Endoplasmic Reticulum Function Research Articles

Calnexin, More Than Just a Molecular Chaperone.

Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein with an N-terminal domain that resides in the lumen of the ER and a C-terminal domain that extends into the cytosol. Calnexin is commonly referred to as a molecular chaperone involved in the folding and quality control of membrane-associated and secreted proteins, a function that is attributed to its ER- localized domain with a structure that bears a strong resemblance to another luminal ER chaperone and Ca2+-binding protein known as calreticulin. Studies have discovered that the cytosolic C-terminal domain of calnexin undergoes distinct post-translational modifications and interacts with a variety of proteins. Here, we discuss recent findings and hypothesize that the post-translational modifications of the calnexin C-terminal domain and its interaction with specific cytosolic proteins play a role in coordinating ER functions with events taking place in the cytosol and other cellular compartments.

Brigatinib-repurposed chemo-photodynamic therapy nanoplatform via effective apoptosis against colorectal cancer

Colorectal cancer (CRC), a malignancy with a relatively high fatality rate, still poses a growing global public health challenge due to the lack of well-established and effective treatment strategies. Brigatinib, an ALK inhibitor approved for the treatment of ALK-positive NSCLC, has been repurposed to suppress the growth of ALK-negative CRC cells via sustained endoplasmic reticulum (ER) stress. Moreover, the photosensitizer Chlorine e6 (Ce6) exhibits a high singlet oxygen quantum yield, which promote the generation of reactive oxygen species (ROS) to exacerbate ER stress and function synergistically with chemotherapeutics. However, the clinical application of both brigatinib and Ce6 is limited by their poor water solubility. Therefore, a repurposed chemo-photodynamic therapy nanoplatform (BCT NPs) utilizing d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) as a nanocarrier for the codelivery of the chemotherapeutics brigatinib and photosensitizer Ce6 is developed, which is capable of disrupting redox homeostasis and enhancing ER stress against CRC. The developed nanoplatform successfully addressed therapeutic drug solubility, improved brigatinib biocompatibility with a lower hemolysis rate, and demonstrated satisfactory CRC suppression effects in vitro and in vivo. Overall, BCT NPs provide a promising strategy to construct repurposed and versatile nanoplatforms against CRC.

Loss of TMCC2 activates endoplasm reticulum stress and causes auditory hair cell death.

As the auditory and balance receptor cells in the inner ear, hair cells are responsible for converting mechanical stimuli into electrical signals, a process referred to as mechano-electrical transduction. Hair cell development and function are tightly regulated, and hair cell deficits are the main reasons for hearing loss and balance disorders. TMCC2 is an endoplasmic reticulum (ER)-residing transmembrane protein whose physiological function largely remains unknown. In the present work, we show that Tmcc2 is specifically expressed in the auditory hair cells of mouse inner ear. Tmcc2 knockout mice were then established to investigate its physiological role in hearing. Auditory brainstem responses measurements show that Tmcc2 knockout mice suffer from congenital hearing loss. Further investigations reveal progressive auditory hair cell loss in the Tmcc2 knockout mice. The general morphology and function of ER are unaffected in Tmcc2 knockout hair cells. However, increased ER stress was observed in Tmcc2 knockout mice and knockdown cells, suggesting that loss of TMCC2 leads to auditory hair cell death through elevated ER stress.

Antioxidants affect endoplasmic reticulum stress-related diseases.

The endoplasmic reticulum (ER) is a complex multifunctional organelle that maintains cell homeostasis. Intrinsic and extrinsic factors alter ER functions, including the rate of protein folding that triggers the accumulation of misfolded proteins and alters homeostasis, thus generating stress in the ER, which activates the unfolded protein response (UPR) pathway to promote cell survival and restore their homeostasis; however, if the damage is not corrected, it could also trigger cell death. In addition, ER stress and oxidative stress are closely related because excessive production of reactive oxygen species (ROS), a well-known inducer of ER stress, promotes the accumulation of misfolded proteins; at the same time, the ER stress enhances ROS production, generating a pathological cycle. Furthermore, it has been described that the dysregulation of the UPR contributes to the progression of various diseases, so the use of compounds capable of regulating ER stress, such as antioxidants, has been used in several experimental models of diseases to alleviate the damage induced by the maladaptive signaling of the UPR, the mechanism of action of antioxidants generally is dose-dependent, and it is specific in each tissue and pathology, could decrease or enhance specific proteins of the UPR to have beneficial or detrimental effects.

