Endoplasmic Reticulum Function Research Articles

FMNL2 regulates actin for endoplasmic reticulum and mitochondria distribution in oocyte meiosis.

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.

FMNL2 regulates actin for endoplasmic reticulum and mitochondria distribution in oocyte meiosis

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.

Biomaterials for Targeting Endoplasmic Reticulum in Cancer.

Endoplasmic reticulum (ER) is one of the most important sub-cellular organelles which controls myriads of biological functions including protein biosynthesis with proper functional folded form, protein misfolding, protein transport into Golgi body for secretion, Ca2+ homeostasis and so on. Subsequently, dysregulation in ER function leads to ER stress followed by disease pathology like cancer. Hence, targeting ER in the cancer cells emerged as one of the futuristic strategies for cancer treatment. However, the major challenge is to selectively and specifically target ER in the sub-cellular milieu in the cancer tissues, due to the lack of ER targeting chemical moieties to recognize the ER markers. To address this, in last decade, numerous biomaterials were explored to selectively impair and image ER in cancer cells to induce ER stress. This review outlines those biomaterials which consists of carbon and silicon materials, lipid nanoparticles (liposomes and micelles), supramolecular self-assembled nanostructures, cell membrane coated nanoparticles and metallic nanoparticles. Moreover, we also discussed the challenges and possible solutions of this promising field to usher the readers towards next-generation ER targeted cancer therapy.

Systematic characterization of all Toxoplasma gondii TBC domain-containing proteins identifies an essential regulator of Rab2 in the secretory pathway.

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii.

Multi-omics analysis identifies sex-specific hepatic protein-metabolite networks in yellow catfish (Pelteobagrus fulvidraco) exposed to chronic hypoxia

Hypoxia disrupts the endocrine system of teleosts. The liver plays important roles in the endocrine system, energy storage, and metabolic processes. The aim of this study was to investigate the sex-specific hepatic response of yellow catfish under chronic hypoxia at the multi-omics level. Common hepatic responses in both sexes included the HIF-1 signaling pathway, glycolysis/gluconeogenesis, and steroid biosynthesis. Hypoxia dysregulated primary bile acid biosynthesis, lipid metabolism, and vitellogenin levels in female fish. Endoplasmic reticulum function in females also tended to be disrupted by hypoxia, as evidenced by significantly enriched pathways, including ribosome, protein processing in the endoplasmic reticulum, and RNA degradation. Other pathways, including the TCA cycle, oxidative phosphorylation, and Parkinson's and Huntington's disease, were highly enriched by hypoxia in male fish, suggesting that mitochondrial function was dysregulated. In both sexes of yellow catfish, the cell cycle was arrested and apoptosis was inhibited under chronic hypoxia. Multi-omics suggested that SLC2A5, CD209, LGMN, and NEDD8 served as sex-specific markers in these fish under chronic hypoxia. Our results provide insights into hepatic adaptation to chronic hypoxia and facilitate our understanding of sex-specific responses in fish.

The Potential Use of Dimethyltryptamine against Ischemia-reperfusion Injury of the Brain

Ischemia-Reperfusion Injury (IRI) is the outcome of two intertwined pathological processes resulting from the shortage of blood flow to tissues and the subsequent restoration of circulation to a previously ischemic area. IRI (sometimes just one side of the dyad) remains one of the most challenging problems in several branches of emergency medicine. Mitochondrial and endoplasmic reticulum dysfunction is a crucial pathological factor involved in the development of IRI. The sigma-1 receptor (Sig1-R) is an intracellular chaperone molecule located between the mitochondria and endoplasmic reticulum with an apparent physiological role in regulating signaling between these cell organelles and serves as a safety mechanism against cellular stress. Therefore, amelioration of IRI is reasonably expected by the activation of the Sig1-R chaperone. Indeed, under cellular stress, Sig1-R agonists improve mitochondrial respiration and optimize endoplasmic reticulum function by sustaining high-energy phosphate synthesis. The discovery that N, N-dimethyltryptamine (DMT) is an endogenous agonist of the Sig1-R may shed light on yet undiscovered physiological mechanisms and therapeutic potentials of this controversial hallucinogenic compound. In this article, the authors briefly overview the function of Sig1-R in cellular bioenergetics with a focus on the processes involved in IRI and summarize the results of their in vitro and in vivo DMT studies aiming at mitigating IRI. The authors conclude that the effect of DMT may involve a universal role in cellular protective mechanisms suggesting therapeutic potentials against different components and types of IRIs emerging in local and generalized brain ischemia after stroke or cardiac arrest.

