Unfolded Protein Response Proteins Research Articles

Increased ER Stress and Unfolded Protein Response Activation in Epithelial and Inflammatory Cells in Hypersensitivity Pneumonitis.

Several types of cytotoxic insults disrupt endoplasmic reticulum (ER) homeostasis, cause ER stress, and activate the unfolded protein response (UPR). The role of ER stress and UPR activation in hypersensitivity pneumonitis (HP) has not been described. HP is an immune-mediated interstitial lung disease that develops following repeated inhalation of various antigens in susceptible and sensitized individuals. The aim of this study was to investigate the lung expression and localization of the key effectors of the UPR, BiP/GRP78, CHOP, and sXBP1 in HP patients compared with control subjects. Furthermore, we developed a mouse model of HP to determine whether ER stress and UPR pathway are induced during this pathogenesis. In human control lungs, we observed weak positive staining for BiP in some epithelial cells and macrophages, while sXBP1 and CHOP were negative. Conversely, strong BiP, sXBP1- and CHOP-positive alveolar and bronchial epithelial, and inflammatory cells were identified in HP lungs. We also found apoptosis and autophagy markers colocalization with UPR proteins in HP lungs. Similar results were obtained in lungs from an HP mouse model. Our findings suggest that the UPR pathway is associated with the pathogenesis of HP.

CREB3 mediates the transcriptional regulation of PGC-1α, a master regulator of energy homeostasis and mitochondrial biogenesis.

Lipid metabolism hinges on a balance between lipogenesis and fatty acid oxidation (FAO). Disruptions in this balance can induce endoplasmic reticulum (ER) stress triggering the unfolded protein response (UPR) and contribute to metabolic diseases. The UPR protein, Luman or CREB3, has recently been implicated in metabolic regulation-CREB3 knockout mice exhibit resistance to diet-induced obesity and altered insulin sensitivity. Here, we show that CREB3 activated PPARGC1A transcription from a 1 kb promoter region. An increase in CREB3 expression correlated inversely with endogenous PPARGC1A mRNA levels and genes involved in FAO. As PGC-1α encoded by PPARGC1A is a master regulator of mitochondrial biogenesis and energy homeostasis, these findings demonstrate that CREB3 is a transcriptional regulator of PGC-1α, underlining the potential role of CREB3 in energy metabolism.

MUCOSAL SINGLE-CELL PROFILING OF CROHN’S-LIKE DISEASE OF THE POUCH REVEALS COMMON PATHOGENICS AND THERAPEUTIC TARGETS

Abstract BACKGROUND & AIMS After restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC), a subset of patients develop Crohn’s-like disease of the pouch (CDP), an inflammatory condition that affects both the ileoanal pouch and extra-pouch organs including pre-pouch ileum. The cellular and molecular identities of CDP are unknown, and its diagnosis and treatment remain challenging. To define the pathophysiology of CDP, we examined mucosal cells from patients with and without CDP using single cell analyses. METHODS Endoscopic samples from the pouch and pre-pouch ileum of 50 patients with an IPAA were collected for single-cell RNA sequencing (scRNA-seq) or mass cytometry (or CyTOF). We analyzed immune and non-immune cells from pouch and ileal tissues of patients with normal pouch/ileum and CDP (n = 5 or 6) using scRNA-seq. CyTOF was performed on mucosal immune cells from independent cohorts of patients with normal pouch/ileum, CDP, and pouchitis (n = 9 or 10). Mucosal samples from patients with familial adenomatous polyposis (FAP) after pouch formation were also analyzed by CyTOF. Some scRNA-seq and CyTOF findings were independently validated using immunohistochemistry (n = 6 or 8). RESULTS We revealed distinct immune and non-immune cell clusters in normal pouch and pre-pouch ileum. Colitogenic immune cells expanded in normal UC pouch compared to normal FAP pouch. Compared to normal pouch/pre-pouch ileum, CDP tissues exhibited expanded TCR clonotypes, elevated Th17 signaling, and diminished T cell exhaustion markers. Elevated frequencies of plasma cells, inflammatory fibroblasts and monocytes were noted in CDP pouch and ileum (Fig 1; only pouch data were shown). CDP also harbored elevated Th17-inducing cytokines such as IL23, IL1B, and IL6 produced by myeloid cells. ScRNA-seq and CyTOF identified increased CD14+TREM1+ pathogenic monocytes in both pouch and pre-pouch ileum of CDP. Ligand-receptor interactions further uncovered an essential role of proinflammatory myeloid cells in the pathogenesis of CDP. In addition, pouch and pre-pouch ileum of CDP showed prominent activation of the unfolded protein response (UPR) across all major cell compartments (Fig 2), which was not present in UC, CD or pouchitis based on integrated analysis of published databases. CONCLUSIONS CDP demonstrates altered immune and non-immune cell populations/states in the pouch and pre-pouch ileum. CDP represents a distinct entity of inflammatory bowel disease with extensive UPR activation, a unique feature that may serve as novel diagnostic markers and therapeutic targets using ER stress-alleviating agents including chemical chaperones. Fig 1 CDP reshapes immune and non-immune landscapes in pouch and pre-pouch ileum. (A) UMAP visualization of all cells from the pouch and ileum of CLDP and normal controls. (B) Diversity of epithelial cells, stromal cells, myeloid cells, T cells, innate lymphoid cells (ILCs), B cells and plasma cells in CLDP vs. normal controls in the pouch estimated by scRNA-seq (n = 5 or 6). *p<0.01. Fig 2 Extensive UPR activation in CDP tissues. (A) UPR gene expression in major cell compartments in CDP and normal controls, highlighted genes including CALR, XBP1, and SERP1 were upregulated in all 5 cell compartments. (B) ER stress/UPR pathways are enriched in CDP. (C) IHC of UPR proteins XBP1 and HSPA5/BiP.

