eIF2α Pathway Research Articles

Aminocarb Exposure Induces Cytotoxicity and Endoplasmic Reticulum Stress-Mediated Apoptosis in Mouse Sustentacular Sertoli Cells: Implications for Male Infertility and Environmental Health.

Exposure to pesticides, poses a significant threat to male fertility by compromising crucial cells involved in spermatogenesis. Aminocarb, is a widely used carbamate insecticide, although its detrimental effects on the male reproductive system, especially on sustentacular Sertoli cells, pivotal for spermatogenesis, remains poorly understood. In this study, we investigated the effects of escalating concentrations of aminocarb on a mouse Sertoli cell line, TM4. Assessments included cytotoxic analysis, mitochondrial biogenesis and membrane potential, expression of apoptotic proteins, caspase-3 activity, and oxidative stress evaluation. Our findings revealed a dose-dependent reduction in the proliferation and viability of TM4 cells following exposure to increasing concentrations of aminocarb. Notably, exposure to 5 μM of aminocarb induced depolarization of mitochondria membrane potential, and a significant decrease in the ratio of phosphorylated eIF2α to total eIF2α, suggesting heightened endoplasmic reticulum stress via the activation of the eIF2α pathway. Moreover, the same aminocarb concentration was demonstrated to increase both caspase-3 protein levels and activity, indicating an apoptotic induction. Collectively, our results demonstrate that aminocarb serves as an apoptotic inducer for mouse sustentacular Sertoli cells in vitro, suggesting its potential to modulate independent pathways of the apoptotic cascade. These findings underscore the deleterious impact of aminocarb on spermatogenic performance and male fertility, highlighting the urgent need for further investigation into its mechanisms of action and mitigation strategies to safeguard male fertility.

Remodeling the cellular stress response for enhanced genetic code expansion in mammalian cells

Genetic code expansion (GCE) reprograms the translational machinery to site-specifically incorporate noncanonical amino acids (ncAAs) into a selected protein. The efficiency of GCE in mammalian cells might be compromised by cellular stress responses, among which, the protein kinase R(PKR)-dependent eIF2α phosphorylation pathway can reduce translation rates. Here we test several strategies to engineer the eIF2α pathway and boost the rate of translation and show that such interventions increase GCE efficiency in mammalian cells. In particular, addition of the N-terminal PKR fragment (1–174) provides a substantial enhancement in cytoplasmic GCE and also in GCE realized by OTOs (orthogonally translating designer organelles), which built on the principle of 2D phase separation to enable mRNA-selective ncAA incorporation. Our study demonstrates an approach for improving the efficiency of GCE and provides a means by which the power of designer organelles can be further optimized to tune protein translation.

Oxidized phospholipids facilitate calcific aortic valve disease by elevating ATF4 through the PERK/eIF2α axis.

In this study we sought to analyze the critical role of oxidized phospholipid (OxPL) in the progression of calcific aortic valve disease (CAVD) with the involvement of activating transcription factor 4 (ATF4). Differentially expressed genes related to CAVD were identified using bioinformatics analysis. Expression of ATF4 was examined in mouse models of aortic valve calcification (AVC) induced by the high cholesterol (HC) diet. Valvular interstitial cells (VICs) were then isolated from mouse non-calcified valve tissues, induced by osteogenic induction medium (OIM) and co-cultured with OxPAPC-stimulated macrophages. The effect of OxPLs regulating ATF4 on the macrophage polarization and osteogenic differentiation of VICs was examined with gain- and loss-of-function experiments in VICs and in vivo. In aortic valve tissues and OIM-induced VICs, ATF4 was highly expressed. ATF4 knockdown alleviated the osteogenic differentiation of VICs, as evidenced by reduced expression of bone morphogenetic protein-2 (BMP2), osteopontin (OPN), and osteocalcin. In addition, knockdown of ATF4 arrested the AVC in vivo. Meanwhile, OxPL promoted M1 polarization of macrophages and mediated osteogenic differentiation of VICs. Furthermore, OxPL up-regulated ATF4 expression through protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2 subunit alpha (eIF2α) pathway. In conclusion, OxPL can potentially up-regulate the expression of ATF4, inducing macrophages polarized to M1 phenotype, osteogenic differentiation of VICs and AVC, thus accelerating the progression of CAVD.