Exercise enhances hepatic mitochondrial structure and function while preventing endoplasmic reticulum stress and metabolic dysfunction-associated steatotic liver disease in mice fed a high-fat diet

Metabolic dysfunction-associated steatotic liver disease (MASLD) has attracted increasing attention from the scientific community because of its severe but silent progression and the lack of specific treatment. Glucolipotoxicity triggers endoplasmic reticulum (ER) stress with decreased beta-oxidation and enhanced lipogenesis, promoting the onset of MASLD, whereas regular physical exercise can prevent MASLD by preserving ER and mitochondrial function. Thus, the hypothesis of this study was that high-intensity interval training (HIIT) could prevent the development of MASLD in high-fat (HF)-fed C57BL/6J mice by maintaining insulin sensitivity, preventing ER stress, and promoting beta-oxidation. Forty male C57BL/6J mice (3 months old) comprised 4 experimental groups: the control (C) diet group, the C diet + HIIT (C-HIIT) group, the HF diet group, and the HF diet + HIIT (HF-HIIT) group. HIIT sessions lasted 12 minutes and were performed 3 times weekly by trained mice. The diet and exercise protocols lasted for 10 weeks. The HIIT protocol prevented weight gain and maintained insulin sensitivity in the HF-HIIT group. A chronic HF diet increased ER stress-related gene and protein expression, but HIIT helped to maintain ER homeostasis, preserve mitochondrial ultrastructure, and maximize beta-oxidation. The increased sirtuin-1/peroxisome proliferator-activated receptor-gamma coactivator 1-alpha expression implies that HIIT enhanced mitochondrial biogenesis and yielded adequate mitochondrial dynamics. High hepatic fibronectin type III domain containing 5/irisin agreed with the antilipogenic and anti-inflammatory effects observed in the HF-HIIT group, reinforcing the antisteatotic effects of HIIT. Thus, we confirmed that practicing HIIT 3 times per week maintained insulin sensitivity, prevented ER stress, and enhanced hepatic beta-oxidation, impeding MASLD development in this mouse model even when consuming high energy intake from saturated fatty acids.

Unraveling the CLCC1 interactome: Impact of the Asp25Glu variant and its interaction with SigmaR1 at the Mitochondrial-Associated ER Membrane (MAM)

The endoplasmic reticulum (ER) plays an indispensable role in cellular processes, including maintenance of calcium homeostasis, and protein folding, synthesized and processing. Disruptions in these processes leading to ER stress and the accumulation of misfolded proteins can instigate the unfolded protein response (UPR), culminating in either restoration of balanced proteostasis or apoptosis. A key player in this intricate balance is CLCC1, an ER-resident chloride channel, whose essential role extends to retinal development, regulation of ER stress, and UPR. The importance of CLCC1 is further underscored by its interaction with proteins localized to mitochondria-associated endoplasmic reticulum membranes (MAMs), where it participates in UPR induction by MAM proteins.In previous research, we identified a p.(Asp25Glu) pathogenic CLCC1 variant associated with retinitis pigmentosa (RP) (CLCC1 hg38 NC_000001.11; NM_001048210.3, c.75C > A; UniprotKB Q96S66). In attempt to decipher the impact of this variant function, we leveraged liquid chromatography-mass spectrometry (LC-MS) to identify likely CLCC1-interacting proteins. We discovered that the CLCC1 interactome is substantially composed of proteins that localize to ER compartments and that the Asp25Glu variant results in noticeable loss and gain of specific protein interactors. Intriguingly, the analysis suggests that the CLCC1Asp25Glu mutant protein exhibits a propensity for increased interactions with cytoplasmic proteins compared to its wild-type counterpart.To corroborate our LC-MS data, we further scrutinized two novel CLCC1 interactors, Calnexin and SigmaR1, chaperone proteins that localize to the ER and MAMs. Through microscopy, we demonstrate that CLCC1 co-localizes with both proteins, thereby validating our initial findings. Moreover, our results reveal that CLCC1 co-localizes with SigmaR1 not merely at the ER, but also at MAMs. These findings reinforce the notion of CLCC1 interacting with MAM proteins at the ER-mitochondria interface, setting the stage for further exploration into how these interactions impact ER or mitochondria function and lead to retinal degenerative disease when impaired.