Heterologous expression of influenza haemagglutinin leads to early and transient activation of the unfolded protein response in Nicotiana benthamiana.

The unfolded protein response (UPR) allows cells to cope with endoplasmic reticulum (ER) stress induced by accumulation of misfolded proteins in the ER. Due to its sensitivity to Agrobacterium tumefaciens, the model plant Nicotiana benthamiana is widely employed for transient expression of recombinant proteins of biopharmaceutical interest, including antibodies and virus surface proteins used for vaccine production. As such, study of the plant UPR is of practical significance, since enforced expression of complex secreted proteins often results in ER stress. After 6 days of expression, we recently reported that influenza haemagglutinin H5 induces accumulation of UPR proteins. Since up-regulation of corresponding UPR genes was not detected at this time, accumulation of UPR proteins was hypothesized to be independent of transcriptional induction, or associated with early but transient UPR gene up-regulation. Using time course sampling, we here show that H5 expression does result in early and transient activation of the UPR, as inferred from unconventional splicing of NbbZIP60 transcripts and induction of UPR genes with varied functions. Transient nature of H5-induced UPR suggests that this response was sufficient to cope with ER stress provoked by expression of the secreted protein, as opposed to an antibody that triggered stronger and more sustained UPR activation. As up-regulation of defence genes responding to H5 expression was detected after the peak of UPR activation and correlated with high increase in H5 protein accumulation, we hypothesize that these immune responses, rather than the UPR, were responsible for onset of the necrotic symptoms on H5-expressing leaves.

Synergistic lethality in chronic myeloid leukemia – targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.

Adaptive changes in tumor cells in response to reductive stress

Reductive stress is characterized by an excess of cellular electron donors and can be linked with various human pathologies including cancer. We developed melanoma cell lines resistant to reductive stress agents: rotenone (ROTR), n-acetyl-L-cysteine, (NACR), or dithiothreitol (DTTR). Resistant cells divided more rapidly and had intracellular homeostatic redox-couple ratios that were shifted towards the reduced state. Resistance caused alterations in general cell morphology, but only ROTR cells had significant changes in mitochondrial morphology with higher numbers that were more isolated, fragmented and swollen, with greater membrane depolarization and decreased numbers of networks. These changes were accompanied by lower basal oxygen consumption and maximal respiration rates. Whole cell flux analyses and mitochondrial function assays showed that NACR and DTTR preferentially utilized tricarboxylic acid (TCA) cycle intermediates, while ROTR used ketone body substrates such as D, L-β-hydroxybutyric acid. NACR and DTTR cells had constitutively decreased levels of reactive oxygen species (ROS), although this was accompanied by activation of nuclear factor erythroid 2–related factor 2 (Nrf2), with concomitant increased expression of the downstream gene products such as glutathione S-transferase P (GSTP). Further adaptations included enhanced expression of endoplasmic reticulum proteins controlling the unfolded protein response (UPR). Although expression patterns of these UPR proteins were distinct between the resistant cells, a trend implied that resistance to reductive stress is accompanied by a constitutively increased UPR phenotype in each line. Overall, tumor cells, although tolerant of oxidative stress, can adapt their energy and survival mechanisms in lethal reductive stress conditions.

