Aberrant Endoplasmic Reticulum Research Articles

Visualizing Endoplasmic Reticulum Stress and Autophagy in Alzheimer's Model Cells by a Peroxynitrite-Responsive AIEgen Fluorescent Probe.

Endoplasmic reticulum (ER) stress and autophagy (ER-phagy) occurring in nerve cells are crucial physiological processes closely associated with Alzheimer's disease (AD). Visualizing the two processes is paramount to advance our understanding of AD pathologies. Among the biomarkers identified, peroxynitrite (ONOO-) emerges as a key molecule in the initiation and aggravation of ER stress and ER-phagy, highlighting its significance in the underlying mechanisms of the two processes. In this work, we designed and synthesized an innovative ONOO--responsive AIEgen-based fluorescent probe (DHQM) with the ability to monitor ER stress and ER-phagy in AD model cells. DHQM demonstrated excellent aggregation-induced emission (AIE) properties, endowing it with outstanding ability for washing-free intracellular imaging. Meanwhile, it exhibited high sensitivity, remarkable selectivity to ONOO-, and exceptional ER-targeting ability. The probe was successfully applied for fluorescence imaging of ER ONOO- fluctuations to assess the ER stress status in aluminum-induced AD model cells. Our findings revealed that aluminum-induced ferroptosis, a regulated cell death process, was pivotal in the excessive ONOO- production, which in turn activated and exacerbated ER stress. Furthermore, the aluminum-stimulated ER-phagy was observed utilizing DHQM, which might be crucial in inhibiting ferroptosis and mitigating aberrant ER stress. Overall, this study not only offers valuable insights into the pathological mechanisms of AD at the ER level but also opens new potential therapeutic avenues targeting these pathways.

Exploring the dual role of endoplasmic reticulum stress in urological cancers: Implications for tumor progression and cell death interactions.

The endoplasmic reticulum (ER) is crucial for maintaining calcium balance, lipid biosynthesis, and protein folding. Disruptions in ER homeostasis, often due to the accumulation of misfolded or unfolded proteins, lead to ER stress, which plays a significant role in various diseases, especially cancer. Urological cancers, which account for high male mortality worldwide, pose a persistent challenge due to their incurability and tendency to develop drug resistance. Among the numerous dysregulated biological mechanisms, ER stress is a key factor in the progression and treatment response of these cancers. This review highlights the dual role of aberrant ER stress activation in urologic cancers, affecting both tumor growth and therapeutic outcomes. While ER stress can support tumor growth through pro-survival autophagy, it primarily inhibits cancer progression via apoptosis and pro-death autophagy. Interestingly, ER stress can paradoxically aid cancer progression through mechanisms such as exosome-mediated immune evasion. Additionally, the review examines how pharmacological interventions, particularly with phytochemicals, can stimulate ER stress-mediated tumor suppression. Key regulators, including PERK, IRE1α, and ATF6, are discussed for their roles in upregulating CHOP levels and triggering apoptosis. In conclusion, a deeper understanding of ER stress in urological cancers not only clarifies the complex interactions between cellular stress and cancer progression but also provides new opportunities for innovative therapeutic strategies.

The potential protective role of carotenoids from saffron: A focus on endoplasmic reticulum stress-related organ damage.