Kinesin KIF3A regulates meiotic progression and spindle assembly in oocyte meiosis

Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule movement. In this study, we characterized the critical roles of KIF3A during mouse oocyte meiosis. We found that KIF3A associated with microtubules during meiosis and depletion of KIF3A resulted in oocyte maturation defects. LC–MS data indicated that KIF3A associated with cell cycle regulation, cytoskeleton, mitochondrial function and intracellular transport-related molecules. Depletion of KIF3A activated the spindle assembly checkpoint, leading to metaphase I arrest of the first meiosis. In addition, KIF3A depletion caused aberrant spindle pole organization based on its association with KIFC1 to regulate expression and polar localization of NuMA and γ-tubulin; and KIF3A knockdown also reduced microtubule stability due to the altered microtubule deacetylation by histone deacetylase 6 (HDAC6). Exogenous Kif3a mRNA supplementation rescued the maturation defects caused by KIF3A depletion. Moreover, KIF3A was also essential for the distribution and function of mitochondria, Golgi apparatus and endoplasmic reticulum in oocytes. Conditional knockout of epithelial splicing regulatory protein 1 (ESRP1) disrupted the expression and localization of KIF3A in oocytes. Overall, our results suggest that KIF3A regulates cell cycle progression, spindle assembly and organelle distribution during mouse oocyte meiosis.

Possible role of endoplasmic reticulum stress in the pathogenesis of chronic adenoiditis and adenoid hypertrophy: A prospective, parallel-group study.

Adenoid tissue is a first-line host defense secondary lymphoid organ, especially in childhood. The endoplasmic reticulum (ER) is required to maintain balanced cellular activity. With impaired ER functions, protein accumulation occurs, resulting in ER stress, which plays a role in the etiopathogenesis of many diseases. We aimed to investigate the relationship between ER stress and adenoid tissue disorders, thereby elucidating the mechanisms of immunity-related diseases. Fifty-four pediatric patients (>3 years old) who underwent adenoidectomy for chronic adenoiditis (CA) or adenoid hypertrophy (AH) were enrolled in this prospective, parallel-group clinical study. Adenoids were divided into two groups (CA or AH) based on their size and evaluated for ER stress pathway and apoptosis pathway markers by Real-time PCR and Western blot analysis. ER stress pathway markers significantly differed between the CA and AH groups. Children with CA had higher ER stress marker levels than the AH group (p < .001 for ATF-4, ATF-6, and GRP78, and p < .05 for EDEM1, CHOP, EIF2AK3, ERNI, and GRP94). Apoptosis pathway marker levels (BAX and BCL-2) were not different between groups. ER stress contributes to the etiopathogenesis of adenoid tissue diseases and the pathogenesis of adenoid tissue disorders, which are part of the immune response. These results may guide the development of new and alternative treatments for immune system disorders.

"Sticki-ER": Functions of the Platelet Endoplasmic Reticulum.

Significance: The primary role of platelets is to generate a thrombus by platelet activation. Platelet activation relies on calcium mobilization from the endoplasmic reticulum (ER). ER resident proteins, which are externalized upon platelet activation, are essential for the function of platelet surface receptors and intercellular interactions. Recent Advances: The platelet ER is a conduit for changes in cellular function in response to the extracellular milieu. ER homeostasis is maintained by an appropriate redox balance, regulated calcium stores and normal protein folding. Alterations in ER function and ER stress results in ER proteins externalizing to the cell surface, including members of the protein disulfide isomerase family (PDIs) and chaperones. Critical Issues: The platelet ER is central to platelet function, but our understanding of its regulation is incomplete. Previous studies have focused on the function of PDIs in the extracellular space, and much less on their intracellular role. How platelets maintain ER homeostasis and how they direct ER chaperone proteins to facilitate intercellular signalling is unknown. Future Directions: An understanding of ER functions in the platelet is essential as these may determine critical platelet activities such as secretion and adhesion. Studies are necessary to understand the redox reactions of PDIs in the intracellular versus extracellular space, as these differentially affect platelet function. An unresolved question is how platelet ER proteins control calcium release. Regulation of protein folding in the platelet and downstream pathways of ER stress require further evaluation. Targeting the platelet ER may have therapeutic application in metabolic and neoplastic disease.