An initial characterisation of the Unfolded Protein Response pathway in haematopoietic canine cancer cell lines - a necessary step for the future development of new therapies in dogs with neoplasia.

New and more effective therapies for canine cancer patients are urgently required and this necessitates advanced experimental research. Dogs are good models for studies in comparative oncology; however, canine cancer cell biology research is currently limited by low availability of validated antibody reagents and techniques. This study characterises the expression of key components of the unfolded protein response (UPR) in a panel of haematopoietic canine cancer cell lines using commercially available antibodies, and validates the methods used to study this pathway. The CLBL-1 canine lymphoma cell line and the GL-1 canine leukaemia cell line sourced externally and two counterparts established in house (CNK-89 and CLB70) were used as models of different lymphoma and leukaemia canine cell lines for the study. The human U2OS cell line served as the control. Antibodies were selected for identifying UPR proteins according to known canine cell reactivity and canine-murine and canine-human homology. Endoplasmic reticulum stress was induced with thapsigargin and MG132 in the cell lines. Etoposide was used to induce DNA damage in the cells. The techniques used for this validation analysis were RNA sequencing to observe the expression of UPR components in canine cell lines, Western blot to observe changes of protein expression levels after inducing ER stress in the cells, and flow cytometry in order to study cell death. Substantial variations in both the basic expression and agonist-induced activation of the UPR pathway were observed in canine cancer cell lines, although the biological significance of these differences requires further investigation. These findings will be a starting point for future studies on cancer biology in dogs. They will also contribute to developing novel anticancer therapies for canine patients and may provide new insights into human oncology.

Abstract P3077: Withdrawal Of High Fat Diet And L-NAME Dampens T Cell Activation And Ameliorates Pathology In Experimental Heart Failure With Preserved Ejection Fraction (HFPEF) Through Modulation Of The T Cell IRE1a-XBP1 Axis

Introduction: Cardiometabolic HFpEF is characterized by T cell systemic inflammation and can be modeled preclinically with high fat diet (HFD) and L-NAME. Metabolic perturbations can alter T cell function through modulation of the unfolded protein response (UPR). How T cells are activated in HFpEF and whether T cell inflammation can be tuned by removing cardiometabolic stress remains unknown. Hypothesis: HFD and L-NAME induce reversible alterations in T cell UPR signaling that modulate T cell dependent cardiac inflammation and pathology in experimental HFpEF. Methods: Male C57/BL6J mice (wild-type, WT), OT-II antigen restricted mice, and CD4 Cre Xbp1 fl/fl mice were fed a high-fat diet (HFD) and L-NAME (H/L) for 5 weeks. WT mice were switched to standard diet (STD) for two additional weeks. WT mice receiving HFD or L-NAME only, or TAC surgery (a model of HF with reduced EF) were used as controls. Left ventricular (LV) end diastolic pressure volume relationship (EDPVR) was assessed using invasive hemodynamic analyses and LV EF by echocardiography. Cardiac and splenic T cells were characterized by flow cytometry and T cell UPR was evaluated by qPCR and Western blot. Results: 5 weeks of H/L was sufficient to induce cardiac T cell infiltration and splenic T cell activation. OTII mice fed H/L showed increased cardiac CD4 + T cell infiltration compared to STD controls, indicating T cell activation is not cardiac antigen restricted. Furthermore, splenic T cell characterization revealed H/L resulted in decreased phosphorylation of the UPR protein inositol-requiring enzyme 1α (IRE1α) and reduced expression of X box-binding protein 1 ( Xbp1s/u ) compared to T cells from mice exposed to HFD or L-NAME alone, STD, or WT TAC controls. CD4 Cre+ Xbp1 fl/fl mice (T cell Xbp1 -/- ) fed with H/L had increased cardiac T cell infiltration compared to Cre - controls. Lastly, Xbp1s expression in splenic CD4 + T cells was recovered after withdrawal of H/L and resulted in partial reduction of cardiac hypertrophy, decreased EDPVR, as well as less cardiac T cell infiltration compared to mice kept on H/L. Conclusions: Reversible downregulation of the T cell IRE1α-XBP1 axis is a unique T cell signature of HFpEF that contributes to T cell inflammation and adverse cardiac remodeling.