The anticancer, antioxidant, and immunomodulatory properties of carotenoids from saffron or apocarotenoids (e.g., crocin, safranal, crocetin, and picrocrocin) have prompted research into their benefits. Apocarotenoids seem to be effective compound for the treatment of chronic diseases, such as neurodegenerative, cardiovascular, cancer, respiratory, and metabolic disorders. Endoplasmic reticulum (ER) is an essential organelle found in the cytoplasm of eukaryotic cells that participates in the biosynthesis of proteins, lipids, and steroid hormones. Given the role of the ER in the regulation of several fundamental biological processes, including metabolic pathways and immune responses, aberrant ER function can have a significant influence on these vital processes and result in serious pathological consequences. Exposure of cell to adverse environmental challenges, such as toxic agents, ischemia, and so on, causes accumulation of unfolded or misfolded proteins in the ER lumen, also called ER stress. There is a growing evidence to suggest that ER disturbance in the form of oxidative/nitrosative stress and subsequent apoptotic cell death plays major roles in the pathogenesis of many human diseases, including cardiovascular diseases, diabetes mellitus, neurodegenerative diseases, and liver diseases. Apocarotenoids with their unique properties can modulate ER stress through PERK/eIF2α/ATF4/CHOP (protein kinase R (PKR)-like ER kinase/eukaryotic initiation factor 2α/activating transcription factor 4/C/EBP /homologous protein) and X-Box Binding Protein 1/activating transcription factor 6 (XBP1/ATF6) pathways. In addition, they suppress apoptosis through inhibition of endoplasmic and mitochondrial-dependent caspase cascade and can stimulate SIRT1 (silent information regulator 1) and Nrf2 (nuclear factor erythroid 2-related factor 2) expression, thereby leading to protection against oxidative stress. This review summarizes the potential benefits of apocarotenoids in various ER-stress-related disorders.

Massive ER protein disposal by reticulophagy receptors and selective disposal by TOLLIP

ABSTRACT Proteostasis of the endoplasmic reticulum (ER) is maintained by coordinated action of two major catabolic pathways: proteasome-dependent ER-associated degradation (ERAD) and less characterized lysosomal pathways. Recent studies on ER-specific autophagy (termed “reticulophagy”) have highlighted the importance of lysosomes for ER proteostasis. Key to this process are proteins termed reticulophagy receptors that connect ER fragments and Atg8-family proteins, facilitating the lysosomal degradation of both native and aberrant ER proteins in a relatively nonselective manner. In contrast, our recent work identified TOLLIP as a novel type of cargo receptor specifically dedicated to the lysosomal degradation of aberrant ER membrane proteins. The clients of TOLLIP include an engineered model substrate, which mimics an ER-retained aberrant membrane protein, and motor neuron disease-linked misfolded mutants of VAPB and BSCL2/Seipin. TOLLIP acts as a receptor to connect these aberrant ER membrane proteins and phosphatidylinositol-3-phosphate (PtdIns3P) by recognizing the former through its misfolding-sensing intrinsically disordered region (IDR) and ubiquitin-binding CUE domain, and the latter through its C2 domain. These interactions enable PtdIns3P-dependent vesicular trafficking of aberrant membrane proteins to lysosomes without promoting reticulophagic turnover of bulk ER.

Endoplasmic reticulum and inner nuclear membrane ubiquitin-conjugating enzymes Ubc6 and Ubc7 confer resistance to hygromycin B in Saccharomyces cerevisiae.

Aberrant endoplasmic reticulum (ER) and inner nuclear membrane (INM) proteins are destroyed through ER-associated degradation (ERAD) and INM-associated degradation (INMAD). We previously showed the Hrd1, Doa10, and Asi ERAD and INMAD ubiquitin ligases (E3s) in Saccharomyces cerevisiae confer resistance to hygromycin B, which distorts the ribosome decoding center. Here, we assessed the requirement of Ubc6 and Ubc7, the primary ERAD and INMAD ubiquitin-conjugating enzymes (E2s) for hygromycin B resistance. Loss of either E2 sensitized cells to hygromycin B, with UBC7 deletion having a greater impact, consistent with characterized roles for Ubc6 and Ubc7 in ER and INM protein quality control.

WASF3 disrupts mitochondrial respiration and may mediate exercise intolerance in myalgic encephalomyelitis/chronic fatigue syndrome