Abstract 1615: NMS-812, a novel potent PERK inhibitor that also inhibits GCN2, exhibits strong anti-tumor activity as single agent and in combination in preclinical models

Abstract PERK and GCN2 are eIF-2α serine-threonine kinases activated by endoplasmic reticulum (ER) stress and amino acid deprivation respectively. These kinases are key effectors of the unfolded protein response (UPR) and amino acid Response (AAR) branches of the integrated stress response (ISR). ISR downregulates protein synthesis while upregulating specific genes in response to various cellular stresses, thus allowing adaptation and survival. PERK and GCN2 cooperate to modulate survival under stress conditions in cancer cells, with GCN2 activation compensating when PERK is inhibited, therefore suggesting a certain degree of redundancy between the two. Multiple myeloma (MM) and other protein-secreting tumors are highly dependent on UPR for survival, due to their high protein load. Therefore, PERK inhibition represents a promising anticancer approach in these tumor types. Notably, in these contexts, dual PERK/GCN2 inhibition may be more effective than inhibiting the single kinase.NMS-03597812 is a novel, very potent, ATP-competitive PERK/GCN2 inhibitor, NMS-812 is preferentially active in vitro on multiple myeloma cell lines; activity has however been observed also on lymphoma and selected solid tumor cell lines, suggesting a broader but still specific role of PERK and GCN2 inhibition depending on cell context. In assays revealing the mechanism of action, as expected, a transient dose-dependent eIF2α pathway activation is observed, suggesting the attempt of GCN2 to compensate for PERK inhibition. However, at higher doses, NMS-812 strongly induces pathway downmodulation and apoptosis compared to prototype PERK selective inhibitors. NMS-812 treatment of a MM xenograft model significantly prolongs the median survival time as single agent and demonstrates synergistic activity with MM standard of care (SOC) agents. In lymphoma, NMS-812 shows activity both as single agent and in combination with SOC.NMS-812 shows a favorable in vitro ADME profile and good PK properties with oral bioavailability in preclinical species and an acceptable toxicity profile. NMS-812 is a novel dual, PERK/GCN2 inhibitor with potential for first-in-class, that validates dual PERK and GCN2 inhibition as a therapeutic approach for MM and other PERK/GCN2-dependent settings. Based on these promising results, a Ph1 study has been started in Multiple Myeloma with plans to expand to other malignancies in the near future. Citation Format: Claudia Perrera, Maurizio Pulici, Patrizia Banfi, Nilla Avanzi, Davide Carenzi, Daniele Casero, Antonella Ciavolella, Laura Gianellini, Fabio Gasparri, Grazia Saturno, Barbara Valsasina, Elena Ardini, Alessia Montagnoli, Arturo P. Galvani, Antonella Isacchi. NMS-812, a novel potent PERK inhibitor that also inhibits GCN2, exhibits strong anti-tumor activity as single agent and in combination in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1615.

Potential Therapeutic Value of the STING Inhibitors.

The stimulator of interferon genes (STING) is a critical protein in the activation of the immune system in response to DNA. It can participate the inflammatory response process by modulating the inflammation-preferred translation program through the STING-PKR-like endoplasmic reticulum kinase (PERK)-eIF2α pathway or by inducing the secretion of type I interferons (IFNs) and a variety of proinflammatory factors through the recruitment of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) or the regulation of the nuclear factor kappa-B (NF-κB) pathway. Based on the structure, location, function, genotype, and regulatory mechanism of STING, this review summarizes the potential value of STING inhibitors in the prevention and treatment of infectious diseases, psoriasis, systemic lupus erythematosus, non-alcoholic fatty liver disease, and other inflammatory and autoimmune diseases.

SERCA2a directs the cardioprotective role of nano-emulsion curcumin against PM2.5-induced cardiac injury in rats by prohibiting PERK-eIF2α pathway

