Canonical Stress Response Research Articles

Definition of the Neurotoxicity-Associated Metabolic Signature Triggered by Berberine and Other Respiratory Chain Inhibitors.

To characterize the hits from a phenotypic neurotoxicity screen, we obtained transcriptomics data for valinomycin, diethylstilbestrol, colchicine, rotenone, 1-methyl-4-phenylpyridinium (MPP), carbaryl and berberine (Ber). For all compounds, the concentration triggering neurite degeneration correlated with the onset of gene expression changes. The mechanistically diverse toxicants caused similar patterns of gene regulation: the responses were dominated by cell de-differentiation and a triggering of canonical stress response pathways driven by ATF4 and NRF2. To obtain more detailed and specific information on the modes-of-action, the effects on energy metabolism (respiration and glycolysis) were measured. Ber, rotenone and MPP inhibited the mitochondrial respiratory chain and they shared complex I as the target. This group of toxicants was further evaluated by metabolomics under experimental conditions that did not deplete ATP. Ber (204 changed metabolites) showed similar effects as MPP and rotenone. The overall metabolic situation was characterized by oxidative stress, an over-abundance of NADH (>1000% increase) and a re-routing of metabolism in order to dispose of the nitrogen resulting from increased amino acid turnover. This unique overall pattern led to the accumulation of metabolites known as biomarkers of neurodegeneration (saccharopine, aminoadipate and branched-chain ketoacids). These findings suggest that neurotoxicity of mitochondrial inhibitors may result from an ensemble of metabolic changes rather than from a simple ATP depletion. The combi-omics approach used here provided richer and more specific MoA data than the more common transcriptomics analysis alone. As Ber, a human drug and food supplement, mimicked closely the mode-of-action of known neurotoxicants, its potential hazard requires further investigation.

The Human Glyoxalase Gene Family in Health and Disease.

The glyoxalase gene family consists of six structurally and functionally diverse enzymes with broad roles in metabolism. The common feature that defines this family is based on structural motifs that coordinate divalent cations which are required for activity. These family members have been implicated in a variety of physiological processes, including amino-acid metabolism (4-hydroxyphenylpyruvate dioxygenase; HPD), primary metabolism (methylmalonyl-CoA epimerase; MCEE), and aldehyde detoxication (glyoxalase 1; GLO1) and therefore have significant associations with disease. A central function of this family is the detoxification of reactive dicarbonyls (e.g., methylglyoxal), which react with cellular nucleophiles, resulting in the modification of lipids, proteins, and DNA. These damaging modifications activate canonical stress responses such as heat shock, unfolded protein, antioxidant, and DNA damage responses. Thus, glyoxalases serve an important role in homeostasis, preventing the pathogenesis of metabolic disease states, including obesity, diabetes, cardiovascular disease, renal failure, and aging. This review presents a thorough overview of the literature surrounding this diverse enzyme class. Although extensive literature exists for some members of this family (e.g., GLO1), little is known about the physiological role of glyoxalase domain-containing protein 4 (GLOD4) and 5 (GLOD5), paving the way for exciting avenues for future research.

Sexual dimorphism in Caenorhabditis elegans stress resistance.

Physiological responses to the environment, disease, and aging vary by sex in many animals, but mechanisms of dimorphism have only recently begun to receive careful attention. The genetic model nematode Caenorhabditis elegans has well-defined mechanisms of stress response, aging, and sexual differentiation. C. elegans has males, but the vast majority of research only uses hermaphrodites. We found that males of the standard N2 laboratory strain were more resistant to hyperosmolarity, heat, and a natural pro-oxidant than hermaphrodites when in mixed-sex groups. Resistance to heat and pro-oxidant were also male-biased in three genetically and geographically diverse C. elegans strains consistent with a species-wide dimorphism that is not specific to domestication. N2 males were also more resistant to heat and pro-oxidant when keep individually indicating that differences in resistance do not require interactions between worms. We found that males induce canonical stress response genes by similar degrees and in similar tissues as hermaphrodites suggesting the importance of other mechanisms. We find that resistance to heat and pro-oxidant are influenced by the sex differentiation transcription factor TRA-1 suggesting that downstream organ differentiation pathways establish differences in stress resistance. Environmental stress influences survival in natural environments, degenerative disease, and aging. Understanding mechanisms of stress response dimorphism can therefore provide insights into sex-specific population dynamics, disease, and longevity.