Abstract 2100: Novel LIPA targeted therapy for treating inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare but incredibly aggressive subtype of breast cancer (BC). IBC accounts for 2-4% of all occurrences of breast cancer and results in 7-10% of breast cancer-related deaths. IBC is only partially treatable with current therapies such as chemotherapy, surgery, and radiotherapy. Identification of novel therapeutic targets is urgently required. The main organelle for the synthesis, folding, and modification of proteins is called the endoplasmic reticulum (ER). Since elevated basal ER stress (ERS) is usually found in many cancers including IBC, our hypothesis was that the high basal ERS in IBC constitutes a serious vulnerability that can be targeted. Recently, we developed a small molecule ERX41 that promotes ER stress by blocking Lysosomal acid lipase A (LIPA) function in ER of cancer cells. The goal of the project is to test the utility of ERX-41 in treating IBC. Methods: To study the effect of ERX-41, we have used three well-established IBC model cells. We evaluated the efficacy of ERX-41 as a novel therapeutic for treating IBC using cell viability assays. The long-term effects of ERX-41 on IBC cell survival were evaluated using colony formation assays. Annexin V assays were used to measure apoptosis. Reporter assays, splicing assays, RT-qPCR, and Western blotting were used in mechanistic investigations. The effectiveness of ERX-41 was examined in vivo using KPL4 cell-based xenografts. Results: ERX-41 significantly decreased the viability of all three IBC model cells (SUM149, SUM190PT, and KPL4), with an IC50 of about 125 nM in MTT assay. Additionally, ERX-41 treatment significantly decreased IBC cells capacity to form colonies and promoted apoptosis. Specificity of the ERX41 mediated actions were confirmed using LIPA KO cells. Using RTqPCR based splicing assays, we demonstrated increased splicing of XBP1 as early as 6 hours after ERX41 treatment. Western analyses showed robust induction of stress markers (CHOP, PERK, ATF4) in IBC cells upon treatment with ERX-41. In KPL4 xenograft studies, ERX-41 showed significant reduction in the tumor volume. IHC analyses confirmed decreased proliferation marker and increased ER stress markers. Conclusions: Collectively, our results suggest that ERX-41 is a novel therapeutic agent that targets the LIPA with a unique mechanism of action and implicate ERX-41 binding to LIPA induces ER stress and promotes apoptosis of IBC cells. Citation Format: Zenaida Fuentes, Rahul Gopalam, Bianca A. Romo, Khaled Mohamed Nassar, Xue Yang, Behnam Ebrahimi, Paulina Ramirez, Chia-Yuan Chen, Scott Elmore, Harika Nagandla, Uday Pratap, Christoforos Thomas, Suryavathi Viswanadhapalli, Jung-Mo Ahn, Ganesh Raj, Ratna K. Vadlamudi. Novel LIPA targeted therapy for treating inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2100.

Abstract 205: Adaptive membrane trafficking activated by epithelial-to-mesenchymal transition drives lung adenocarcinoma progression