AimsActivation of endoplasmic reticulum stress (ERS) by particulate matter 2.5 (PM2.5) has recently been linked to an increased risk of heart problems. Although the PERK- eIF2α pathway is known to be involved in ERS, its crucial role in the pathogenesis of PM2.5-induced cardiotoxicity remains unclear. With the expected potentiality of SERCA2a to modulate ERS via inhibiting the PERK-eIF2α axis, this study intended to assess the possible cardioprotective efficacy of nano-emulsion curcumin (NEC), as a SERCA2a activator, against PM2.5-induced heart damage. Main methodsThirty rats were specified into: Vehicle (V); Blank Filter (BF); NEC; PM and NEC + PM. Cardiac biomarkers as PTX3 and cTnI were assayed. The oxidant/antioxidant status was evaluated via detecting Nrf2/HO-1 axis, as well as MDA and TAC. In addition, the protein expressions of PLN, DWORF, SERCA2a, PERK/eIF2α/ATF4/CHOP, and PI3K/AKT/mTOR in the heart were assessed by western blotting. The levels of inflammatory cytokines (TNF-α and IL-1β) and apoptotic markers were also determined. For detecting PM and NEC particles, cardiac tissues were examined using TEM. Key findingsThe results revealed that NEC markedly alleviated the oxidative stress following PM2.5 exposure, up-regulated DWORF, and produced a significant improvement in cardiac functions. Through activating SERCA2a, NEC could hinder ERS, ameliorate PM2.5-associated cardiac inflammation and myocardial apoptosis by suppressing Bax/Bcl-2 ratio and caspase-3 expression, as well as inhibiting autophagy. SignificanceThese findings revealed that NEC may have a SERCA2a-dependent cardioprotective effect against PM2.5-induced cardiac injury via inhibiting the PERK-eIF2 pathway.

Stress granule assembly in vivo is deficient in the CNS of mutant TDP-43 ALS mice.

Responding effectively to external stress is crucial for neurons. Defective stress granule dynamics has been hypothesized as one of the pathways that renders motor neurons in amyotrophic lateral sclerosis (ALS) more prone to early death. Specifically, it is thought that stress granules seed the cytoplasmic TDP-43 inclusions that are observed in the neurons of most ALS patients, as well as ~50% of all frontotemporal dementia (FTD) patients. In this study, we tested this hypothesis in an intact mammalian nervous system. We established an in vivo heat stress paradigm in mice that effectively triggers the eIF2α pathway and the formation of stress granules in the CNS. In non-transgenic mice, we report an age-dependent decline in the formation of heat-induced stress granules, with 18-month-old animals showing a significant impairment. Furthermore, although neuronal stress granules were robustly observed in non-transgenic mice and SOD1G93A mice, they were largely absent in age-matched TDP-43M337V animals. The observed defect in stress granule formation in TDP-43M337V mice correlated with deficits in expression of key protein components typically required for phase separation. Lastly, while TDP-43 was not localized to stress granules, we observed complete nuclear depletion of TDP-43 in a subset of neurons, with the highest proportion being in the TDP-43M337V mice. Overall, our results indicate that mutant TDP-43 expression is associated with defective stress granule assembly and increased TDP-43 nuclear depletion in the mammalian nervous system, which could be relevant to ALS/FTD pathogenesis.

Glucose controls co-translation of structurally related mRNAs via the mTOR and eIF2 pathways in human pancreatic beta cells.

Pancreatic beta cell response to glucose is critical for the maintenance of normoglycemia. A strong transcriptional response was classically described in rodent models but, interestingly, not in human cells. In this study, we exposed human pancreatic beta cells to an increased concentration of glucose and analysed at a global level the mRNAs steady state levels and their translationalability. Polysome profiling analysis showed an early acute increase in protein synthesis and a specific translation regulation of more than 400 mRNAs, independently of their transcriptional regulation. We clustered the co-regulated mRNAs according to their behaviour in translation in response to glucose and discovered common structural and sequence mRNA features. Among them mTOR- and eIF2-sensitive elements have a predominant role to increase mostly the translation of mRNAs encoding for proteins of the translational machinery. Furthermore, we show that mTOR and eIF2α pathways are independently regulated in response to glucose, participating to a translational reshaping to adapt beta cell metabolism. The early acute increase in the translation machinery components prepare the beta cell for further protein demand due to glucose-mediated metabolism changes.