Betulinic acid and Drummondin E: Potential inhibitors of Unfolded Protein Response Pathway of Candida auris

Candida auris is a rapidly emerging global public health concern. The increasing mortality in immunocompromised patients is mostly attributed to the rise of drug-resistant clinical isolates. Low bioavailability and toxicity of the existing antifungals further exacerbate the condition. Unfolded protein response (UPR) has been linked to fungal pathogenesis in previous studies. In this study the two hallmark proteins of the UPR pathway, Hac1p and Ire1p, were targeted to identify novel antifungals. Different phytochemicals showing various therapeutic potential were selected. Using various bioinformatics tools, the molecular property, bioactivity, toxicity, drug-likeness of these compounds were determined. The compounds showing the best properties were analyzed for their ability to interact with UPR proteins by molecular docking study. Finally, the molecular dynamics simulation analysis was performed to determine the stability of the interactions between the phytochemicals and the target protein. Flinderole-B, Drummondin-E, Betulinic acid, Ursolic acid, Oleanolic acid, Stigmasterol showed good drug-likeness scores. They were also found to be non-carcinogenic, and non-toxic; and followed Lipinski’s rule of five. Based on the simulation analysis Betulinic acid showed the best potential to target Hac1p while Drummondin-E showed the best potential to target Ire1p. Betulinic acid and Drummondin E could be potential inhibitors of the UPR pathway in C. auris. However, further in vitro and in vivo studies are needed to corroborate their antifungal potential.

Valosin-containing protein (VCP/p97) is prognostically unfavorable in pediatric AML, and negatively correlates with unfolded protein response proteins IRE1 and GRP78: A report from the Children's Oncology Group.

The endoplasmic reticulum (ER) is the major site of protein synthesis and folding in the cell. ER-associated degradation (ERAD) and unfolded protein response (UPR) are the main mechanisms of ER-mediated cell stress adaptation. Targeting the cell stress response is a promising therapeutic approach in acute myeloid leukemia (AML). Protein expression levels of valosin-containing protein (VCP), a chief element of ERAD, were measured in peripheral blood samples from in 483 pediatric AML patients using reverse phase protein array methodology. Patients participated in the Children's Oncology Group AAML1031 phase 3 clinical trial that randomized patients to standard chemotherapy (cytarabine (Ara-C), daunorubicin, and etoposide [ADE]) versus ADE plus bortezomib (ADE+BTZ). Low-VCP expression was significantly associated with favorable 5-year overall survival (OS) rate compared to middle-high-VCP expression (81%versus 63%, p<0.001), independent of additional bortezomib treatment. Multivariable Cox regression analysis identified VCP as independent predictor of clinical outcome. UPR proteins IRE1 and GRP78 had significant negative correlation with VCP. Five-year OS in patients characterized by low-VCP, moderately high-IRE1 and high-GRP78 improved after treatment with ADE+BTZ versus ADE (66%versus 88%, p=0.026). Our findings suggest the potential of the protein VCP as biomarker in prognostication prediction in pediatric AML.

The interplay between tauopathy and aging through interruption of UPR/Nrf2/autophagy crosstalk in the Alzheimer’s disease transgenic experimental models

Purpose Alzheimer’s disease (AD) is the most common form of tauopathy that usually occursduring aging and unfolded protein response (UPR), oxidative stress and autophagy play a crucialrole in tauopathy-induced neurotoxicity. The aim of this study was to investigate the effects oftauopathy on normal brain aging in a Drosophila model of AD. Method We investigated the interplay between aging (10, 20, 30, and 40 days) and human tauR406W (htau)-induced cell stress in transgenic fruit flies. Results Tauopathy caused significant defects in eye morphology, a decrease in motor function and olfactory memory performance (after 20 days), and an increase in ethanol sensitivity (after 30 days). Our results showed a significant increase in UPR (GRP78 and ATF4), redox signalling (p-Nrf2, total GSH, total SH, lipid peroxidation, and antioxidant activity), and regulatory associated protein of mTOR complex 1 (p-Raptor) activity in the control group after 40 days, while the tauopathy model flies showed an advanced increase in the above markers at 20 days of age. Interestingly, only the control flies showed reduced autophagy by a significant decrease in the autophagosome formation protein (dATG1)/p-Raptor ratio at 40 days of age. Our results were also confirmed by bioinformatic analysis of microarray data from tauPS19 transgenic mice (3, 6, 9, and 12 months), in which tauopathy increased expression of heme oxygenase 1, and glutamate-cysteine ligase catalytic subunit and promote aging in transgenic animals. Conclusions Overall, we suggest that the neuropathological effects of tau aggregates may be accelerated brain aging, where redox signaling and autophagy efficacy play an important role.

