Induction of the unfolded protein response and cell death pathway in Alzheimer's disease, but not in aged Tg2576 mice.
ABSTRACT: The endoplasmic reticulum (ER) stress results from disrupted protein folding triggered by protein mutation or oxidation, reduced proteasome activity, and altered Ca2+ homeostasis. ER stress is accompanied by activation of the unfolded protein response (UPR) and cell death pathway. We examined if the UPR and cell death pathway would be activated in Alzheimer's disease (AD). RT-PCR experiments revealed increased splicing of X-box binding protein-1 (XBP-1), an UPR transcription factor, in AD compared with age-matched control. Among target genes of XBP-1, expression of protein disulfide isomerase (PDI), but not glucose-regulated protein 78 (GRP78), was increased in AD, suggesting disturbed activation of the UPR in AD. C/EBP homologous protein (CHOP), caspase-3, caspase-4, and caspase-12, downstream mediators of cell death pathway, were activated in AD. Neither the UPR nor cell death pathway was induced in aged Tg2576 mice, a transgenic mouse model of Alzheimer's disease that reveals both plaque pathology and some cognitive deficits. The present study suggests that disturbed induction of the UPR and activation of the pro-apoptotic proteins contribute to neuropathological process in AD irrespective of amyloid beta and senile plaque.
Project description:3-O-trans-p-coumaroyl-alphitolic acid (3OTPCA), a triterpenoid isolated from the plant Zizyphus jujuba (ZJ), is known to be cytotoxic to cancer cells; however, the molecular mechanism underlying 3OTPCA-induced cell death remains unknown. Here, we provide novel evidence that 3OTPCA induces apoptotic cell death in human leukemia cells. We found that 3OPTCA induces DNA fragmentation within 24 h after treatment in U937 cells, which was also observed in other leukemia cell lines, including Molt-4 and Jurkat cells. We then investigated other parameters involved in apoptosis, including phosphatidylserine externalization and caspase-3 cleavage in U937 cells treated with 3OTPCA. 3OTPCA caused significant DNA fragmentation, annexin-V binding, and caspase-3 cleavage, indicating that 3OTPCA exerts cytotoxicity through apoptosis induction. RNA-seq analysis revealed that the expression of transcripts associated with the unfolded protein response (UPR), such as spliced XBP-1 and CHOP, were up-regulated by 3OTPCA treatment. 3OTPCA-induced UPR activation may be due to endoplasmic reticulum (ER) stress because both 3OTPCA and thapsigargin, an endoplasmic Ca2+ transport ATPase inhibitor, increased intracellular calcium levels. 3OTPCA down-regulated the expression of Bcl-2, a target of CHOP, and led to the loss of the mitochondrial membrane, indicating that the intrinsic (mitochondrial) apoptotic pathway was triggered by 3OTPCA, likely through UPR activation. Furthermore, we found that 3OTPCA induced superoxide anion generation and, following p38 MAPK phosphorylation, caspase-8 cleavage without affecting Fas expression. It also induced subsequent Bid cleavage, which may enhance the apoptosis triggered by the intrinsic pathway. These findings reveal for the first time that 3OTPCA induces apoptotic cell death through the generation of reactive oxygen species and activation of UPR.
Project description:The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). We have investigated here the contribution of the UPR transcription factors XBP-1, ATF6alpha, and ATF6beta to UPR target gene expression. Gene profiling of cell lines lacking these factors yielded several XBP-1-dependent UPR target genes, all of which appear to act in the ER. These included the DnaJ/Hsp40-like genes, p58(IPK), ERdj4, and HEDJ, as well as EDEM, protein disulfide isomerase-P5, and ribosome-associated membrane protein 4 (RAMP4), whereas expression of BiP was only modestly dependent on XBP-1. Surprisingly, given previous reports that enforced expression of ATF6alpha induced a subset of UPR target genes, cells deficient in ATF6alpha, ATF6beta, or both had minimal defects in upregulating UPR target genes by gene profiling analysis, suggesting the presence of compensatory mechanism(s) for ATF6 in the UPR. Since cells lacking both XBP-1 and ATF6alpha had significantly impaired induction of select UPR target genes and ERSE reporter activation, XBP-1 and ATF6alpha may serve partially redundant functions. No UPR target genes that required ATF6beta were identified, nor, in contrast to XBP-1 and ATF6alpha, did the activity of the UPRE or ERSE promoters require ATF6beta, suggesting a minor role for it during the UPR. Collectively, these results suggest that the IRE1/XBP-1 pathway is required for efficient protein folding, maturation, and degradation in the ER and imply the existence of subsets of UPR target genes as defined by their dependence on XBP-1. Further, our observations suggest the existence of additional, as-yet-unknown, key regulators of the UPR.