Induction and modulation of the unfolded protein response during porcine deltacoronavirus infection

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that has the potential for cross-species infection. Many viruses have been reported to induce endoplasmic reticulum stress (ERS) and activate the unfolded protein response (UPR). To date, little is known about whether and, if so, how the UPR is activated by PDCoV infection. Here, we investigated the activation state of UPR pathways and their effects on viral replication during PDCoV infection. We found that PDCoV infection induced ERS and activated all three known UPR pathways (inositol-requiring enzyme 1 [IRE1], activating transcription factor 6 [ATF6], and PKR-like ER kinase [PERK]), as demonstrated by IRE1-mediated XBP1 mRNA cleavage and increased mRNA expression of XBP1s, ATF4, CHOP, GADD34, GRP78, and GRP94, as well as phosphorylated eIF2α expression. Through pharmacologic treatment, RNA interference, and overexpression experiments, we confirmed the negative role of the PERK–eIF2α pathway and the positive regulatory role of the ATF6 pathway, but found no obvious effect of IRE1 pathway, on PDCoV replication. Taken together, our results characterize, for the first time, the state of the ERS response during PDCoV infection and identify the PERK and ATF6 pathways as potential antiviral targets.

A non-canonical cGAS-STING-PERK pathway facilitates the translational program critical for senescence and organ fibrosis.

Innate DNA sensing via the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) mechanism surveys microbial invasion and cellular damage and thus participates in various human infectious diseases, autoimmune diseases and cancers. However, how DNA sensing rapidly and adaptively shapes cellular physiology is incompletely known. Here we identify the STING-PKR-like endoplasmic reticulum kinase (PERK)-eIF2α pathway, a previously unknown cGAS-STING mechanism, enabling an innate immunity control of cap-dependent messenger RNA translation. Upon cGAMP binding, STING at the ER binds and directly activates the ER-located kinase PERK via their intracellular domains, which precedes TBK1-IRF3 activation and is irrelevant to the unfolded protein response. The activated PERK phosphorylates eIF2α, forming an inflammatory- and survival-preferred translation program. Notably, this STING-PERK-eIF2α pathway is evolutionarily primitive and physiologically critical to cellular senescence and organ fibrosis. Pharmacologically or genetically targeting this non-canonical cGAS-STING pathway attenuated lung and kidney fibrosis. Collectively, the findings identify an alternative innate immune pathway and its critical role in organ fibrosis, report an innate immunity-directed translation program and suggest the therapeutic potential for targeting the STING-PERK pathway in treating fibrotic diseases.

Bixin Prevents Colorectal Cancer Development through AMPK-Activated Endoplasmic Reticulum Stress.

Chemicals isolated from natural products have been broadly applied in the treatment of colorectal cancer (CRC). Bixin, an apocarotenoid from the seeds of Bixa orellana, exerts multiple pharmacological properties, including neuroprotective, anti-inflammatory, cardioprotective, and antitumor effects; yet, the therapeutic effects of Bixin on CRC are still unknown. Here, we described that Bixin treatment significantly inhibited the proliferation and motility of two CRC cell lines (CaCO2 and SW480) in vitro and in vivo. In addition, Bixin administration has sensitized CRC cells to TNF-related apoptosis-inducing ligand- (TRAIL-) induced cell apoptosis. Moreover, we showed that Bixin treatment initiated the activation of PERK/eIF-2α signal in CaCO2 and SW480 cells, leading to endoplasmic reticulum stress-associated apoptosis. Pharmacological inhibition of AMP-activated protein kinase (AMPK) abrogated the Bixin-induced activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2 alpha (eIF-2α) pathway, as well as reversed the inhibitory effects of Bixin on CRC development. In conclusion, this study indicated that Bixin treatment inhibits the progression of CRC through activating the AMPK/PERK/eIF-2α pathway, providing a novel potential strategy for clinical prevention of CRC.

Post-treatment with apocynin at a lower dose regulates the UPR branch of eIF2α and XBP-1 pathways after stroke