Abstract Cancer cells can become metastatic and develop resistance to targeted therapies through epithelial-to-mesenchymal transition (EMT), which is regulated by epigenetic factors. Our ultimate objective is to understand the molecular mechanisms behind EMT-driven cancer progression and identify potential targets for preventing cancer metastasis. Recently, we made groundbreaking discoveries indicating that EMT can modify membrane trafficking machinery to coordinate cancer cell invasion and immunosuppression in the tumor microenvironment (TME) of lung adenocarcinoma (LUAD). To dissect the “adaptive” membrane trafficking program, we initiated a CRISPRi in vivo screen to assess more than 2000 trafficking-related genes in syngeneic mouse LUAD models for their requirements for tumorigenesis both with and without immune selective pressure. We identified a panel of membrane trafficking regulators that specifically “dropped out” in immunocompetent mice, suggesting their essential roles in the immunosuppressive TME. Of particular interest is REEP2. REEPs are an evolutionarily conserved protein family that is critical to the endoplasmic reticulum (ER) function and potentially regulates secretory trafficking. We discovered that REEP2 is specifically linked to poor prognosis in LUAD patients and is highly correlated with a z-normalized, 16-gene EMT score (P = 1.32e-27) that was previously reported. Our previous studies demonstrated that the EMT activator, ZEB1, accelerates membrane trafficking dynamics by silencing a microRNA network that targets multiple membrane trafficking components. Analysis of REEP2 3’-untranslated regions (3’-UTRs) identified predicted binding sites for several microRNAs, which are silenced by ZEB1. Thus, we hypothesize that ZEB1 coordinates a microRNA network to activate REEP2-driven ER trafficking. We show that REEP2 depletion significantly reduces the invasion of cancer cells but does not affect their migration and proliferation. These findings suggest that REEP2 plays a significant role in extracellular matrix (ECM) modification to enhance the invasion of metastatic cells, conferring a vulnerability in EMT-driven LUAD metastasis. Citation Format: Oluwafunminiyi E. Obaleye, Kevin L. Fulp, Guan-Yu Xiao. Adaptive membrane trafficking activated by epithelial-to-mesenchymal transition drives lung adenocarcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 205.

Luminal transport through intact endoplasmic reticulum limits the magnitude of localized Ca2+ signals

The endoplasmic reticulum (ER) forms an interconnected network of tubules stretching throughout the cell. Understanding how ER functionality relies on its structural organization is crucial for elucidating cellular vulnerability to ER perturbations, which have been implicated in several neuronal pathologies. One of the key functions of the ER is enabling Ca[Formula: see text] signaling by storing large quantities of this ion and releasing it into the cytoplasm in a spatiotemporally controlled manner. Through a combination of physical modeling and live-cell imaging, we demonstrate that alterations in ER shape significantly impact its ability to support efficient local Ca[Formula: see text] releases, due to hindered transport of luminal content within the ER. Our model reveals that rapid Ca[Formula: see text] release necessitates mobile luminal buffer proteins with moderate binding strength, moving through a well-connected network of ER tubules. These findings provide insight into the functional advantages of normal ER architecture, emphasizing its importance as a kinetically efficient intracellular Ca[Formula: see text] delivery system.

Abnormal phosphorylation / dephosphorylation and Ca2+ dysfunction in heart failure.

Heart failure (HF) can be caused by a variety of causes characterized by abnormal myocardial systole and diastole. Ca2+ current through the L-type calcium channel (LTCC) on the membrane is the initial trigger signal for a cardiac cycle. Declined systole and diastole in HF are associated with dysfunction of myocardial Ca2+ function. This disorder can be correlated with unbalanced levels of phosphorylation / dephosphorylation of LTCC, endoplasmic reticulum (ER), and myofilament. Kinase and phosphatase activity changes along with HF progress, resulting in phased changes in the degree of phosphorylation / dephosphorylation. It is important to realize the phosphorylation / dephosphorylation differences between a normal and a failing heart. This review focuses on phosphorylation / dephosphorylation changes in the progression of HF and summarizes the effects of phosphorylation / dephosphorylation of LTCC, ER function, and myofilament function in normal conditions and HF based on previous experiments and clinical research. Also, we summarize current therapeutic methods based on abnormal phosphorylation / dephosphorylation and clarify potential therapeutic directions.

Doxorubicin‑induced cardiomyopathy is mitigated by empagliflozin via the modulation of endoplasmic reticulum stress pathways.

Doxorubicin (Dox) exhibits a high efficacy in the treatment of numerous types of cancer. However, the beneficial cytotoxic effects of Dox are often accompanied by an increase in the risk of cardiotoxicity. Oxidative stress (OS) plays a key role in Dox‑induced cardiomyopathy (DIC). OS in cardiomyocytes disrupts endoplasmic reticulum (ER) function, leading to the accumulation of misfolded/unfolded proteins known as ER stress. ER stress acts as an adaptive mechanism; however, prolonged ER stress together with OS may lead to the initiation of cardiomyocyte apoptosis. The present study aimed to explore the potential of an anti‑diabetic drug, empagliflozin (EMPA), in mitigating Dox‑induced ER stress and cardiomyocyte apoptosis. In the present study, the effects of 1h pretreatment of EMPA on Dox‑treated cardiomyocytes isolated from Sprague‑Dawley rats were investigated. After 24h, EMPA pre‑treatment promoted cell survival in the EMPA + Dox group compared with the Dox group. Results of the present study also demonstrated that EMPA mitigated overall ER stress, as the increased expression of ER stress markers was reduced in the EMPA + Dox group. Additionally, OS, inflammation and expression of ER stress apoptotic proteins were also significantly reduced following EMPA pre‑treatment in the EMPA + Dox group. Thus, EMPA may exert beneficial effects on Dox‑induced ER stress and may exhibit potential changes that can be utilised to further evaluate the role of EMPA in mitigating DIC.