Project description:The increased demand on protein folding in the endoplasmic reticulum (ER) during bacterial infection activates the unfolded protein response (UPR). OCTR-1--a G protein-coupled catecholamine receptor expressed in neurons--suppresses innate immunity by downregulating a non-canonical UPR pathway and the p38 MAPK pathway. Here, we show that OCTR-1 also regulates the canonical UPR pathway, which is controlled by XBP-1, at the organismal level. Importantly, XBP-1 is not under OCTR-1 control during development, only at the adult stage. Our results indicate that the nervous system temporally controls the UPR pathway to maintain ER homeostasis during development and immune activation.
Project description:Adaptation to endoplasmic reticulum (ER) stress depends on the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). Bax inhibitor-1 (BI-1) is an evolutionarily conserved ER-resident protein that suppresses cell death. Here we have investigated the role of BI-1 in the UPR. BI-1 expression suppressed IRE1alpha activity in fly and mouse models of ER stress. BI-1-deficient cells displayed hyperactivation of the ER stress sensor IRE1alpha, leading to increased levels of its downstream target X-box-binding protein-1 (XBP-1) and upregulation of UPR target genes. This phenotype was associated with the formation of a stable protein complex between BI-1 and IRE1alpha, decreasing its ribonuclease activity. Finally, BI-1 deficiency increased the secretory activity of primary B cells, a phenomenon regulated by XBP-1. Our results suggest a role for BI-1 in early adaptive responses against ER stress that contrasts with its known downstream function in apoptosis.
Project description:Evidence implicating dysregulation of the IRE1/XBP-1s arm of the unfolded protein response (UPR) in cancer pathogenesis (e.g., multiple myeloma) has prompted the development of IRE1 RNase inhibitors. Here, effects of cyclin-dependent kinase (CDK) inhibitor SCH727965 (dinaciclib) on the IRE1 arm of the UPR were examined in human leukemia and myeloma cells. Exposure of cells to extremely low (e.g., nmol/L) concentrations of SCH727965, a potent inhibitor of CDKs 1/2/5/9, diminished XBP-1s and Grp78 induction by the endoplasmic reticulum (ER) stress-inducers thapsigargin and tunicamycin, while sharply inducing cell death. SCH727965, in contrast to IRE1 RNase inhibitors, inhibited the UPR in association with attenuation of XBP-1s nuclear localization and accumulation rather than transcription, translation, or XBP-1 splicing. Notably, in human leukemia cells, CDK1 and 5 short hairpin RNA (shRNA) knockdown diminished Grp78 and XBP-1s upregulation while increasing thapsigargin lethality, arguing for a functional role for CDK1/5 in activation of the cytoprotective IRE1/XBP-1s arm of the UPR. In contrast, CDK9 or 2 inhibitors or shRNA knockdown failed to downregulate XBP-1s or Grp78. Furthermore, IRE1, XBP-1, or Grp78 knockdown significantly increased thapsigargin lethality, as observed with CDK1/5 inhibition/knockdown. Finally, SCH727965 diminished myeloma cell growth in vivo in association with XBP-1s downregulation. Together, these findings demonstrate that SCH727965 acts at extremely low concentrations to attenuate XBP-1s nuclear accumulation and Grp78 upregulation in response to ER stress inducers. They also highlight a link between specific components of the cell-cycle regulatory apparatus (e.g., CDK1/5) and the cytoprotective IRE1/XBP-1s/Grp78 arm of the UPR that may be exploited therapeutically in UPR-driven malignancies.