Stroke leads to disturbance in the physiology of the ER (Endoplasmic Reticulum) that triggers UPR (Unfolded Protein Response) pathways aimed to compensate neuronal cell damage. However, sustained UPR causes stressful conditions in the ER lumen forming abnormal protein aggregates. Stroke-induced oxidative stress also amalgamates with UPR to safeguard and ensure the proper functioning of brain cells. Thus we tested the effect of apocynin (a potent antioxidant) post-treatment in experimental stroke on the outcome of ER stress and UPR branch pathways. We administered a low dose of apocynin at 1 mg/kg (intraperitoneal) to adult Sprague-Dawley rats subjected to Middle Cerebral Artery Occlusion (MCAO) for two-time points. The first dose immediately after re-establishing the blood flow and another at 6 h of reperfusion. Apocynin post-treatment significantly reduced ROS (Reactive Oxygen Species) generation at an early reperfusion time point of 4 h. It preserved neuronal morphology, dendritic spine density, reduced protein aggregation, and brain damage after 24 h of reperfusion. Apocynin post-treatment regulates the two UPR branch pathways in our experimental paradigm. 1) Down-regulation of eIF2α (Eukaryotic Initiation Factor 2α) phosphorylation, and CHOP (C/EBP homologous protein) 2) by reducing the XBP-1 (X-Box binding Protein-1) mRNA splicing downstream to PERK (Protein Kinase RNA-Like ER Kinase) and IRE1α (Inositol Requiring Enzyme 1alpha) UPR pathways, respectively. Bioinformatics prediction showed that apocynin has binding sites for PERK (Protein Kinase RNA-Like ER Kinase) and IRE1α proteins. The amino acid residues interacting with apocynin were Cys891 and Gln889 (for PERK), and the amino acids Ser726, Arg722, and Ala719 (for IRE1α) lying within their activation loop. Overall, these studies indicate that apocynin post-treatment might regulate ER stress/UPR pathways and minimize stroke brain damage, thus having implications for developing newer strategies for stroke treatment.

Linking endoplasmic reticulum stress to depression: involvement of eIF2α pathway

Linking endoplasmic reticulum stress to depression: involvement of eIF2α pathway

Therapeutic Role of Sirtuins Targeting Unfolded Protein Response, Coagulation, and Inflammation in Hypoxia-Induced Thrombosis.

Thrombosis remains one of the leading causes of morbidity and mortality across the world. Many pathological milieus in the body resulting from multiple risk factors escort thrombosis. Hypoxic condition is one such risk factor that disturbs the integrity of endothelial cells to cause an imbalance between anticoagulant and procoagulant proteins. Hypoxia generates reactive oxygen species (ROS) and triggers inflammatory pathways to augment the coagulation cascade. Hypoxia in cells also activates unfolded protein response (UPR) signaling pathways in the endoplasmic reticulum (ER), which tries to restore ER homeostasis and function. But the sustained UPR linked with inflammation, generation of ROS and apoptosis stimulates the severity of thrombosis in the body. Sirtuins, a group of seven proteins, play a vast role in bringing down inflammation, oxidative and ER stress and apoptosis. As a result, sirtuins might provide a therapeutic approach towards the treatment or prevention of hypoxia-induced thrombosis. Sirtuins modulate hypoxia-inducible factors (HIFs) and counteract ER stress-induced apoptosis by attenuating protein kinase RNA-like endoplasmic reticulum kinase (PERK)/Eukaryotic translation initiation factor 2α (eIF2α) pathway activation. It prevents ER-stress mediated inflammation by targeting X-Box Binding Protein 1 (XBP1) and inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ) signaling through deacetylation. Sirtuins also obstruct nucleotide-binding domain, leucine-rich-containing family, pyrin domain containing 3 (NLRP3) inflammasome activation to reduce the expression of several pro-inflammatory molecules. It protects cells against oxidative stress by targeting nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione (GSH), forkhead box O3 (FOXO3), superoxide dismutase (SOD), catalase (CAT), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), glucose-6-phosphate dehydrogenase (G6PD), phosphoglucomutase-2 (PGAM2), and NF-κB, to name few. This review, thus, discusses the potential role of sirtuins as a new treatment for hypoxia-induced thrombosis that involves an intersection of UPR and inflammatory pathways in its pathological manifestation.

Gambogenic Acid Induces Endoplasmic Reticulum Stress in Colorectal Cancer via the Aurora A Pathway.

Colorectal cancer (CRC) is one of the most common malignancies in the world and has a poor prognosis. In the present research, gambogenic acid (GNA), isolated from the traditional Chinese medicine gamboge, markedly induced apoptosis and inhibited the proliferation of CRC in vitro and in vivo. Furthermore, GNA triggered endoplasmic reticulum (ER) stress, which subsequently activated inositol-requiring enzyme (IRE) 1α and the eukaryotic translation initiation factor (eIF) 2α pathway. Pretreatment with salubrinal (an eIF2α inhibitor) rescued GNA-induced cell death. Furthermore, GNA downregulated the expression of Aurora A. The Aurora A inhibitor alisertib decreased ER stress. In human colorectal adenocarcinoma tissue, Aurora A was upregulated compared to normal colorectal epithelial nuclei. Furthermore, GNA ameliorated mouse colitis-associated cancer models. Our findings demonstrated that GNA significantly inhibited the proliferation of CRC through activation of ER stress by regulating Aurora A, which indicates the potential of GNA for preventing the progression of CRC.