Non-additive gene interactions underpin molecular and phenotypic responses in honey bee larvae exposed to imidacloprid and thymol

Understanding the cumulative risk of chemical mixtures at environmentally realistic concentrations is a key challenge in honey bee ecotoxicology. Ecotoxicogenomics, including transcriptomics, measures responses in individual organisms at the molecular level which can provide insights into the mechanisms underlying phenotypic responses induced by one or more stressors and link impacts on individuals to populations. Here, fifth instar honey bee larvae were sampled from a previously reported field experiment exploring the phenotypic impacts of environmentally realistic chronic exposures of the pesticide imidacloprid (5 μg.kg−1 for six weeks) and the acaricide thymol (250 g.kg−1 applied via Apiguard gel in-hive for four weeks), both separately and in combination. RNA-seq was used to discover individual and interactive chemical effects on larval gene expression and to uncover molecular mechanisms linked to reported adult and colony phenotypes. The separate and combined treatments had distinct gene expression profiles which represented differentially affected signaling and metabolic pathways. The molecular signature of the mixture was characterised by additive interactions in canonical stress responses associated with oxidative stress and detoxification, and non-additive interactions in secondary responses including developmental, neurological, and immune pathways. Novel emergent impacts on eye development genes correlated with long-term defects in visual learning performance as adults. This is consistent with these chemicals working through independent modes of action that combine to impact common downstream pathways, and highlights the importance of establishing mechanistic links between molecular and phenotypic responses when predicting effects of chemical mixtures on ecologically relevant population outcomes.

Microbiota regulate social behaviour via stress response neurons in the brain.

Social interactions among animals mediate essential behaviours, including mating, nurturing, and defence1,2. The gut microbiota contribute to social activity in mice3,4, but the gut-brain connections that regulate this complex behaviour and its underlying neural basis are unclear5,6. Here we show that the microbiome modulates neuronal activity in specific brain regions of male mice to regulate canonical stress responses and social behaviours. Social deviation in germ-free and antibiotic-treated mice is associated with elevated levels of the stress hormone corticosterone, which is primarily produced by activation of the hypothalamus-pituitary-adrenal (HPA) axis. Adrenalectomy, antagonism of glucocorticoid receptors, or pharmacological inhibition of corticosterone synthesis effectively corrects social deficits following microbiome depletion. Genetic ablation of glucocorticoid receptors in specific brain regions or chemogenetic inactivation of neurons in the paraventricular nucleus of the hypothalamus that produce corticotrophin-releasing hormone (CRH) reverse social impairments in antibiotic-treated mice. Conversely, specific activation of CRH-expressing neurons in the paraventricular nucleus induces social deficits in mice with a normal microbiome. Via microbiome profiling and in vivo selection, we identify a bacterial species, Enterococcus faecalis, that promotes social activity and reduces corticosterone levels in mice following social stress. These studies suggest that specific gut bacteria can restrain the activation of the HPA axis, and show that the microbiome can affect social behaviours through discrete neuronal circuits that mediate stress responses in the brain.

Responses to social evaluative stress in regular cannabis smokers.