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by various disabling symptoms including exercise intolerance and is diagnosed in the absence of a specific cause, making its clinical management challenging. A better understanding of the molecular mechanism underlying this apparent bioenergetic deficiency state may reveal insights for developing targeted treatment strategies. We report that overexpression of Wiskott-Aldrich Syndrome Protein Family Member 3 (WASF3), here identified in a 38-y-old woman suffering from long-standing fatigue and exercise intolerance, can disrupt mitochondrial respiratory supercomplex formation and is associated with endoplasmic reticulum (ER) stress. Increased expression of WASF3 in transgenic mice markedly decreased their treadmill running capacity with concomitantly impaired respiratory supercomplex assembly and reduced complex IV levels in skeletal muscle mitochondria. WASF3 induction by ER stress using endotoxin, well known to be associated with fatigue in humans, also decreased skeletal muscle complex IV levels in mice, while decreasing WASF3 levels by pharmacologic inhibition of ER stress improved mitochondrial function in the cells of the patient with chronic fatigue. Expanding on our findings, skeletal muscle biopsy samples obtained from a cohort of patients with ME/CFS showed increased WASF3 protein levels and aberrant ER stress activation. In addition to revealing a potential mechanism for the bioenergetic deficiency in ME/CFS, our study may also provide insights into other disorders associated with fatigue such as rheumatic diseases and long COVID.

Fatty Acid Excess Dysregulates CARF to Initiate the Development of Hepatic Steatosis.

CARF (CDKN2AIP) regulates cellular fate in response to various stresses. However, its role in metabolic stress is unknown. We found that fatty livers from mice exhibit low CARF expression. Similarly, overloaded palmitate inhibited CARF expression in HepG2 cells, suggesting that excess fat-induced stress downregulates hepatic CARF. In agreement with this, silencing and overexpressing CARF resulted in higher and lower fat accumulation in HepG2 cells, respectively. Furthermore, CARF overexpression lowered the ectopic palmitate accumulation in HepG2 cells. We were interested in understanding the role of hepatic CARF and underlying mechanisms in the development of NAFLD. Mechanistically, transcriptome analysis revealed that endoplasmic reticulum (ER) stress and oxidative stress pathway genes significantly altered in the absence of CARF. IRE1α, GRP78, and CHOP, markers of ER stress, were increased, and the treatment with TUDCA, an ER stress inhibitor, attenuated fat accumulation in CARF-deficient cells. Moreover, silencing CARF caused a reduction of GPX3 and TRXND3, leading to oxidative stress and apoptotic cell death. Intriguingly, CARF overexpression in HFD-fed mice significantly decreased hepatic steatosis. Furthermore, overexpression of CARF ameliorated the aberrant ER function and oxidative stress caused by fat accumulation. Our results further demonstrated that overexpression of CARF alleviates HFD-induced insulin resistance assessed with ITT and GTT assay. Altogether, we conclude that excess fat-induced reduction of CARF dysregulates ER functions and lipid metabolism leading to hepatic steatosis.

Axonal ER Ca2+ Release Selectively Enhances Activity-Independent Glutamate Release in a Huntington Disease Model.

Action potential-independent (miniature) neurotransmission occurs at all chemical synapses, but remains poorly understood, particularly in pathological contexts. Axonal endoplasmic reticulum (ER) Ca2+ stores are thought to influence miniature neurotransmission, and aberrant ER Ca2+ handling is implicated in progression of Huntington disease (HD). Here, we report elevated miniature excitatory postsynaptic current (mEPSC) frequencies in recordings from YAC128 mouse (HD-model) neurons (from cortical cultures and striatum-containing brain slices - both from male and female animals). Pharmacological experiments suggest this is mediated indirectly by enhanced tonic ER Ca2+ release. Calcium imaging, using an axon-localized sensor, revealed slow action potential (AP)-independent ER Ca2+ release waves in both YAC128 and wild-type cultures. These Ca2+ waves occurred at similar frequencies in both genotypes, but spread less extensively and were of lower amplitude in YAC128 axons, consistent with axonal ER Ca2+ store depletion. Surprisingly, basal cytosolic Ca2+ levels were lower in YAC128 boutons and YAC128 mEPSCs were less sensitive to intracellular Ca2+ chelation. Together, these data suggest elevated miniature glutamate release in YAC128 cultures is associated with axonal ER Ca2+ depletion but not directly mediated by ER Ca2+ release into the cytoplasm. In contrast to increased mEPSC frequencies, cultured YAC128 cortical neurons showed less frequent AP-dependent (spontaneous) Ca2+ events in soma and axons, although evoked glutamate release detected by iGluSnFR in brain slices was similar between genotypes. Our results indicate axonal ER dysfunction selectively elevates miniature glutamate release from cortical terminals in HD. This, together with reduced spontaneous cortical neuron firing, may cause a shift from activity-dependent to -independent glutamate release in HD, with potential implications for fidelity and plasticity of cortical excitatory signaling.Significance statementMiniature neurotransmitter release persists at all chemical neuronal synapses in the absence of action potential firing but remains poorly understood, particularly in disease states. We show enhanced miniature glutamate release from cortical neurons in the YAC128 mouse Huntington disease model. This effect is mediated by axonal ER Ca2+ store depletion, but is not obviously due to elevated ER-to-cytosol Ca2+ release. Conversely, YAC128 cortical pyramidal neurons fired fewer action potentials and evoked cortical glutamate release was similar between WT an YAC128 preparations, indicating axonal ER depletion selectively enhances miniature glutamate release in YAC128 mice. These results extend our understanding of action potential independent neurotransmission and highlight a potential involvement of elevated miniature glutamate release in Huntington disease pathology.