Different regulation of IRE1α and eIF2α pathways by oxygen and insulin in ACH-3P trophoblast model.

Endoplasmic reticulum (ER)-stress activates the unfolded protein response (UPR), which plays a (patho)physiological role in the placenta. Oxygen and hyperinsulinemia are major regulators of placental development. Thus, we hypothesized that oxygen, insulin and their interplay modulate ER-stress in early pregnancy. Using the human first-trimester trophoblast cell line ACH-3P, we quantified mRNA and protein of several members of UPR by RT-qPCR and Western blotting, respectively. ER-stress induction using tunicamycin and brefeldin A resulted in increased CHOP (4.6-fold change; P ≤ 0.001), XBP1 expression (1.7- and 1.3-fold change, respectively; P ≤ 0.001 and P < 0.05) and XBP1 splicing (7.9- and 12.8-fold change, respectively; P ≤ 0.001). We subsequently analyzed the effect of oxygen (6.5%, 2.5%), insulin (0.1-10 nM) and their interaction using ANCOVA adjusted for cell passage as co-variate. Although GRP78 protein remained unaffected, low oxygen (2.5% O2) increased IRE1α phosphorylation (+52%; P < 0.05) and XBP1 splicing (1.8-fold change; P ≤ 0.001) after 24 h, while eIF2α protein and CHOP expression were downregulated (-28%; P < 0.05 and -24%; P ≤ 0.001; respectively). eIF2α phosphorylation was also reduced after 48 h by low oxygen (-61%; P < 0.05) but increased in the presence of insulin (+46%; P ≤ 0.01). These changes were not PERK-mediated, since PERK phosphorylation and total protein were not altered. Overall, our results suggest that IRE1α and eIF2α UPR-pathways are differentially regulated by oxygen and insulin in early pregnancy.

Effect of ammonia stress on transcriptome and endoplasmic reticulum stress pathway for common carp (Cyprinus carpio) hepatopancreas

Ammonia is the mainly environmental pollutant in the field of aquaculture in association with negative impact on the healthy cultivation of common carp. Gene expression profiles in the hepatopancreas of common carp treated with ammonia stress using an RNA-seq technique, and22,431,825 and 23,592,342 clean reads from the control and treatment groups by a 96-h ammonia challenge (0.85 mg/L unionized ammonia, NH3) are obtained. Total amount of 3367 unigenes are identified as differentially expressed genes (DEGs) related to ammonia stress. The 1724 DEGs are down-regulated and 1643 DEGs are upregulated. The metabolic pathways of these DEGs are classified into 49 different terms by KEGG pathway analysis. Protein processing in the endoplasmic reticulum (ko04141) pathway is a significantly enriched pathway of DEGs. Many DEGs are enriched in the endoplasmic reticulum stress (ERS) pathway and unfolded protein response (UPR) signal pathway in the ko04141 pathway. ERS and UPR pathway are involved in the response of hepatopancreas cells by ammonia stress. The RT-PCR and transcriptomic results indicate that PERK- eIF2α and IRE1-xbp1 pathways take participate in the response to ERS in hepatopancreas cells induced by ammonia stress. The TUNEL assays results confirmed that ammonia stress induced apoptosis in hepatopancreatic cells. Furthermore, apoptosis of hepatopancreas cells under ammonia-stress is definitely induced by ERS through the PERK-eIF2α-CHOP and IRE1-XBP1-CHOP signaling pathways.

EIF2AK2 Missense Variants Associated with Early Onset Generalized Dystonia.