Golgi apparatus, endoplasmic reticulum and mitochondrial function implicated in Alzheimer’s disease through polygenic risk and RNA sequencing

Polygenic risk scores (PRS) have been widely adopted as a tool for measuring common variant liability and they have been shown to predict lifetime risk of Alzheimer’s disease (AD) development. However, the relationship between PRS and AD pathogenesis is largely unknown. To this end, we performed a differential gene-expression and associated disrupted biological pathway analyses of AD PRS vs. case/controls in human brain-derived cohort sample (cerebellum/temporal cortex; MayoRNAseq). The results highlighted already implicated mechanisms: immune and stress response, lipids, fatty acids and cholesterol metabolisms, endosome and cellular/neuronal death, being disrupted biological pathways in both case/controls and PRS, as well as previously less well characterised processes such as cellular structures, mitochondrial respiration and secretion. Despite heterogeneity in terms of differentially expressed genes in case/controls vs. PRS, there was a consensus of commonly disrupted biological mechanisms. Glia and microglia-related terms were also significantly disrupted, albeit not being the top disrupted Gene Ontology terms. GWAS implicated genes were significantly and in their majority, up-regulated in response to different PRS among the temporal cortex samples, suggesting potential common regulatory mechanisms. Tissue specificity in terms of disrupted biological pathways in temporal cortex vs. cerebellum was observed in relation to PRS, but limited tissue specificity when the datasets were analysed as case/controls. The largely common biological mechanisms between a case/control classification and in association with PRS suggests that PRS stratification can be used for studies where suitable case/control samples are not available or the selection of individuals with high and low PRS in clinical trials.

THE ROLE OF THE ENDOPLASMIC RETICULUM UNFOLDED PROTEIN STRESS RESPONSES IN A C. ELEGANS MODEL OF QUIESCENCE

Abstract Cells devote extraordinary energy to translate and fold approximately 20,000 proteins encoded by the human genome. About one third of these proteins are transmembrane proteins—correctly folded in the endoplasmic reticulum (ER). The ER Unfolded Protein Response (UPRER) is a response to disturbances at the ER. As an organism ages, cells lose the ability to activate the UPRER. Our lab uses C. elegans—a microscopic roundworm—as a model organism to determine how loss of ER function can contribute to aging. The UPRER plays a key role in maintenance of functional quiescence. Quiescent cells exist in a reversible state of dormancy and can receive specific environmental cues to proliferate. The developmental state known as L1 arrest resembles the same features as quiescent cells. IRE-1 is required for worms to exit their quiescent state after starvation. Understanding the genetic pathways that are best at suppressing ire-1 terminal L1 arrest could indicate ways to improve functional quiescence. We forced the worms into an L1 arrest state; The unfolded protein response in the endoplasmic reticulum maintains functional quiescence within the cell. The worm’s ability to develop once they exit this quiescent stage effectively, indicated the ability of the worm to maintain protein homeostasis despite non-ideal conditions. It was found that all tested mutations were unable to demonstrate improvement in ability to maintain development when arrested long-term. Our data indicates that our worms were counted prematurely. In the future, we will allow for a longer growth period as well as larger quantities of food.

Mitochondria and Endoplasmic Reticulum Stress in Retinal Organoids from Patients with Vision Loss

Activating transcription factor 6 (ATF6), a key regulator of the unfolded protein response, plays a key role in endoplasmic reticulum function and protein homeostasis. Variants of ATF6 that abrogate transcriptional activity cause morphologic and molecular defects in cones, clinically manifesting as the human vision loss disease achromatopsia (ACHM). ATF6 is expressed in all retinal cells. However, the effect of disease-associated ATF6 variants on other retinal cell types remains unclear. Herein, this was investigated by analyzing bulk RNA-sequencing transcriptomes from retinal organoids generated from patients with ACHM, carrying homozygous loss-of-function ATF6 variants. Marked dysregulation in mitochondrial respiratory complex gene expression and disrupted mitochondrial morphology in ACHM retinal organoids were identified. This indicated that loss of ATF6 leads to previously unappreciated mitochondrial defects in the retina. Next, gene expression from control and ACHM retinal organoids were compared with transcriptome profiles of seven major retinal cell types generated from recent single-cell transcriptomic maps of nondiseased human retina. This indicated pronounced down-regulation of cone genes and up-regulation in Müller glia genes, with no significant effects on other retinal cells. Overall, the current analysis of ACHM patient retinal organoids identified new cellular and molecular phenotypes in addition to cone dysfunction: activation of Müller cells, increased endoplasmic reticulum stress, disrupted mitochondrial structure, and elevated respiratory chain activity gene expression.