Lithium Provides Broad Therapeutic Benefits in an Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder with a progressive loss of cognitive function. Currently, no effective treatment regimen is available. Lithium, a mood stabilizer for bipolar disorder, exerts broad neuroprotective and neurotrophic actions and improves cognitive function. The study investigated if lithium stabilizes Ca2+ signaling abnormalities in hippocampal neurons and subsequently normalize downstream effects on AD neuropathology and synaptic plasticity in young AD mice. Four-month-old 3xTg-AD mice were treated with a LiCl diet chow for 30 days. At the end of the lithium treatment, a combination of two-photon Ca2+ imaging, electrophysiology, and immunohistochemistry assays were used to assess the effects of the LiCl treatment on inositol trisphosphate receptor (IP3R)-dependent endoplasmic reticulum (ER) Ca2+ and voltage-gated Ca2+ channel (VGCC)-mediated Ca2+ signaling in CA1 neurons, neuronal nitric oxide synthase (nNOS) and hyperphosphorylated tau (p-tau) levels and synaptic plasticity in the hippocampus and overlying cortex from 3xTg-ADmice. Thirty-day LiCl treatment reduced aberrant IP3R-dependent ER Ca2+ and VGCC-mediated Ca2+ signaling in CA1 pyramidal neurons from 3xTg-AD mice and restored neuronal nitric oxide synthase (nNOS) and hyperphosphorylated tau (p-tau) levels to control levels in the hippocampal subfields and overlying cortex. The LiCl treatment enhanced post-tetanic potentiation (PTP), a form of short-term plasticity in the hippocampus. The study found that lithium exerts therapeutic effects across several AD-associated early neuronal signaling abnormalities including aberrant Ca2+ signaling, nNOS, and p-tau formation and enhances short-term synaptic plasticity. Lithium could serve as an effective treatment or co-therapeutic for AD.

Consequences of a peroxiredoxin 4 (Prdx4) deficiency on learning and memory in mice

Peroxiredoxin 4 (Prdx4) is responsible for the oxidative folding of new proteins that are synthesized in the endoplasmic reticulum (ER). It has recently been suggested that increased ER stress is associated with neurodegenerative diseases, including Alzheimer's disease. Prdx4 is widely distributed throughout the brain, and is also expressed in hippocampal neurons and oligodendrocytes, suggesting that it is associated with learning and memory. We previously established Prdx4-knockout (KO) mice but did not examine the behavioral phenotypes. In the present study, we report on the learning and memory abilities of Prdx4-KO mice based on Morris water maze and the Y-maze tests. The findings indicate that Prdx4-KO mice showed a lower spatial memory ability in both tests. In contrast, the results of the open field test indicated that locomotor activity is significantly increased in Prdx4-KO mice. We then performed mRNA analyses of the brains of Prdx4-KO mice and found an increased expression of genes related to the ER-associated degradation (ERAD) mechanism, which is an important protein quality control system for the maintenance of ER homeostasis. Finally, proteomic analyses of the brains of Prdx4-KO mice showed an aberrant expression in the proteins, which have been suggested to be related to calcium homeostasis and synaptogenesis in neurons. Our collective results suggest that the Prdx4 ablation perturbs oxidative protein folding in the ER, thus leading to aberrant ER homeostasis in neuronal cells, ultimately leading to impaired spatial memory formation.