Effects of acute ammonia exposure on oxidative stress, endoplasmic reticulum stress and apoptosis in the kuruma shrimp (Marsupenaeus japonicus)

Ammonia is one of the main toxic substances in aquaculture water and causes serious physiological harm to aquatic animals. In the present study, we investigated the effects of ammonia exposure on antioxidant defense, endoplasmic reticulum stress and apoptosis in the Marsupenaeus japonicus hepatopancreas. Shrimp in three experimental groups (control, 15 mg/L and 45 mg/L ammonia) were sampled at 6, 12, 24, 48 and 96 h. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX), and the corresponding relative mRNA expression levels in the hepatopancreas, showed slight or obvious increases during the early stage of ammonia exposure, and then significantly decreased after 48 h or 96 h of exposure, while the malondialdehyde (MDA) content increased continuously during ammonia exposure. In addition, the mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) showed a similar regulation pattern to the antioxidant enzymes, whereas Kelch-like ECH associated protein 1 (Keap1), a negative regulator of Nrf2, showed the opposite pattern. Moreover, the relative mRNA expression levels of heat shock proteins (HSP70 and HSP90), endoplasmic reticulum stress marker (GRP78) gene and key unfolded protein response (UPR)-related genes (eIF2α, ATF4, IRE1 and XBP1) increased significantly during the early stage of ammonia exposure. Taken together, these results indicate that the antioxidant system, Nrf2-Keap1 signaling pathway, UPR and heat shock proteins play protective roles in ammonia-induced oxidative stress and apoptosis in M. japonicus. However, ammonia exposure also induced up-regulation of apoptosis genes (Caspase 3 and ASK1) levels, indicating that cell apoptotic may relate to oxidative stress. After 96 h of exposure to 45 mg/L ammonia, down-regulation of HSP70 and HSP90 gene transcription levels suggests that the protein synthesis system may be destroyed under severe ammonia-induced oxidative stress. Our results provide new insights into the mechanism through which crustaceans resist ammonia stress.

Spinal Stroke: Outcome Attenuation by Erythropoietin and Carbamylated Erythropoietin and Its Prediction by Sphingosine-1-Phosphate Serum Levels in Mice.

Spinal strokes may be associated with tremendous spinal cord injury. Erythropoietin (EPO) improves the neurological outcome of animals after spinal cord ischemia (SCI) and its effects on ischemia-induced endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are considered possible molecular mechanisms. Furthermore, sphingosin-1-phosphate (S1P) is suggested to correlate with SCI. In this study, the effect of recombinant human EPO (rhEPO) and carbamylated EPO (cEPO-Fc) on the outcome of mice after SCI and a prognostic value of S1P were investigated. SCI was induced in 12-month-old male mice by thoracic aortal cross-clamping after administration of rhEPO, cEPO-Fc, or a control. The locomotory behavior of mice was evaluated by the Basso mouse scale and S1P serum levels were measured by liquid chromatography-tandem mass spectrometry. The spinal cord was examined histologically and the expressions of key UPR proteins (ATF6, PERK, and IRE1a, caspase-12) were analyzed utilizing immunohistochemistry and real-time quantitative polymerase chain reaction. RhEPO and cEPO-Fc significantly improved outcomes after SCI. The expression of caspase-12 significantly increased in the control group within the first 24 h of reperfusion. Animals with better locomotory behavior had significantly higher serum levels of S1P. Our data indicate that rhEPO and cEPO-Fc have protective effects on the clinical outcome and neuronal tissue of mice after SCI and that the ER is involved in the molecular mechanisms. Moreover, serum S1P may predict the severity of impairment after SCI.

TGFβ1 regulates HRas-mediated activation of IRE1α through the PERK-RPAP2 axis in keratinocytes.

