory RNAs about 22 nt long, post-transcriptional and transcriptional regulators of gene expression that stabilize the cellular phenotype and play an important role in differentiation, development, and apoptosis. MicroRNA biogenesis includes several precisely controlled post-transcriptional stages of processing and transport, including cytoplasmic cleavage of pre-miRNA by type III ribonuclease DICER with the formation of a mature duplex included in the RISC complex. The role of miRNA and its biogenesis are not well understood in such an important process as cellular stress. Cellular stress is a non-specific cellular response to non-physiological stimuli that can switch a cell to death or cellular senescence. The global decrease in microRNA levels is a key feature of cancer cells and an important reason for the formation of a malignant phenotype. In this work, using flow cytometry and high-throughput analysis of gene expression, we showed that chronic endoplasmic reticulum (ER) stress, one of the types of cellular stress associated with impaired protein folding in the ER, leads to the formation of a cellular aging phenotype in fibroblast-like FRSN cells. Despite the fact that acute ER stress can reduce miRNA biogenesis, chronic stress does not lead to a significant drop in global miRNA expression and is accompanied by only a slight decrease in DICER1 mRNA expression. Under chronic ER stress, we found an increase in cell population heterogeneity in terms of lysosomal beta-galactosidase activity, which does not exclude induced or initial cell heterogeneity and in terms of expression of microRNA biogenesis pathway components.