Diabetes-Associated Mutations in Proinsulin Provide a "Molecular Rheostat" of Nascent Foldability.

Diabetes mellitus (DM) due to toxic misfolding of proinsulin variants provides a monogenic model of endoplasmic reticulum (ER) stress. The mutant proinsulin syndrome (also designated MIDY; Mutant INS-gene-induced Diabetes of Youth or Maturity-onset diabetes of the young 10 (MODY10)) ordinarily presents as permanent neonatal-onset DM, but specific amino-acid substitutions may also present later in childhood or adolescence. This review highlights structural mechanisms of proinsulin folding as inferred from phenotype-genotype relationships. MIDY mutations most commonly add or remove a cysteine, leading to a variant polypeptide containing an odd number of thiol groups. Such variants are associated with aberrant intermolecular disulfide pairing, ER stress, and neonatal β-cell dysfunction. Non-cysteine-related (NCR) mutations (occurring in both the B and A domains of proinsulin) define distinct determinants of foldability and vary in severity. The range of ages of onset, therefore, reflects a "molecular rheostat" connecting protein biophysics to quality-control ER checkpoints. Because in most mammalian cell lines even wild-type proinsulin exhibits limited folding efficiency, molecular barriers to folding uncovered by NCR MIDY mutations may pertain to β-cell dysfunction in non-syndromic type 2 DM due to INS-gene overexpression in the face of peripheral insulin resistance. Recent studies of MIDY mutations and related NCR variants, combining molecular and cell-based approaches, suggest that proinsulin has evolved at the edge of non-foldability. Chemical protein synthesis promises to enable comparative studies of "non-foldable" proinsulin variants to define key steps in wild-type biosynthesis. Such studies may create opportunities for novel therapeutic approaches to non-syndromic type 2 DM.

Down-regulation of AMPK/PPARδ signalling promotes endoplasmic reticulum stress-induced endothelial dysfunction in adult rat offspring exposed to maternal diabetes.

Exposure to maternal diabetes is associated with increased prevalence of hypertension in the offspring. The mechanisms underlying the prenatal programming of hypertension remain unclear. Because endoplasmic reticulum (ER) stress plays a key role in vascular endothelial dysfunction in hypertension, we investigated whether aberrant ER stress causes endothelial dysfunction and high blood pressure in the offspring of dams with diabetes. Pregnant Sprague-Dawley rats were intraperitoneally injected with streptozotocin (35 mg/kg) or citrate buffer at Day 0 of gestation. Compared with control mother offspring (CMO), the diabetic mother offspring (DMO) had higher blood pressure and impaired endothelium-dependent relaxation in mesenteric arteries, accompanied by decreased AMPK phosphorylation and PPARδ expression, increased ER stress markers, and reactive oxygen species (ROS) levels. The inhibition of ER stress reversed these aberrant changes in DMO. Ex vivo treatment of mesenteric arteries with an AMPK agonist (A769662) or a PPARδ agonist (GW1516) improved the impaired EDR in DMO and reversed the tunicamycin-induced ER stress, ROS production, and EDR impairment in mesenteric arteries from CMO. The effects of A769662 were abolished by co-treatment with GSK0660 (PPARδ antagonist), whereas the effects of GW1516 were unaffected by Compound C (AMPK inhibitor). These results suggest an abnormal foetal programming of vascular endothelial function in offspring of rats with maternal diabetes that is associated with increased ER stress, which can be ascribed to down-regulation of AMPK/PPARδ signalling cascade.

3-Ketodihydrosphingosine reductase maintains ER homeostasis and unfolded protein response in leukemia.