Transforming Growth Factor β1 (TGFβ1) is a critical regulator of tumor progression in response to HRas. Recently, TGFβ1 has been shown to trigger ER stress in many disease models; however, its role in oncogene-induced ER stress is unclear. Oncogenic HRas induces the unfolded protein response (UPR) predominantly via the Inositol-requiring enzyme 1α (IRE1α) pathway to initiate the adaptative responses to ER stress, with importance for both proliferation and senescence. Here, we show a role of the UPR sensor proteins IRE1α and (PKR)-like endoplasmic reticulum kinase (PERK) to mediate the tumor-suppressive roles of TGFβ1 in mouse keratinocytes expressing mutant forms of HRas. TGFβ1 suppressed IRE1α phosphorylation and activation by HRas both in in vitro and in vivo models while simultaneously activating the PERK pathway. However, the increase in ER stress indicated an uncoupling of ER stress and IRE1α activation by TGFβ1. Pharmacological and genetic approaches demonstrated that TGFβ1-dependent dephosphorylation of IRE1α was mediated by PERK through RNA Polymerase II Associated Protein 2 (RPAP2), a PERK-dependent IRE1α phosphatase. In addition, TGFβ1-mediated growth arrest in oncogenic HRas keratinocytes was partially dependent on PERK-induced IRE1α dephosphorylation and inactivation. Together, these results demonstrate a critical cross-talk between UPR proteins that is important for TGFβ1-mediated tumor suppressive responses.

Abstract P3063: Downregulation Of The T Cell Unfolded Protein Response Precedes T Cell Cardiac Infiltration In Experimental Heart Failure With Preserved Ejection Fraction

Introduction: Diastolic dysfunction in Heart Failure with Preserved Ejection Fraction (HFpEF) is associated with T cell systemic inflammation and downregulation of myocardial unfolded protein response (UPR) genes. The T cell UPR can modulate T cell effector function and immune responses, yet this is largely unexplored in HFpEF. Hypothesis: We hypothesized T cells contribute to cardiometabolic HFpEF and that metabolic and nitrosative stress-induced alterations of the T cell UPR modulate T cell activation and diastolic function. Methods: Male C57/BL6 (wild-type, WT) or T cell receptor alpha-deficient ( Tcra-/-) were fed a high-fat diet (HFD) and L-NAME for 1, 3, and 5 weeks, or standard chow (STD). We assessed diastolic function by invasive hemodynamic analyses. Immune cells were characterized in the heart and spleen by flow cytometry, and cardiac pathology was assessed by histology. The UPR gene expression was evaluated in splenic CD4 + T cells over time by qPCR. Results: Unlike WT mice, Tcra-/- mice fed HFD/L-NAME for 5 weeks did not develop diastolic dysfunction or cardiomyocyte hypertrophy, demonstrating a critical role for T cells in experimental cardiometabolic HFpEF. Indeed, cardiac CD4 + T cells were increased in WT mice fed HFD/L-NAME for 5 weeks compared to STD, concordant with an expansion of splenic CD44 hi CD62l lo interferon-gamma (IFNγ) + effector cells. Strikingly, splenic T cells also experienced downregulation of the UPR proteins activating transcription factor 4 ( Atf4 ), Atf6 , and X box-binding protein 1 ( Xbp1s/u ). We found that the Atf4 arm of the T cell UPR is the earliest responder to HFD/L-NAME, with observed downregulation of Atf4 and its downstream effector C/EBP homologous protein ( Chop ) as early as 3 weeks, prior to onset of diastolic dysfunction, followed by the subsequent downregulation of Atf6 and Xbp1s/u . Conclusions: T cells contribute to diastolic dysfunction and cardiomyocyte hypertrophy in experimental cardiometabolic HFpEF. Early modulation of T cell Atf4 precedes diastolic dysfunction, enhanced IFNγ + effector T cell activation, and global T cell UPR downregulation. Ongoing studies are focused on understanding the functional consequences of impaired T cell UPR in cardiometabolic HFpEF.

Why antidiabetic drugs are potentially neuroprotective during the Sars-CoV-2 pandemic: The focus on astroglial UPR and calcium-binding proteins.

We are living in a terrifying pandemic caused by Sars-CoV-2, in which patients with diabetes mellitus have, from the beginning, been identified as having a high risk of hospitalization and mortality. This viral disease is not limited to the respiratory system, but also affects, among other organs, the central nervous system. Furthermore, we already know that individuals with diabetes mellitus exhibit signs of astrocyte dysfunction and are more likely to develop cognitive deficits and even dementia. It is now being realized that COVID-19 incurs long-term effects and that those infected can develop several neurological and psychiatric manifestations. As this virus seriously compromises cell metabolism by triggering several mechanisms leading to the unfolded protein response (UPR), which involves endoplasmic reticulum Ca2+ depletion, we review here the basis involved in this response that are intimately associated with the development of neurodegenerative diseases. The discussion aims to highlight two aspects—the role of calcium-binding proteins and the role of astrocytes, glial cells that integrate energy metabolism with neurotransmission and with neuroinflammation. Among the proteins discussed are calpain, calcineurin, and sorcin. These proteins are emphasized as markers of the UPR and are potential therapeutic targets. Finally, we discuss the role of drugs widely prescribed to patients with diabetes mellitus, such as statins, metformin, and calcium channel blockers. The review assesses potential neuroprotection mechanisms, focusing on the UPR and the restoration of reticular Ca2+ homeostasis, based on both clinical and experimental data.