Aspects of the canonical stress response differ in stimulant, opioid, and alcohol users relative to controls, and dysregulated responses to stress may contribute to continued use of these drugs. Little prior research has focused on stress responses in regular cannabis smokers. We assessed responses to a standardized laboratory social stress assay (the Trier Social Stress Task; TSST) in regular cannabis smokers (CANs) compared with controls (CONs). Healthy, non-treatment-seeking adult CANs (⩾4×/week; smoking cannabis as usual) and demographically matched CONs completed the TSST. Outcome measures were subjective mood, heart rate, and salivary cortisol. Nineteen CANs (1 female) and 20 CONs (2 female) participated; groups were matched on trauma exposure, sex, race, and age. CANs smoked cannabis 6.4 ± 1.1 days/week. Eight CANs and one CON smoked tobacco cigarettes daily. Overall, the TSST produced expected increases in anxiety, negative mood states, cortisol, and heart rate. CANs had blunted subjective response to stress relative to CONs, but they did not differ in physiological (cortisol and cardiovascular) stress responding. These results indicate that CANs have blunted mood responses to social stress, but normative physiological stress responding. Observed differences could be due to residual effects of cannabis, reluctance to endorse negative mood states, or to issues related to identifying (i.e., emotional identification) or feeling (i.e., interoception) stress-related affective states. Further research is warranted to characterize the mechanisms of these differences and assess implications for daily functioning and treatment outcomes.

The Interaction of Aging and Cellular Stress Contributes to Pathogenesis in Mouse and Human Huntington Disease Neurons.

Huntington disease (HD) is a fatal, inherited neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene. While mutant HTT is present ubiquitously throughout life, HD onset typically occurs in mid-life. Oxidative damage accumulates in the aging brain and is a feature of HD. We sought to interrogate the roles and interaction of age and oxidative stress in HD using primary Hu97/18 mouse neurons, neurons differentiated from HD patient induced pluripotent stem cells (iPSCs), and the brains of HD mice. We find that primary neurons must be matured in culture for canonical stress responses to occur. Furthermore, when aging is accelerated in mature HD neurons, mutant HTT accumulates and sensitivity to oxidative stress is selectively enhanced. Furthermore, we observe HD-specific phenotypes in neurons and mouse brains that have undergone accelerated aging, including a selective increase in DNA damage. These findings suggest a role for aging in HD pathogenesis and an interaction between the biological age of HD neurons and sensitivity to exogenous stress.

Aneuploidy-induced proteotoxic stress can be effectively tolerated without dosage compensation, genetic mutations, or stress responses

BackgroundThe protein homeostasis (proteostasis) network maintains balanced protein synthesis, folding, transport, and degradation within a cell. Failure to maintain proteostasis is associated with aging and disease, leading to concerted efforts to study how the network responds to various proteotoxic stresses. This is often accomplished using ectopic overexpression of well-characterized, model misfolded protein substrates. However, how cells tolerate large-scale, diverse burden to the proteostasis network is not understood. Aneuploidy, the state of imbalanced chromosome content, adversely affects the proteostasis network by dysregulating the expression of hundreds of proteins simultaneously. Using aneuploid haploid yeast cells as a model, we address whether cells can tolerate large-scale, diverse challenges to the proteostasis network.ResultsHere we characterize several aneuploid Saccharomyces cerevisiae strains isolated from a collection of stable, randomly generated yeast aneuploid cells. These strains exhibit robust growth and resistance to multiple drugs which induce various forms of proteotoxic stress. Whole genome re-sequencing of the strains revealed this was not the result of genetic mutations, and transcriptome profiling combined with ribosome footprinting showed that genes are expressed and translated in accordance to chromosome copy number. In some strains, various facets of the proteostasis network are mildly upregulated without chronic activation of environmental stress response or heat shock response pathways. No severe defects were observed in the degradation of misfolded proteins, using model misfolded substrates of endoplasmic reticulum-associated degradation or cytosolic quality control pathways, and protein biosynthesis capacity was not impaired.ConclusionsWe show that yeast strains of some karyotypes in the genetic background studied here can tolerate the large aneuploidy-associated burden to the proteostasis machinery without genetic changes, dosage compensation, or activation of canonical stress response pathways. We suggest that proteotoxic stress, while common, is not always an obligate consequence of aneuploidy, but rather certain karyotypes and genetic backgrounds may be able to tolerate the excess protein burden placed on the protein homeostasis machinery. This may help clarify how cancer cells are paradoxically both highly aneuploid and highly proliferative at the same time.

Redefining proteostasis transcription factors in organismal stress responses, development, metabolism, and health.