Sphingolipids and their metabolic pathways have been implicated in disease development and therapeutic response; however, the detailed mechanisms remain unclear. Using a sphingolipid network focused CRISPR/Cas9 library screen, we identified an endoplasmic reticulum (ER) enzyme, 3-Ketodihydrosphingosine reductase (KDSR), to be essential for leukemia cell maintenance. Loss of KDSR led to apoptosis, cell cycle arrest, and aberrant ER structure. Transcriptomic analysis revealed the indispensable role of KDSR in maintaining the unfolded protein response (UPR) in ER. High-density CRISPR tiling scan and sphingolipid mass spectrometry pinpointed the critical role of KDSR’s catalytic function in leukemia. Mechanistically, depletion of KDSR resulted in accumulated 3-ketodihydrosphingosine (KDS) and dysregulated UPR checkpoint proteins PERK, ATF6, and ATF4. Finally, our study revealed the synergism between KDSR suppression and pharmacologically induced ER-stress, underscoring a therapeutic potential of combinatorial targeting sphingolipid metabolism and ER homeostasis in leukemia treatment.

Dextromethorphan Dampens Neonatal Astrocyte Activation and Endoplasmic Reticulum Stress Induced by Prenatal Exposure to Buprenorphine.

Prenatal exposure to buprenorphine renders offspring vulnerable to cerebral impairments. In this study, our data demonstrate, for the first time, that prenatal exposure to buprenorphine escalates astrocyte activation concurrent with indications of endoplasmic reticulum (ER) stress in the hippocampi of neonates, and this can be prevented by the coadministration of dextromethorphan with buprenorphine. Furthermore, dextromethorphan can inhibit the accumulation of GPR37 in the hippocampus of newborns caused by buprenorphine and is accompanied by the proapoptotic ER stress response that involves the procaspase-3/CHOP pathway. Primary astrocyte cultures derived from the neonates of the buprenorphine group also displayed aberrant ER calcium mobilization and elevated basal levels of cyclooxygenase-2 (COX-2) at 14 days in vitro while showing sensitivity to lipopolysaccharide-activated expression of COX-2. Similarly, these long-lasting defects in the hippocampus and astrocytes were abolished by dextromethorphan. Our findings suggest that prenatal exposure to buprenorphine might instigate long-lasting effects on hippocampal and astrocytic functions. The beneficial effects of prenatal coadministration of dextromethorphan might be, at least in part, attributed to its properties in attenuating astrocyte activation and hippocampal ER stress in neonates.

Legionella pneumophila LegC7 effector protein drives aberrant endoplasmic reticulum:endosome contacts in yeast.

Legionella pneumophila is a facultative intracellular bacterial pathogen, causing the severe form of pneumonia known as Legionnaires' disease. Legionella actively alters host organelle trafficking through the activities of "effector" proteins secreted via a type-IVB secretion system, in order to construct the bacteria-laden Legionella-containing vacuole (LCV) and prevent lysosomal degradation. The LCV is created with membrane derived from host endoplasmic reticulum (ER), secretory vesicles and phagosomes, although the precise molecular mechanisms that drive its synthesis remain poorly understood. In an effort to characterize the in vivo activity of the LegC7/YlfA SNARE-like effector protein from Legionella in the context of eukaryotic membrane trafficking in yeast, we find that LegC7 interacts with the Emp46p/Emp47p ER-to-Golgi glycoprotein cargo adapter complex, alters ER morphology and induces aberrant ER:endosome interactions, as measured by visualization of ER cargo degradation, reconstitution of split-GFP proteins and enhanced oxidation of the ER lumen. LegC7-dependent toxicity, disruption of ER morphology and ER:endosome fusion events were dependent upon endosomal VPS class C tethering complexes and the endosomal t-SNARE, Pep12p. This work establishes a model in which LegC7 functions to recruit host ER material to the bacterial phagosome during infection by driving ER:endosome contacts, potentially through interaction with host membrane tethering complexes and/or cargo adapters.

Clinical Implications of HBV PreS/S Mutations and the Effects of PreS2 Deletion on Mitochondria, Liver Fibrosis, and Cancer Development.