Project description:Tumors encounter endoplasmic reticulum stress during tumor growth and activate an adaptive pathway known as the unfolded protein response (UPR). Because this pathway is induced by the tumor microenvironment, it is a promising target for cancer therapy. We have previously demonstrated that X-box binding protein 1 (XBP-1), a key regulator of the UPR, was required for survival under hypoxia and critical for tumor growth in tumor xenografts. In this study, we investigated the role of XBP-1 in regulating tumor angiogenesis.We used an intradermal angiogenesis model to quantify the effect of XBP-1 on angiogenesis. We also used a human tumor xenograft model to assay for tumor growth delay. We determined vascular endothelial growth factor (VEGF) expression by quantitative polymerase chain reaction and ELISA. Finally, we stained human pancreatic adenocarcinoma specimens for XBP-1 expression and correlated the expression pattern of XBP-1 with CD31 (endothelial cell marker) expression.We demonstrated that XBP-1 is essential for angiogenesis during early tumor growth. Inhibiting XBP-1 expression by short-hairpin RNA sequence specific for XBP-1 reduced blood vessel formation in tumors from mouse embryonic fibroblast cells and human fibrosarcoma tumor cells (HT1080). Expressing a dominant-negative form of IRE1alpha also reduced blood vessel formation in tumors. Moreover, expression of spliced XBP-1 (XBP-1s) restored angiogenesis in IRE1alpha dominant-negative expressing cells. We further demonstrated that XBP-1-mediated angiogenesis does not depend on VEGF.We propose that the IRE1alpha-XBP-1 branch of the UPR modulates a complex proangiogenic, VEGF-independent response that depends on signals received from the tumor microenvironment.
Project description:Alzheimer's disease (AD) is characterized by the deposition of aggregated beta-amyloid (Abeta), which triggers a cellular stress response called the unfolded protein response (UPR). The UPR signaling pathway is a cellular defense system for dealing with the accumulation of misfolded proteins but switches to apoptosis when endoplasmic reticulum (ER) stress is prolonged. ER stress is involved in neurodegenerative diseases including AD, but the molecular mechanisms of ER stress-mediated Abeta neurotoxicity still remain unknown. Here, we show that treatment of Abeta triggers the UPR in the SK-N-SH human neuroblastoma cells. Abeta mediated UPR pathway accompanies the activation of protective pathways such as Grp78/Bip and PERK-eIF2alpha pathway, as well as the apoptotic pathways of the UPR such as CHOP and caspase-4. Knockdown of PERK enhances Abeta neurotoxicity through reducing the activation of eIF2alpha and Grp8/Bip in neurons. Salubrinal, an activator of the eIF2alpha pathway, significantly increased the Grp78/Bip ER chaperone resulted in attenuating caspase-4 dependent apoptosis in Abeta treated neurons. These results indicate that PERK-eIF2alpha pathway is a potential target for therapeutic applications in neurodegenerative diseases including AD.
Project description:Heat stress can be acutely cytotoxic, and heat stress-induced apoptosis is a prominent pathological feature of heat-related illnesses, although the precise mechanisms by which heat stress triggers apoptosis are poorly defined.The percentages of viability and cell death were assessed by WST-1 and LDH release assays. Apoptosis was assayed by DNA fragmentation and caspase activity. Expression of cleaved PARP, Apaf-1, phospho-PERK, Phospho-eIF2a, ATF4, XBP-1s, ATF6, GRP78, phospho-IP3R, RYR and SERCA was estimated by Western blot. The effect of calcium overload was determined using flow cytometric analysis with the fluorescent probe Fluo-3/AM. The generation of ROS (O2-, H2O2, NO) was labeled by confocal laser scanning microscopy images of fluorescently and flow cytometry.In this study, we found that heat stress in HUVEC cells activated initiators of three major unfolded protein response (UPR) signaling transduction pathways: PERK-eIF2a-ATF4, IRE1-XBP-1S and ATF6 to protect against ER stress, although activation declined over time following cessation of heat stress. Furthermore, we show that intense heat stress may induce apoptosis in HUVEC cells through the calcium-mediated mitochondrial apoptotic pathway, as indicated by elevation of cytoplasmic Ca2+, expression of Apaf-1, activation of caspase-9 and caspase-3, PARP cleavage, and ultimately nucleosomal DNA fragmentation; Reactive oxygen species (ROS) appear to act upstream in this process. In addition, we provide evidence that IP3R upregulation may promote influx of Ca2+ into the cytoplasm after heat stress.Our findings describe a novel mechanism for heat stress-induced apoptosis in HUVEC cells: following elevation of cytoplasm Ca2+, activation of the mitochondrial apoptotic pathway via the IP3R upregulation, with ROS acting as an upstream regulator of the process.