P472: THE FLT3-ITD RECEPTOR CAN PROMOTE PROTEIN FOLDING IN THE ENDOPLASMIC RETICULUM THROUGH THE OXIDOREDUCTASE ERO1

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy and the most common myeloid tumour in adults. Internal tandem duplications (ITD) in FLT3 receptor appear in 30% of cases and is associated with poor disease outcome. This receptor is synthesized and processed in the endoplasmic reticulum (ER) before being transported to the plasma membrane, where it is activated by ligand binding. Upon activation, it regulates cell differentiation, proliferation, and survival. Mutations in FLT3, such as ITD mutations, not only constitutively activate this receptor, but also prevent its correct processing in the endoplasmic reticulum. Therefore, it is mostly retained in this organelle. Aims: We decided to study if the aberrant accumulation of FLT3-ITD affects the functioning of the ER. Methods: Experiments were performed on wild-type and FLT3-ITD AML cell lines, as well as on stably transfected FLT3 and FLT3-ITD Ba/F3 cell lines. Protein aggregation, reactive oxygen species (ROS) and glutathione (GSH) levels were determined by Thioflavin T, DCFH-DA and Thioltracker Violet fluorescence, respectively. Protein detection was performed by Western Blot. Cell viability and death was analysed by cell count and Trypan Blue exclusion. Results: Protein folding in the ER generates reactive oxygen species (ROS). Antioxidant molecules such as GSH are needed to maintain redox homeostasis. We found that FLT3-ITD cell lines have lower levels of protein aggregates, as well as ROS and GSH. This data could suggest that mutant cell lines have more efficient protein folding mechanisms. In fact, among the ER proteins that are involved in the protein folding process, we found the oxidoreductase ERO1 to be increased in FLT3-ITD cell lines. Supporting our findings, we saw that FLT3-ITD cell lines were less sensitive to protein folding blocking agents, DTT and 2-mercaptoethanol. This was reflected by a slight decrease in cell proliferation compared to wild-type cell lines. We confirmed that the decrease in cell viability in FLT3-ITD cell lines was not accompanied by cell death, as opposed to wild-type cell lines. Under ER stress conditions, such as an accumulation of misfolded proteins in the ER, the unfolded protein response (UPR) is activated. We decided to study if there were differences in the basal levels of UPR by western blot. We found FLT3-ITD cell lines to have lower levels of UPR, reflected by lower protein expression of the active forms of PERK and IRE (p-PERK and p-IRE respectively), together with the protein BiP, the major chaperone found in the ER and binds to misfolded proteins. Altogether, our data suggest that FLT3-ITD cell lines have less ER stress, reflected by lower levels of chaperones and UPR proteins, possibly because they rely on more efficient protein folding mechanisms. To confirm the importance of protein folding process in acute myeloid leukemia, we tested the effect of ERO1 inhibitor (EN460), and found it to have a greater impact in FLT3-ITD cell lines. In order to determine a possible relation between the ITD mutation in the FLT3 receptor and ERO1, we used stably transfected Ba/F3 cell lines carrying the normal or mutated FLT3 receptor. We found ERO1 to be overexpressed on FLT3-ITD Ba/F3 cell lines. Summary/Conclusion: Current AML treatments are mainly based on the administration of FLT3 inhibitors, with limited success. Our data suggest that FLT3-ITD cell lines, with greater resistance to treatments, have more efficient protein folding mechanisms. This process should be studied in more detail to determine its potential as a therapeutic target for AML treatment.

Transcription factor CREB3 is a potent regulator of high-fat diet-induced obesity and energy metabolism.