Bisphenol S Impairs Behaviors through Disturbing Endoplasmic Reticulum Function and Reducing Lipid Levels in the Brain of Zebrafish.

The number of neurotoxic pollutants is increasing, but their mechanism of action is unclear. Here, zebrafish were exposed to 0, 1, 10, and 100 μg/L bisphenol S (BPS) for different durations beginning at 2 h postfertilization (hpf) to explore the neurotoxic mechanisms of BPS. Zebrafish larvae exposed to BPS displayed abnormal neurobehaviors. At 48 and 120 hpf, BPS inhibited yolk lipid consumption and reduced the lipid distribution in the zebrafish brain. Moreover, BPS downregulated the mRNA levels of genes involved in fatty acid elongation in the endoplasmic reticulum (ER) and activated ER stress pathways at 48 and 120 hpf, and KEGG analysis after RNA-seq showed that the protein processing pathway in the ER was significantly enriched after BPS exposure. Exposure to ER toxicants (thapsigargin and tunicamycin), two positive controls, induced neurotoxic effects on zebrafish embryos and larvae similar to those of BPS exposure. These data suggested that BPS and ER toxicants disturbed ER function and reduced brain lipid levels. Continued exposure to BPS into adulthood not only inhibited brain fatty acid elongation and ER function but also caused abnormal swelling of the ER in zebrafish. Our data provide new insights into the neurotoxic mechanism of BPS.

ER-associated RNA silencing promotes ER quality control

The endoplasmic reticulum (ER) coordinates mRNA translation and processing of secreted and endomembrane proteins. ER-associated degradation (ERAD) prevents the accumulation of misfolded proteins in the ER, but the physiological regulation of this process remains poorly characterized. Here, in a genetic screen using an ERAD model substrate in Caenorhabditis elegans, we identified an anti-viral RNA interference pathway, referred to as ER-associated RNA silencing (ERAS), which acts together with ERAD to preserve ER homeostasis and function. Induced by ER stress, ERAS is mediated by the Argonaute protein RDE-1/AGO2, is conserved in mammals and promotes ER-associated RNA turnover. ERAS and ERAD are complementary, as simultaneous inactivation of both quality-control pathways leads to increased ER stress, reduced protein quality control and impaired intestinal integrity. Collectively, our findings indicate that ER homeostasis and organismal health are protected by synergistic functions of ERAS and ERAD.

AA147 ameliorates post-cardiac arrest cerebral ischemia/reperfusion injury through the co-regulation of the ATF6 and Nrf2 signaling pathways.

Ischemia/reperfusion caused by cardiac arrest (CA) disturbs endoplasmic reticulum (ER) homeostasis and redox balance in neurons. AA147, originally developed as a pharmacologic activator of the activating transcription factor 6 (ATF6), can protect multiple tissues from ischemia/reperfusion injury (IRI) by decreasing reactive oxygen species (ROS) and restoring ER function. However, it is unclear whether pharmacologic treatment of AA147 could ameliorate post-CA cerebral IRI and whether it is associated with proteostasis regulation and anti-oxidative stress mechanism. In the present study, mice were subjected to 9 min-CA surgery followed by cardiopulmonary resuscitation (CPR). AA147 or vehicle was administered 1 day before the operation and 15min after the return of spontaneous circulation. We found that AA147 restored neurological function and reduced dead neurons in mice suffering from CA. Moreover, AA147 inhibited CA/CPR-caused neuronal apoptosis and ER stress, indicated by reduced TUNEL-positive neurons, surged expression of Bcl-2/Bax, and down expression of cleaved caspase-3, caspase-12, C/EBP homologous protein (CHOP). The expression of ATF6 and its regulated gene glucose-regulated protein 78 (GRP78) increased significantly after the administration of AA147, suggesting the activation of the ATF6 pathway. In addition, AA147 also alleviated the upsurge of the ROS generation and MDA levels as well as increased SOD activity, accompanied by enhancement of the nuclear factor E2-related factor 2 (Nrf2) and its modulated heme-oxygenase-1 (HO-1) expressions. Cotreatment of AA147 with inhibitors of the ATF6 or Nrf2 significantly suppressed AA147-dependent reductions in ROS scavenging and neuronal death after CA/CPR. The results suggested that AA147 could confer neuroprotection against post-CA cerebral IRI through inhibition of oxidative stress along with ER stress-associated apoptosis, which is attributed to the coregulation of both ATF6 and Nrf2 signaling pathways activity. Our findings support the potential for AA147 as a therapeutic approach to improve post-CA brain injury.