Therapeutic Potential of Targeting the PERK Signaling Pathway in Ischemic Stroke.

Many pathologic states can lead to the accumulation of unfolded/misfolded proteins in cells. This causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR), which encompasses three main adaptive branches. One of these UPR branches is mediated by protein kinase RNA-like ER kinase (PERK), an ER stress sensor. The primary consequence of PERK activation is the suppression of global protein synthesis, which reduces ER workload and facilitates the recovery of ER function. Ischemic stroke induces ER stress and activates the UPR. Studies have demonstrated the involvement of the PERK pathway in stroke pathophysiology; however, its role in stroke outcomes requires further clarification. Importantly, considering mounting evidence that supports the therapeutic potential of the PERK pathway in aging-related cognitive decline and neurodegenerative diseases, this pathway may represent a promising therapeutic target in stroke. Therefore, in this review, our aim is to discuss the current understanding of PERK in ischemic stroke, and to summarize pharmacologic tools for translational stroke research that targets PERK and its associated pathways.

Deciphering the role of PGRMC2 in the human endometrium during the menstrual cycle and in vitro decidualization using an in vitro approach.

What is the human endometrial non-classical progesterone receptor (PGR) membrane component 2 (PGRMC2) expression pattern throughout the menstrual cycle and what role does it play during decidualization? Endometrial PGRMC2 expression fluctuates during the human menstrual cycle and is abundantly expressed in human endometrial stromal cells (hEnSCs) during in vitro decidualization, process where PGRMC2 is involved in embryo implantation-related pathways. The endometrial response to progesterone is mediated by the classical and non-classical PGRs. We previously demonstrated that PGR membrane component 1 (PGRMC1) is critical for endometrial function, embryo implantation, and future placentation, however, the role(s) of PGRMC2, which is structurally similar to PGRMC1, have not been studied in the human endometrium. This prospective study comprehensively evaluated the endometrial expression of PGRMC2 throughout the human menstrual cycle and during in vitro decidualization of hEnSCs (isolated from 77 endometrial biopsies that were collected from 66 oocyte donors), using immunohistochemistry, RT-qPCR, western blot, transcriptomic, and proteomic analyses. In addition, functional analysis was carried out to validate the implication of PGRMC2 in hEnSCs during embryo invasion using an in vitro outgrowth model. In vitro decidualization of hEnSCs was induced using co-treatment with cAMP and medroxyprogesterone 17-acetate progestin, and evaluated by measuring prolactin by ELISA and F-actin immunostaining. RT-qPCR was employed to compare expression with other PGRs. To reveal the function of PGRMC2 during the decidualization process, we specifically knocked down PGRMC2 with siRNAs and performed RNA-seq and quantitative proteomics techniques (SWATH-MS). The common differentially expressed genes (DEGs) and proteins (DEPs) were considered for downstream functional enrichment analysis. Finally, to verify its implication in the trophoblast invasion, an outgrowth model was carried out where hEnSCs with silenced PGRMC2 were co-cultured with human trophoblastic spheroids (JEG-3) following in vitro decidualization. In contrast to PGRMC1 and classical PGRs, endometrial PGRMC2 gene expression was significantly lower during the late- versus mid-secretory phase (P < 0.05). Accordingly, the elevated PGRMC2 protein abundance observed in the endometrial epithelial glands throughout the menstrual cycle dropped in the late secretory phase, when abundance decreased in all endometrial compartments. Nevertheless, PGRMC2 protein increased during the mid-secretory phase in stromal and glandular cells, and PGRMC2 mRNA (P < 0.0001) and protein (P < 0.001) levels were significantly enhanced in the membranes/organelles of decidualized hEnSCs, compared to non-decidualized hEnSCs. Notably, PGRMC1 and PGRMC2 mRNA were significantly more abundant than classical PGRs throughout menstrual cycle phases and in decidualized and non-decidualized hEnSCs (P < 0.05). RNA-seq and proteomics data revealed 4687 DEGs and 28 DEPs, respectively, in decidualized hEnSCs after