Project description:The detection and compensatory response to the accumulation of unfolded proteins in the endoplasmic reticulum (ER), termed the unfolded protein response (UPR), represents a conserved cellular homeostatic mechanism with important roles in normal development and in the pathogenesis of disease. The IRE1-XBP1/Hac1 pathway is a major branch of the UPR that has been conserved from yeast to human. X-box binding protein 1 (XBP1) is required for the differentiation of the highly secretory plasma cells of the mammalian adaptive immune system, but recent work also points to reciprocal interactions between the UPR and other aspects of immunity and inflammation. We have been studying innate immunity in the nematode Caenorhabditis elegans, having established a principal role for a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway in mediating resistance to microbial pathogens. Here we show that during C. elegans development, XBP-1 has an essential role in protecting the host during activation of innate immunity. Activation of the PMK-1-mediated response to infection with Pseudomonas aeruginosa induces the XBP-1-dependent UPR. Whereas a loss-of-function xbp-1 mutant develops normally in the presence of relatively non-pathogenic bacteria, infection of the xbp-1 mutant with P. aeruginosa leads to disruption of ER morphology and larval lethality. Unexpectedly, the larval lethality phenotype on pathogenic P. aeruginosa is suppressed by loss of PMK-1-mediated immunity. Furthermore, hyperactivation of PMK-1 causes larval lethality in the xbp-1 mutant even in the absence of pathogenic bacteria. Our data establish innate immunity as a physiologically relevant inducer of ER stress during C. elegans development and indicate that an ancient, conserved role for XBP-1 may be to protect the host organism from the detrimental effects of mounting an innate immune response to microbes.
Project description:Cerebral hypometabolism and amyloid accumulation are principal neuropathological manifestations of Alzheimer's disease (AD). Whether and how brain/neuronal activity might modulate certain pathological processes of AD are interesting topics of recent clinical and basic research in the field, and may be of potential medical relevance in regard to both the disease etiology and intervention. Using the Tg2576 transgenic mouse model of AD, this study characterized a promotive effect of neuronal hypoactivity associated with functional deprivation on amyloid plaque pathogenesis in the olfactory pathway. Unilateral naris-occlusion caused beta-secretase-1 (BACE1) elevation in neuronal terminals in the deprived relative to the non-deprived bulb and piriform cortex in young adult mice. In parallel with the overall age-related plaque development in the forebrain, locally increased BACE1 immunoreactivity co-occurred with amyloid deposition first in the piriform cortex then within the bulb, more prominent on the deprived relative to the non-deprived side. Biochemical analyses confirmed elevated BACE1 protein levels, enzymatic activity and products in the deprived relative to non-deprived bulbs. Plaque-associated BACE1 immunoreactivity in the bulb and piriform cortex was localized preferentially to swollen/sprouting glutamatergic axonal terminals, with Abeta immunoreactivity occurring inside as well as around these terminals. Together, these findings suggest that functional deprivation or neuronal hypoactivity facilitates amyloid plaque formation in the forebrain in a transgenic model of AD, which operates synergistically with age effect. The data also implicate an intrinsic association of amyloid accumulation and plaque formation with progressive axonal pathology.