ER Stress and the Unfolded Protein Response: Homeostatic Regulation Coordinate Plant Survival and Growth

The endoplasmic reticulum (ER), a eukaryotic organelle, is the major site of protein biosynthesis. The disturbance of ER function by biotic or abiotic stress triggers the accumulation of misfolded or unfolded proteins in the ER. The unfolded protein response (UPR) is the best-studied ER stress response. This transcriptional regulatory system senses ER stress, activates downstream genes that function to mitigate stress, and restores homeostasis. In addition to its conventional role in stress responses, recent reports indicate that the UPR is involved in plant growth and development. In this review, we summarize the current knowledge of ER stress sensing and the activation and downstream regulation of the UPR. We also describe how the UPR modulates both plant growth and stress tolerance by maintaining ER homeostasis. Lastly, we propose that the UPR is a major component of the machinery that balances the trade-off between plant growth and survival in a dynamic environment.

Endoplasmic reticulum stress increases exosome biogenesis and packaging relevant to sperm maturation in response to oxidative stress in obese mice

BackgroundMammalian sperm maturation in the epididymis is mainly modulated by exosomes that are secreted into the epididymal lumen from epididymal epithelial cells (EECs). Exposure to oxidative stress (OS) resulting from being fed a high fat diet (HFD) reduces sperm fertility, which is one of the cause inducing male infertility. Thus, we hypothesize that stress-induced changes in exosome content play a critical role in mediating this detrimental process. MethodsAn obese mouse model was established by feeding a HFD. Then oxidative stress status was measured in the mouse caput epididymis, epididymal fluid and spermatozoa. Meanwhile, epididymis-derived purified exosomes were isolated and validated. Subsequently, liquid chromatography tandem mass spectrometry (LC-MS) was used to perform proteomic analysis of purified exosomes. Gene Ontology (GO) analysis was performed along with pathway enrichment to identify differentially expressed proteins (DEPs).ResultsTwo hundred and two DEPs mostly related to endoplasmic reticulum (ER) function were identified in the exosomes separated from the epididymis of control mice and obese mice. The ER stress and CD63 (an exosome marker), both increased in the caput epididymis of obese mice. Furthermore, an in vitro study showed that palmitic acid (PA), an-oxidative stress inducer, increased exosome biogenesis and secretion in the EECs.ConclusionOxidative stress in the epididymal microenvironment induces ER stress in the EECs. This effect alters the epididymis-derived exosome content, profile and amounts of their differentially expressed ER proteins. Such changes may affect exosome biogenesis and cargo packaging, finally leading to abnormalities in sperm maturation and fertility.

ER stress and calcium-dependent arrhythmias.

The sarcoplasmic reticulum (SR) plays the key role in cardiac function as the major source of Ca2+ that activates cardiomyocyte contractile machinery. Disturbances in finely-tuned SR Ca2+ release by SR Ca2+ channel ryanodine receptor (RyR2) and SR Ca2+ reuptake by SR Ca2+-ATPase (SERCa2a) not only impair contraction, but also contribute to cardiac arrhythmia trigger and reentry. Besides being the main Ca2+ storage organelle, SR in cardiomyocytes performs all the functions of endoplasmic reticulum (ER) in other cell types including protein synthesis, folding and degradation. In recent years ER stress has become recognized as an important contributing factor in many cardiac pathologies, including deadly ventricular arrhythmias. This brief review will therefore focus on ER stress mechanisms in the heart and how these changes can lead to pro-arrhythmic defects in SR Ca2+ handling machinery.