Eukaryotic organisms have evolved complex and robust cellular stress response pathways to ensure maintenance of proteostasis and survival during fluctuating environmental conditions. Highly conserved stress response pathways can be triggered and coordinated at the cell-autonomous and cell-nonautonomous level by proteostasis transcription factors, including HSF1, SKN-1/NRF2, HIF1, and DAF-16/FOXO that combat proteotoxic stress caused by environmental challenges. While these transcription factors are often associated with a specific stress condition, they also direct "noncanonical" transcriptional programs that serve to integrate a multitude of physiological responses required for development, metabolism, and defense responses to pathogen infections. In this review, we outline the established function of these key proteostasis transcription factors at the cell-autonomous and cell-nonautonomous level and discuss a newly emerging stress responsive transcription factor, PQM-1, within the proteostasis network. We look beyond the canonical stress response roles of proteostasis transcription factors and highlight their function in integrating different physiological stimuli to maintain cytosolic organismal proteostasis.

Nrf2 metagene as a prognostic biomarker across all stages of colorectal cancer (CRC).

557 Background: Keap1/Nrf2 is an important intracellular canonical stress response pathway, with Nrf2 acting as a potent transcription factor. Lung cancer is known to acquire constitutive activation of the pathway thus promoting survival, resisting chemo-radiation and dysregulating metabolism. Mechanisms of pathway activation include methylation of KEAP1, somatic mutation and direct activation by KRAS, BRAF and MYC. Its role in CRC is unknown. Due to its role as a transcription factor, activated in many ways, we hypothesise a metagene of Nrf2 regulated genes would act as a prognostic biomarker in CRC. Methods: Using a candidate gene approach and publicly available data (GSE17536), we derived and trained a 36 gene metagene to aggregate Nrf2 pathway expression, using principal component analysis and cox proportional hazard models. GSE14333 was used for validation in stage I/II/III CRC. The first line metastatic FOCUS trial was used for validation in Stage IV disease. Results: 601 patients are included in the validation analysis. Nrf2 metagene expression is associated with worse DFS outcomes in stage I/II/III disease in Cox PH model comparisons (LRT, p = 0.00175) and worse OS in stage IV disease (LRT, p = 0.00574). On multivariate analysis, Nrf2 expression remained significant when adjusted for known prognostic factors of adjuvant chemotherapy and Duke’s stage in stage I/II/III disease (ANOVA, p = 0.03), BRAF V600E and sidedness in stage IV disease (ANOVA, p = 0.00255). Using the median cut point, high expression has a HR 2.36 (C.I 1.27-4.38) in stage I/II/III disease and HR 1.39 (C.I 1.12-1.72) in metastatic disease. Conclusions: Nrf2 expression is a novel, robust prognostic biomarker of poor prognosis across all stages of colorectal cancer. Greater understanding of its mechanistic role could lead to targeted strategies to improve outcomes in CRC. [Table: see text]

Heat Shock Factor 1 Reprograms the DLBCL Microenvironment to Evade Immune Surveillance and Support Tumor Growth