PreS mutants of HBV have been reported to be associated with HCC. We conducted a longitudinal study of the role of HBV preS mutations in the development of HCC, particularly in patients with chronic hepatitis B (CHB) having low HBV DNA or alanine aminotransferase (ALT) levels, and investigated the effects of secretion-defective preS2 deletion mutant (preS2ΔMT) on hepatocyte damage in vitro and liver fibrosis in vivo. Association of preS mutations with HCC in 343 patients with CHB was evaluated by a retrospective case-control follow-up study. Effects of preS2ΔMT on HBsAg retention, endoplasmic reticulum (ER) stress, calcium accumulation, mitochondrial dysfunction, and liver fibrosis were examined. Multivariate analysis revealed a significant association of preS mutations with HCC (HR, 3.210; 95% CI, 1.072-9.613; P=0.037) including cases with low HBV DNA or ALT levels (HR, 2.790; 95% CI, 1.133-6.873; P=0.026). Antiviral therapy reduced HCC risk, including cases with preS mutations. PreS2ΔMT expression promoted HBsAg retention in the ER and unfolded protein response (UPR). Transmission electron microscopic examination, MitoTracker staining, real-time ATP assay, and calcium staining of preS2ΔMT-expressing cells revealed aberrant ER and mitochondrial ultrastructure, reduction of mitochondrial membrane potential and ATP production, and calcium overload. Serum HBV secretion levels were ~100-fold lower in preS2ΔMT-infected humanized Fah-/-/ Rag2-/-/Il2rg-/- triple knockout mice than in wild-type HBV-infected mice. PreS2ΔMT-infected mice displayed up-regulation of UPR and caspase-3 and enhanced liver fibrosis. PreS mutations were significantly associated with HCC development in patients with CHB, including those with low HBV DNA or ALT levels. Antiviral therapy reduced HCC occurrence in patients with CHB, including those with preS mutations. Intracellular accumulation of mutated HBsAg induced or promoted ER stress, calcium overload, mitochondrial dysfunction, impaired energy metabolism, liver fibrosis, and HCC.

Nuclear factor-kappa B signaling in endometriotic stromal cells is not inhibited by progesterone owing to an aberrant endoplasmic reticulum stress response: a possible role for an altered inflammatory process in endometriosis.

Endoplasmic reticulum (ER) stress serves as a key modulator of the inflammatory response by controlling nuclear factor-kappaB (NF-κB) signaling. Previous studies from our laboratory have reported an abnormal induction of ER stress linked to progesterone resistance in human endometriotic cells. Therefore, an aberrant ER stress response to progesterone might contribute to the altered inflammatory response observed in endometriotic tissues. To evaluate this hypothesis, we investigated whether ER stress is involved in regulation of NF-κB in endometrial stromal cells and whether induction of aberrant ER stress in endometriotic stromal cells affects pro-inflammatory cytokine production. We found that tunicamycin-induced ER stress inhibited NF-κB activation and pro-inflammatory cytokine (IL-6 and COX2) production in TNF-α- or IL-1β-treated normal endometrial stromal cells (NECSs). Tunicamycin increased the expression of A20 and C/EBPβ, which are negative regulators of NF-κB, and this increase inhibited NF-κB activity in NESCs incubated with TNF-α or IL-1β. Similarly, progesterone increased A20 and C/EBPβ expression through upregulation of ER stress in NESCs, resulting in inhibition of NF-κB activity and IL-6 and COX2 production. However, progesterone had no significant effects on induction of ER stress, A20 or C/EBPβ expression, NF-κB activity or IL-6 or COX2 production in ovarian endometriotic cyst stromal cells (ECSCs). In contrast, upregulation of ER stress by tunicamycin significantly reduced IL-6 and COX2 production by inhibiting NF-κB activity in ECSCs. In conclusion, our results suggest that NF-κB activity in endometriotic stromal cells was not inhibited because of an aberrant ER stress response to progesterone, resulting in an increase in pro-inflammatory cytokine production.

S-Nitroso-L-Cysteine Ameliorated Pulmonary Hypertension in the MCT-Induced Rats through Anti-ROS and Anti-Inflammatory Pathways.