IER3IP1 is critical for maintaining glucose homeostasis through regulating the endoplasmic reticulum function and survival of β cells

Recessive mutations in IER3IP1 (immediate early response 3 interacting protein 1) cause a syndrome of microcephaly, epilepsy, and permanent neonatal diabetes (MEDS). IER3IP1 encodes an endoplasmic reticulum (ER) membrane protein, which is crucial for brain development; however, the role of IER3IP1 in β cells remains unknown. We have generated two mouse models with either constitutive or inducible IER3IP1 deletion in β cells, named IER3IP1-βKO and IER3IP1-iβKO, respectively. We found that IER3IP1-βKO causes severe early-onset, insulin-deficient diabetes. Functional studies revealed a markedly dilated β-cell ER along with increased proinsulin misfolding and elevated expression of the ER chaperones, including PDI, ERO1, BiP, and P58IPK. Islet transcriptome analysis confirmed by qRT-PCR revealed decreased expression of genes associated with β-cell maturation, cell cycle, and antiapoptotic genes, accompanied by increased expression of antiproliferation genes. Indeed, multiple independent approaches further demonstrated that IER3IP1-βKO impaired β-cell maturation and proliferation, along with increased condensation of β-cell nuclear chromatin. Inducible β-cell IER3IP1 deletion in adult (8-wk-old) mice induced a similar diabetic phenotype, suggesting that IER3IP1 is also critical for function and survival even after β-cell early development. Importantly, IER3IP1 was decreased in β cells of patients with type 2 diabetes (T2D), suggesting an association of IER3IP1 deficiency with β-cell dysfunction in the more-common form of diabetes. These data not only uncover a critical role of IER3IP1 in β cells but also provide insight into molecular basis of diabetes caused by IER3IP1 mutations.

PMON312 A De Novo Heterozygous Nonsense Variant In The SEC31A Gene Associated With Pituitary Hormone Deficiency And Disorders Of Sex Development.

Abstract Introduction XYdisorders of sex development (DSD) result from variants in many different human genes but frequently have no detectable molecular cause. In approximately 25% of cases of XY DSD, the index case may have associated malformations. Genetic disorders of endoplasmic reticulum (ER) function are increasingly being recognised but have not been associated with DSD or pituitary disorders. Clinical case Three siblings (with unaffected non-consanguineous parents) were reviewed at the tertiary endocrine clinic. Child I was noted at birth to have cliteromegaly. Imaging and examination under anaesthetic revealed a normal vagina and uterus but gonads of indeterminate origin. She was 46,XY and basal endocrine investigations at the age of 4 years showed a low AMH for male but otherwise normal gonadal and thyroid function and normal IGF-1. She had a laparoscopic bilateral gonadectomy aged 5 years. Pathology demonstrated bilateral testicular tissue, with substantial fibrotic atrophic change and occasional placental alkaline phosphatase (PLAP) positive cells, suggestive of germ cell tumours. Aged 8 years she developed obesity and later hypertension. Child II was reviewed due to short stature and diagnosed with GH deficiency aged 2 years. She has normal adrenal and thyroid function and gonadotrophins. MRI demonstrated an ectopic posterior pituitary. Child III presented with perineal hypospadias, a small phallus, bilateral undescended testes and craniofacial abnormalities. Endocrine investigations revealed hypogonadotrophic hypogonadism, with no testosterone response to hCG stimulation, a low normal AMH and no response of LH or FSH on LHRH stimulation. He has panhypopituitarism with an ectopic posterior pituitary gland on MRI and is currently on treatment with GH, hydrocortisone and levothyroxine. His BP is on the 98th centile for age and height. Child I and Child III have mild developmental delay but are in mainstream school with additional educational support. High-throughput DNA sequencing revealed, in all three siblings, a heterozygous truncating variant in the SEC31A gene that encodes a component of the COPII-complex that coats the vesicles mediating ER to Golgi transport. CRISPR-Cas9 targeted knockout of the corresponding Sec31a region resulted in embryonic lethality in homozygous mice. mRNA phenotyping of ER-related genes demonstrated increased mRNA expression of ATF4 and CHOP in the affected children, genes encoding key ER stress-related proteins, associated with defective protein transport. Conclusions Dysregulation ofanterograde and retrograde COPII-coated-vesicle ER-Golgi transport is increasingly recognised to underlie human developmental disorders, including Craniolenticulosutural dysplasia (OMIM 607812) and Saul-Wilson syndrome (OMIM 618150). The de novo SEC31A nonsense variant in all three affected siblings, the ER stress response, plus reported developmental syndromes with dysfunction of this transport mechanism and evidence from the preclinical mouse model suggest that SEC31A might underlie a previously unrecognised clinical syndrome comprising DSD, endocrine abnormalities, dysmorphic features and developmental delay. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

The Role of the GRP78/PERK/ATF4 Pathway in the Ability of Gua Lou Gui Zhi Decoction to Attenuate Apoptosis by Inhibiting Endoplasmic Reticulum Stress after Ischemia-Reperfusion Injury.