The primary components of the stromal lymphoma microenvironment (LME) are the cancer-associated fibroblasts (CAF) and the extracellular matrix (ECM) they produce. CAFs are derived from healthy fibroblasts that have been hijacked and transcriptionally reprogrammed by cancer cells into a novel biological entity to promote tumorigenesis. While the key effectors of these programs are largely unknown, recent data indicates that in a variety of solid tumors and T-acute lymphoblastic leukemias an important transcriptional programmer of the microenvironment is the Heat Shock Factor 1 (HSF1). Hence, we sought to determine the contribution of HSF1 within the stromal LME to the acquisition of lymphomagenic features in diffuse large B-cell lymphoma (DLBCL).We first analyzed the activity of HSF1 in 80 DLBCL patients by RNA-sequencing. Patients were segregated into having “high” and “low” HSF1 activity based on the expression of the canonical stress response target genes. We found distinct patterns of stromal-associated genes between DLBCL having “low” vs. “high” HSF1 activity. Furthermore, ChIP-sequencing data of lymphoma cells and CAFs showed the emergence of distinct transcriptional programs that are differentially orchestrated by HSF1. To delve deeper into the mechanism of these effects, we co-cultured lymphoma cells with fibroblasts having either HSF1 WT or HSF1 null in 3D collagen-containing lymphoma organoids. We found that only organoids harboring HSF1 WT fibroblasts were supportive of DLBCL cell proliferation. Moreover, DLBCL cells responsive and non-responsive to HSF1 downregulation in a cell-autonomous manner, failed to proliferate when cultured in HSF1-deficient LME organoids.To further elucidate this mechanism, we implanted a murine HSF1-positive DLBCL cell line (A20) into HSF1 WT (n=13) and HSF1 null (n=11) syngeneic mice and monitored lymphoma development for up to 14 days. We observed a continuous growth of HSF1-positive murine DLBCL in HSF1 WT mice; however, DLBCL in HSF1 null mice underwent spontaneous collapse after day 7, leading to almost complete tumor eradication. We further investigated the lymphoma microarchitecture, ECM biomechanics and composition in relation to the cellular components in serial tumor sections at several time points. At day 7, when no significant difference in tumor sizes exists between HSF1 WTs and nulls, we found significant differences in tissue stiffness (p=0.017), collagen microarchitectureby fiber alignment (p<0.001) and elastic fiber area (p<0.001). ECM proteomics by HPLC-MS/MS of the ECM-enriched fractions of these murine DLBCLs at days 5, 7 and 13 followed by network analysis demonstrated highly dynamic changes in ECM composition. Specifically, comparing the “matrisomes” of HSF1 WT and null tumors at day 7, we identified differentially regulated ECM modules that facilitate collagen functionalization and abundance of small leucine-rich proteoglycans; suggesting a failure of the HSF1 null stroma in efficiently remodeling the ECM. We identified dynamic changes in specific matrisome components with known roles in tumor proliferation and immune activity. These biochemical and biophysical changeswere in turn capable of modulating the cellular composition of DLBCL tumors. By day 7, the LME in HSF1 null was less supportive of lymphoma growth as measured by lower cellularity (p=4.5e-18) and proliferation of lymphoma cells by Ki67 (p=7.6e-6); and increased apoptosis by TUNEL (p=5.6e-5). In regards to CD31 positivity based on endothelial cells and vascularization, we observed no significant differences between HSF1 WT and HSF1 null tumors, however, there were significant changes in the repertoire of immune infiltrating cells. Specifically, HSF1 null LME harbored higher fractions of effector cd3+ T-cells (p=3.0e-6) and mac2+macrophages (p=9.4e-11); and a decrease in the suppressive cells cd3+foxp3+Tregs (p=6.6e-19) and cd11b+ gr1+myeloid-derived suppressor cells (p=0.0467).Taken together, our data in DLBCL specimens and cell lines, lymphoma-fibroblasts organoids, and a murine DLBCL model, all demonstrate that HSF1 activity in DLBCL drives an ECM program that confers microarchitectural and biophysical properties to the LME that are supportive of lymphoma cell proliferation and infiltration by immune suppressive populations. DisclosuresCerchietti:Celgene: Research Funding; Weill Cornell Medicine: Employment.

3:36 PM Abstract No. 316 Developing TACE 2.0: targeting hepatocellular carcinoma epigenetic and ischemia survival pathways