ObjectiveThe study was undertaken to identify a monogenic cause of early onset, generalized dystonia.MethodsMethods consisted of genome‐wide linkage analysis, exome and Sanger sequencing, clinical neurological examination, brain magnetic resonance imaging, and protein expression studies in skin fibroblasts from patients.ResultsWe identified a heterozygous variant, c.388G>A, p.Gly130Arg, in the eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2) gene, segregating with early onset isolated generalized dystonia in 5 patients of a Taiwanese family. EIF2AK2 sequencing in 191 unrelated patients with unexplained dystonia yielded 2 unrelated Caucasian patients with an identical heterozygous c.388G>A, p.Gly130Arg variant, occurring de novo in one case, another patient carrying a different heterozygous variant, c.413G>C, p.Gly138Ala, and one last patient, born from consanguineous parents, carrying a third, homozygous variant c.95A>C, p.Asn32Thr. These 3 missense variants are absent from gnomAD, and are located in functional domains of the encoded protein. In 3 patients, additional neurological manifestations were present, including intellectual disability and spasticity. EIF2AK2 encodes a kinase (protein kinase R [PKR]) that phosphorylates eukaryotic translation initiation factor 2 alpha (eIF2α), which orchestrates the cellular stress response. Our expression studies showed abnormally enhanced activation of the cellular stress response, monitored by PKR‐mediated phosphorylation of eIF2α, in fibroblasts from patients with EIF2AK2 variants. Intriguingly, PKR can also be regulated by PRKRA (protein interferon‐inducible double‐stranded RNA‐dependent protein kinase activator A), the product of another gene causing monogenic dystonia.InterpretationWe identified EIF2AK2 variants implicated in early onset generalized dystonia, which can be dominantly or recessively inherited, or occur de novo. Our findings provide direct evidence for a key role of a dysfunctional eIF2α pathway in the pathogenesis of dystonia. ANN NEUROL 2021;89:485–497

Pathogenesis and promising therapeutics of Alzheimer disease through eIF2α pathway and correspondent kinases.

Eukaryotic initiation factor 2 (eIF2α) pathway is overactivated in Alzheimer disease and is probably associated with synaptic and memory deficiencies. EIF2α protein is principally in charge of the regulation of protein synthesis in eukaryotic cells. Four kinases responsible for eIF2α phosphorylation at ser-51 are: General control non-derepressible-2 kinase (GCN2), double-stranded RNA-activated protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and heme-regulated inhibitor kinase (HRI) are the four kinases. They lead to reduced levels of general translation and paradoxical increase of stress-responsive mRNAs expression including the B-secretase (BACE1) and the transcriptional modulator activating transcription factor 4 (ATF4), which in turn accelerates the beta-amyloidogenesis, tau phosphorylation, proapoptotic pathway induction and autophagy elements formation leading to the main pathological hallmarks of AD. Findings suggest that genetic or pharmacological inhibition of correspondent kinases can restore memory and prevent neurodegeneration. This implies that inhibition of eIF2α phosphorylation through respondent kinases is indeed a feasible prospect of clinical application. This review discusses recent therapeutic approaches targeting eIF2α pathway and provides an overview of the links between correspondent kinases overactivation with neurodegeneration in AD.

Enhanced phosphorylation of PERK in primary cultured neurons as an autonomous neuronal response to prion infection.

Conversion of cellular prion protein (PrPC) into the pathogenic isoform of prion protein (PrPSc) in neurons is one of the key pathophysiological events in prion diseases. However, the molecular mechanism of neurodegeneration in prion diseases has yet to be fully elucidated because of a lack of suitable experimental models for analyzing neuron-autonomous responses to prion infection. In the present study, we used neuron-enriched primary cultures of cortical and thalamic mouse neurons to analyze autonomous neuronal responses to prion infection. PrPSc levels in neurons increased over the time after prion infection; however, no obvious neuronal losses or neurite alterations were observed. Interestingly, a finer analysis of individual neurons co-stained with PrPSc and phosphorylated protein kinase RNA-activated-like endoplasmic reticulum (ER) kinase (p-PERK), the early cellular response of the PERK-eukaryotic initiation factor 2 (eIF2α) pathway, demonstrated a positive correlation between the number of PrPSc granular stains and p-PERK granular stains, in cortical neurons at 21 dpi. Although the phosphorylation of PERK was enhanced in prion-infected cortical neurons, there was no sign of subsequent translational repression of synaptic protein synthesis or activations of downstream unfolded protein response (UPR) in the PERK-eIF2α pathway. These results suggest that PrPSc production in neurons induces ER stress in a neuron-autonomous manner; however, it does not fully activate UPR in prion-infected neurons. Our findings provide insights into the autonomous neuronal responses to prion propagation and the involvement of neuron-non-autonomous factor(s) in the mechanisms of neurodegeneration in prion diseases.