Endoplasmic reticulum stress (ERS) is a key part of the apoptotic cascade that is initiated after cerebral ischemia-reperfusion injury and is very important for research on poststroke rehabilitation. In addition, the unfolded protein response (UPR) plays an important role in ERS because it activates downstream apoptotic signal transduction and induces apoptosis through the glucose-regulated protein 78 (GRP78)/protein kinase R (PKR)-like ER kinase (PERK)/activating transcription factor 4 (ATF4) pathway. The Gua Lou Gui Zhi Decoction (GLGZD) ameliorated neuronal apoptosis of ischemia-reperfusion injury caused by middle cerebral artery occlusion (MCAO) had been proved in our previous study. The present study aims to underly the regulatory ability of GLGZD in ERS-induced apoptosis mediated by the GRP78/PERK/ATF4 pathway. GLGZD was analyzed by HPLC. The effects of GLGZD were obversed on MCAO-induced ischemic rats. The cerebral infarct volume was detected by 2,3,5-Triphenyl-2H-Tetrazolium Chloride (TTC) Staining. Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End Labeling (TUNEL) were used to detect apoptosis. Transmission Electron Microscopy (TEM), Ca2+ levels and reactive oxygen species (ROS) detection were used to determine the function of endoplasmic reticulum. The GRP78/PERK/ATF4 signaling pathway was assessed by western blotting and immunohistochemistry. Our results showed that GLGZD exerted its effects on ischemia-reperfusion injury by significantly promoting the restoration of the quantity and morphology of the rough ER and reducing the neuronal apoptosis rate in the ischemic cortex. Moreover, both of the intracellular ROS and Ca2+ levels in ischemic cortical cells were found significantly reduced by GLGZD. The GLGZD-treated group showed increased levels of phosphorylation in both of PERK and eukaryotic translation initiation factor 2α (eIF2α), activation of cysteinyl aspartate-specific proteinase-3 (Caspase-3), upregulation of the total protein levels of sarcoplasmic/endoplasmic Ca2+ ATPase 2α (SERCA 2α) and B-cell leukemia/lymphoma gene 2 (Bcl-2). These findings suggest that GLGZD reduces oxidative stress-induced injury and promotes a dynamic calcium balance, thereby inhibiting ERS and exerting an antiapoptotic effect on neuronal ischemic injury, which are closely related to the activation of GRP78/PERK/ATF4 signaling pathway.

Emerging links between endoplasmic reticulum stress responses and acute kidney injury.

All cell types must maintain homeostasis under periods of stress. To prevent the catastrophic effects of stress, all cell types also respond to stress by inducing protective pathways. Within the cell, the endoplasmic reticulum (ER) is exquisitely stress-sensitive, primarily because this organelle folds, posttranslationally processes, and sorts one-third of the proteome. In the 1990s, a specialized ER stress response pathway was discovered, the unfolded protein response (UPR), which specifically protects the ER from damaged proteins and toxic chemicals. Not surprisingly, UPR-dependent responses are essential to maintain the function and viability of cells continuously exposed to stress, such as those in the kidney, which have high metabolic demands, produce myriad protein assemblies, continuously filter toxins, and synthesize ammonia. In this mini-review, we highlight recent articles that link ER stress and the UPR with acute kidney injury (AKI), a disease that arises in ∼10% of all hospitalized individuals and nearly half of all people admitted to intensive care units. We conclude with a discussion of prospects for treating AKI with emerging drugs that improve ER function.

Beneficial effects of neuronal ATF6 activation in permanent ischemic stroke.