Pulmonary hypertension (PH) is a progressive and life-threatening chronic disease in which increased pulmonary artery pressure (PAP) and pulmonary vasculature remodeling are prevalent. Inhaled nitric oxide (NO) has been used in newborns to decrease PAP in the clinic; however, the effects of NO endogenous derivatives, S-nitrosothiols (SNO), on PH are still unknown. We have reported that S-nitroso-L-cysteine (CSNO), one of the endogenous derivatives of NO, inhibited RhoA activity through oxidative nitrosation of its C16/20 residues, which may be beneficial for both vasodilation and remodeling. In this study, we presented data to show that inhaled CSNO attenuated PAP in the monocrotaline- (MCT-) induced PH rats and, moreover, improved right ventricular (RV) hypertrophy and fibrosis induced by RV overloaded pressure. In addition, aerosolized CSNO significantly inhibited the hyperactivation of signal transducers and activators of transduction 3 (STAT3) and extracellular regulated protein kinases (ERK) pathways in the lung of MCT-induced rats. CSNO also regulated the expression of smooth muscle contractile protein and improved aberrant endoplasmic reticulum (ER) stress and mitophagy in lung tissues following MCT induction. On the other hand, CSNO inhibited reactive oxygen species (ROS) production in vitro, which is induced by angiotensin II (AngII) as well as interleukin 6 (IL-6). In addition, CSNO inhibited excessive ER stress and mitophagy induced by AngII and IL-6 in vitro; finally, STAT3 and ERK phosphorylation was inhibited by CSNO in a concentration-dependent manner. Taken together, CSNO led to pulmonary artery relaxation and regulated pulmonary circulation remodeling through anti-ROS and anti-inflammatory pathways and may be used as a therapeutic option for PH treatment.

ATF4 leads to glaucoma by promoting protein synthesis and ER client protein load

The underlying pathological mechanisms of glaucomatous trabecular meshwork (TM) damage and elevation of intraocular pressure (IOP) are poorly understood. Here, we report that the chronic endoplasmic reticulum (ER) stress-induced ATF4-CHOP-GADD34 pathway is activated in TM of human and mouse glaucoma. Expression of ATF4 in TM promotes aberrant protein synthesis and ER client protein load, leading to TM dysfunction and cell death. These events lead to IOP elevation and glaucomatous neurodegeneration. ATF4 interacts with CHOP and this interaction is essential for IOP elevation. Notably, genetic depletion or pharmacological inhibition of ATF4-CHOP-GADD34 pathway prevents TM cell death and rescues mouse models of glaucoma by reducing protein synthesis and ER client protein load in TM cells. Importantly, glaucomatous TM cells exhibit significantly increased protein synthesis along with induction of ATF4-CHOP-GADD34 pathway. These studies indicate a pathological role of ATF4-CHOP-GADD34 pathway in glaucoma and provide a possible treatment for glaucoma by targeting this pathway.

Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells.

Enteroviruses manipulate host membranes to form replication organelles, which concentrate viral and host factors to allow for efficient replication. However, this process has not been well-studied in living cells throughout the course of infection. To define the dynamic process of enterovirus membrane remodeling of major secretory pathway organelles, we have developed plasmid-based reporter systems that utilize viral protease-dependent release of a nuclear-localized fluorescent protein from the endoplasmic reticulum (ER) membrane during infection, while retaining organelle-specific fluorescent protein markers such as the ER and Golgi. This system thus allows for the monitoring of organelle-specific changes induced by infection in real-time. Using long-term time-lapse imaging of living cells infected with coxsackievirus B3 (CVB), we detected reporter translocation to the nucleus beginning ~4 h post-infection, which correlated with a loss of Golgi integrity and a collapse of the peripheral ER. Lastly, we applied our system to study the effects of a calcium channel inhibitor, 2APB, on virus-induced manipulation of host membranes. We found that 2APB treatment had no effect on the kinetics of infection or the percentage of infected cells. However, we observed aberrant ER structures in CVB-infected cells treated with 2APB and a significant decrease in viral-dependent cell lysis, which corresponded with a decrease in extracellular virus titers. Thus, our system provides a tractable platform to monitor the effects of inhibitors, gene silencing, and/or gene editing on viral manipulation of host membranes, which can help determine the mechanism of action for antivirals.