The endoplasmic reticulum senses alterations to cellular homeostasis that activates the unfolded protein response (UPR). UPR proteins are known to aid in regulating glucose and lipid metabolism. CREB3 is a UPR-associated transcription factor whose potential role in regulating energy metabolism remains unclear. Eight-week-old wild-type (WT) and Creb3+/- mice were placed on control and high-fat diets (HFD) for 8 weeks, and metabolic phenotypes characterized by weekly weighing, indirect calorimetry, body composition scans, glucose tolerance tests, plasma analysis, tissue lipid quantifications and gene/protein expression analysis. HFD weight gain in Creb3+/- males was reduced by 34% (p < 0.0001) and females by 39.5% (p = 0.014) from their WT counterparts. No differences were found in HFD food intake or total fecal lipids between genotypes. Creb3+/- mice had increased energy expenditure and respiratory exchange ratios (p = 0.002) relative to WT. Creb3+/- mice had significant reductions in absolute fat and lean tissue, while Creb3+/- females had significant reductions in body fat% and increased lean% composition (p < 0.0001) compared to WT females. Creb3+/- mice were protected from HFD-induced basal hyperglycemia (males p < 0.0001; females p = 0.0181). Creb3+/- males resisted HFD-induced hepatic lipid accumulation (p = 0.025) and glucose intolerance compared to WT (p < 0.0001) while Creb3+/- females were protected from lipid accumulation in skeletal muscle (p = 0.001). Despite the metabolic differences of Creb3+/- mice on HFD, lipid plasma profiles did not significantly differ from WT. Fasted Creb3+/- mice additionally revealed upregulation of hepatic energy expenditure and gluconeogenic genes such as Pgc-1a and Gr (glucocorticoid receptor) (p < 0.05), respectively. Reduced expression of CREB3 increased energy expenditure and the respiratory exchange ratio, and protected mice from HFD-induced weight gain, basal hyperglycemia, and sex-specific tissue lipid accumulation. We postulate that CREB3 is a novel key regulator of diet-induced obesity and energy metabolism that warrants further investigation as a potential therapeutic target in metabolic disorders.

Activating ATF6 in spinal muscular atrophy promotes SMN expression and motor neuron survival through the IRE1α-XBP1 pathway.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by survival of motor neuron (SMN) deficiency that induces motor neuron (MN) degeneration and severe muscular atrophy. Gene therapies that increase SMN have proven their efficacy but not for all patients. Here, we explored the unfolded protein response (UPR) status in SMA pathology and explored whether UPR modulation could be beneficial for SMA patients. We analysed the expression and activation of key UPR proteins by RT-qPCR and by western blots in SMA patient iPSC-derived MNs and one SMA cell line in which SMN expression was re-established (rescue). We complemented this approach by using myoblast and fibroblast SMA patient cells and SMA mouse models of varying severities. Finally, we tested in vitro and in vivo the effect of IRE1α/XBP1 pathway restoration on SMN expression and subsequent neuroprotection. We report that the IRE1α/XBP1 branch of the unfolded protein response is disrupted in SMA, with a depletion of XBP1s irrespective of IRE1α activation pattern. The overexpression of XBP1s in SMA fibroblasts proved to transcriptionally enhance SMN expression. Importantly, rebalancing XBP1s expression in severe SMA-like mice, induced SMN expression and spinal MN protection. We have identified XBP1s depletion as a contributing factor in SMA pathogenesis, and the modulation of this transcription factor proves to be a plausible therapeutic avenue in the context of pharmacological interventions for patients.

Mechanisms of liver injury in high fat sugar diet fed mice that lack hepatocyte X-box binding protein 1.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of liver diseases in the United States and can progress to cirrhosis, end-stage liver disease and need for liver transplantation. There are limited therapies for NAFLD, in part, due to incomplete understanding of the disease pathogenesis, which involves different cell populations in the liver. Endoplasmic reticulum stress and its adaptative unfolded protein response (UPR) signaling pathway have been implicated in the progression from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH). We have previously shown that mice lacking the UPR protein X-box binding protein 1 (XBP1) in the liver demonstrated enhanced liver injury and fibrosis in a high fat sugar (HFS) dietary model of NAFLD. In this study, to better understand the role of liver XBP1 in the pathobiology of NAFLD, we fed hepatocyte XBP1 deficient mice a HFS diet or chow and investigated UPR and other cell signaling pathways in hepatocytes, hepatic stellate cells and immune cells. We demonstrate that loss of XBP1 in hepatocytes increased inflammatory pathway expression and altered expression of the UPR signaling in hepatocytes and was associated with enhanced hepatic stellate cell activation after HFS feeding. We believe that a better understanding of liver cell-specific signaling in the pathogenesis of NASH may allow us to identify new therapeutic targets.