Objective: Brain ischemia leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen and consequently, ER stress. To help cells restore ER function, a series of adaptive stress response pathways, collectively termed the unfolded protein response (UPR), are activated. We have previously demonstrated that the UPR pathway initiated by ATF6 is pro-survival in transient ischemic stroke. However, the effect of ATF6 activation on the outcome after permanent ischemic stroke remains unknown. Here, we addressed this knowledge gap.Method: sATF6-KI mice with functional short-form ATF6 (sATF6) predominantly expressed in forebrain neurons were subjected to two ischemic stroke models: photothrombotic stroke and permanent middle cerebral artery occlusion (pMCAO). Both short-term and long-term functional outcomes were evaluated. Changes in neuroinflammation and cerebrovascular density after pMCAO were also assessed.Results: Compared to littermate controls, sATF6-KI mice performed significantly better in open field, cylinder, and foot fault tests on day 1 or 3 after photothrombotic stroke. However, on days 7 and 14 after stroke, the performance of these functional tests was not significantly different between groups, which is likely related to mild brain damage associated with this stroke model. Thus, to evaluate the long-term effects of ATF6 activation in permanent stroke, we turned to our pMCAO model. We first found that on day 4 after pMCAO, functional outcome was better, and infarct volumes were smaller in sATF6-KI mice vs controls. Next, the 15-day stroke outcome study indicated that compared to control mice, sATF6-KI mice consistently exhibited improved performance in neurologic scoring, tight rope test, and tape removal test, after pMCAO. Moreover, sATF6-KI mice showed higher vascular density and lower activation of both astrocytes and microglia around stroke regions on day 16 after pMCAO.Conclusions: Here, we presented the first evidence that activation of the ATF6 UPR branch is protective in permanent ischemic stroke, which further supports the therapeutic potential of targeting the ATF6 pathway in stroke.

Diverse Sphingolipid Species Harbor Different Effects on Ire1 Clustering

Endoplasmic reticulum (ER) function is dedicated to multiple essential processes in eukaryotes, including the processing of secretory proteins and the biogenesis of most membrane lipids. These roles implicate a heavy burden to the organelle, and it is thus prone to fluctuations in the homeostasis of molecules which govern these processes. The unfolded protein response (UPR) is a general ER stress response tasked with maintaining the ER for optimal function, mediated by the master activator Ire1. Ire1 is an ER transmembrane protein that initiates the UPR, forming characteristic oligomers in response to irregularities in luminal protein folding and in the membrane lipid environment. The role of lipids in regulating the UPR remains relatively obscure; however, recent research has revealed a potent role for sphingolipids in its activity. Here, we identify a major role for the oxysterol-binding protein Kes1, whose activity is of consequence to the sphingolipid profile in cells resulting in an inhibition of UPR activity. Using an mCherry-tagged derivative of Ire1, we observe that this occurs due to inhibition of Ire1 to form oligomers. Furthermore, we identify that a sphingolipid presence is required for Ire1 activity, and that specific sphingolipid profiles are of major consequence to Ire1 function. In addition, we highlight cases where Ire1 oligomerization is absent despite an active UPR, revealing a potential mechanism for UPR induction where Ire1 oligomerization is not necessary. This work provides a basis for the role of sphingolipids in controlling the UPR, where their metabolism harbors a crucial role in regulating its onset.

Endoplasmic Reticulum Homeostasis Regulates TLR4 Expression and Signaling in Mast Cells

The endoplasmic reticulum (ER) is a dynamic organelle that responds to demand in secretory proteins by undergoing expansion. The mechanisms that control the homeostasis of ER size and function involve the activation of the unfolded protein response (UPR). The UPR plays a role in various effector functions of immune cells. Mast cells (MCs) are highly granular tissue-resident cells and key drivers of allergic inflammation. Their diverse secretory functions in response to activation through the high-affinity receptor for IgE (FcεRI) suggest a role for the UPR in their function. Using human cord blood-derived MCs, we found that FcεRI triggering elevated the expression level and induced activation of the UPR transducers IRE1α and PERK, accompanied by expansion of the ER. In mouse bone marrow-derived MCs and peritoneal MCs, the ER underwent a more moderate expansion, and the UPR was not induced following MC activation. The deletion of IRE1α in mouse MCs did not affect proliferation, survival, degranulation, or cytokine stimulation following FcεRI triggering, but it did diminish the surface expression of TLR4 and the consequent response to LPS. A similar phenotype was observed in human MCs using an IRE1α inhibitor. Our data indicate that the ER of MCs, primarily of humans, undergoes a rapid remodeling in response to activation that promotes responses to TLR4. We suggest that IRE1α inhibition can be a strategy for inhibiting the hyperactivation of MCs by LPS over the course of allergic responses.