Epigenetic alterations in hepatocellular carcinoma (HCC) may facilitate cell survival under transarterial chemoembolization-induced ischemia by potentiating the function of canonical stress response pathways including the unfolded protein response (UPR), Hypoxia-Inducible Factor (HIF), and Autophagy. Moreover, the redundancy in function of these pathways makes inhibition of any single pathway insufficient to abrogate their function entirely. We hypothesize that HCC cells surviving severe TACE-like ischemia are susceptible to inhibition of these pathways and that combination of the inhibition of these pathways would lead to synergistic effects. Viability assays and cytotoxicity profiles of Huh-7, SNU-387 and SNU-449 HCC cell lines were studied under standard (21% O2 with complete medium) and compared to severely ischemic conditions (1% O2, 1% serum, 1 mM glucose) with an inhibitor of the UPR (GSK2606141), a HIF-1 alpha inhibitor (PX-478 or BAY87-2243) and an inhibitor of autophagy (Hydroxychloroquine). Cytotoxicity measurements were derived from measured dose-response curves using the WST-1 cytotoxicity assay. The t-test was used to compare standard to severely ischemic conditions. Each of the cell lines tested demonstrated decreased cellular viability with incubation of either of the inhibitory agents (EC50 GSK2606141, PX-478, BAY87-2243, Hydroxcychloroquine of 75-150μM, 50-100 μM, 300-600uM, and 100-200μM respectively). Ischemia potentiated the cytotoxicity (EC50 GSK2606141, PX-478, BAY87-2243, Hydroxcychloroquine of 25-50μM, 50-75 μM, 200-400uM, and 50-80μM respectively; with p<0.05 except for BAY87-2243). Combination inhibition of the three pathways under TACE-like ischemia lead to a synergistic response showing cell death at concentrations below any single drug alone (EC50 of BAY87-2243 in combination of 100-200uM compared to 400-500uM as a single agent). Inhibition of the UPR, HIF, and autophagy independently lead to cytotoxicity of HCC cells. Combination of the three drugs under TACE-like ischemia lead to a synergistic response and should be considered as potential chemotherapeutics for TACE.

A huntingtin-mediated fast stress response halting endosomal trafficking is defective in Huntington's disease.

Cellular stress is a normal part of the aging process and is especially relevant in neurodegenerative disease. Canonical stress responses, such as the heat shock response, activate following exposure to stress and restore proteostasis through the action of isomerases and chaperones within the cytosol. Through live-cell imaging, we demonstrate involvement of the Huntington's disease (HD) protein, huntingtin, in a rapid cell stress response that lies temporally upstream of canonical stress responses. This response is characterized by the formation of distinct cytosolic puncta and reversible localization of huntingtin to early endosomes. The formation of these puncta, which we have termed huntingtin stress bodies (HSBs), is associated with arrest of early-to-recycling and early-to-late endosomal trafficking. The critical domains for this response have been mapped to two regions of huntingtin flanking the polyglutamine tract, and we observe polyglutamine-expanded huntingtin-expressing cells to be defective in their ability to recover from this stress response. We propose that HSB formation rapidly diverts high ATP use from vesicular trafficking during stress, thus mobilizing canonical stress responses without relying on increased energy metabolism, and that restoration from this response is defective in HD.

Short- and long-term adaptation to ethanol stress and its cross-protective consequences in Lactobacillus plantarum.

This paper describes the molecular responses of Lactobacillus plantarum WCFS1 toward ethanol exposure. Global transcriptome profiling using DNA microarrays demonstrated adaptation of the microorganism to the presence of 8% ethanol over short (10-min and 30-min) and long (24-h) time intervals. A total of 57 genes were differentially expressed at all time points. Expression levels of an additional 859 and 873 genes were modulated after 30 min and 24 h of exposure to the solvent, respectively. Ethanol exposure led to induced expression of genes involved in citrate metabolism and cell envelope architecture, as well as canonical stress response pathways controlled by the central stress regulators HrcA and CtsR. Correspondingly, cells grown for 24 h in medium containing 8% ethanol exhibited higher levels of citrate consumption and modified cell membrane fatty acid composition and showed invaginating septa compared with cells grown in liquid medium without ethanol. In addition, these physiological changes resulted in cross-protection against high temperatures but not against several other stresses tested. To evaluate the role of HrcA and CtsR in ethanol tolerance, ctsR and hrcA gene deletion mutants were constructed. The growth rate of the L. plantarum ΔctsR::cat strain was impaired in de Man-Rogosa-Sharpe (MRS) medium containing 8% ethanol, whereas growth of the L. plantarum ΔhrcA::cat and ΔctsR ΔhrcA::cat mutants was indistinguishable from that of wild-type cells. Overall, these results suggest that the induction of CtsR class III stress responses provides cross-protection against heat stress.