PGRMC2 silencing. While functional enrichment analysis showed that the 2420 upregulated genes were mainly associated with endoplasmic reticulum function, vesicular transport, morphogenesis, angiogenesis, cell migration, and cell adhesion, the 2267 downregulated genes were associated with aerobic respiration and protein biosynthesis. The protein enrichment analysis showed that 4 upregulated and 24 downregulated proteins were related to aerobic respiration, cellular response, metabolism, localization of endoplasmic reticulum proteins, and ribonucleoside biosynthesis routes. Finally, PGRMC2 knockdown significantly compromised the ability of the decidualized hEnSCs to support trophoblast expansion in an outgrowth model (P < 0.05). Transcriptomic data are available via NCBI's Gene Expression Omnibus (GEO) under GEO Series accession number GSE251843 and proteomic data via ProteomeXchange with identifier PXD048494. The functional analyses were limited by the discrete number of human endometrial biopsies. A larger sample size is required to further investigate the potential role(s) of PGRMC2 during embryo implantation and maintenance of pregnancy. Further, the results obtained in the present work should be taken with caution, as the use of a pure primary endometrial stromal population differentiated in vitro does not fully represent the heterogeneity of the endometrium in vivo, nor the paracrine communications occurring between the distinct endometrial cell types. The repression of endometrial PGRMC2 during the late- versus mid-secretory phase, together with its overexpression during decidualization and multiple implications with embryo implantation not only highlighted the unknown roles of PGRMC2 in female reproduction but also the potential to exploit PGRMC2 signaling pathways to improve assisted reproduction treatments in the future. This research was funded by Instituto de Salud Carlos III (ISCIII) granted to F.D. (PI20/00405 and PI23/00860), co-funded by the European Union. Y.M.-L. was supported by a predoctoral research grant from Generalitat Valenciana (ACIF/2019/262). R.G.-M. was supported by Generalitat Valenciana (CIAPOT/2022/15). P.d.C. was supported by a predoctoral grant for training in research into health (PFIS FI20/00086) from the Instituto de Salud Carlos III. I.D.-H. was supported by the Spanish Ministry of Science, Innovation and Universities (FPU18/01550). A.P. was supported by the Instituto de Salud Carlos III (PFIS FI18/00009). This research was also supported by IVI Foundation-RMA Global (1911-FIVI-103-FD). The authors declare no conflict of interest.

Loss of Grp170 results in catastrophic disruption of endoplasmic reticulum function.

GRP170 (Hyou1) is required for mouse embryonic development, and its ablation in kidney nephrons leads to renal failure. Unlike most chaperones, GRP170 is the lone member of its chaperone family in the ER lumen. However, the cellular requirement for GRP170, which both binds nonnative proteins and acts as nucleotide exchange factor for BiP, is poorly understood. Here, we report on the isolation of mouse embryonic fibroblasts obtained from mice in which LoxP sites were engineered in the Hyou1 loci (Hyou1LoxP/LoxP). A doxycycline-regulated Cre recombinase was stably introduced into these cells. Induction of Cre resulted in depletion of Grp170 protein which culminated in cell death. As Grp170 levels fell we observed a portion of BiP fractionating with insoluble material, increased binding of BiP to a client with a concomitant reduction in its turnover, and reduced solubility of an aggregation-prone BiP substrate. Consistent with disrupted BiP functions, we observed reactivation of BiP and induction of the unfolded protein response (UPR) in futile attempts to provide compensatory increases in ER chaperones and folding enzymes. Together, these results provide insights into the cellular consequences of controlled Grp170 loss and provide hypotheses as to why mutations in the Hyou1 locus are linked to human disease.

Endoplasmic reticulum stress and its role in various neurodegenerative diseases

The Endoplasmic reticulum (ER), a critical cellular organelle, maintains cellular homeostasis by regulating calcium levels and orchestrating essential functions such as protein synthesis, folding, and lipid production. A pivotal aspect of ER function is its role in protein quality control. When misfolded proteins accumulate within the ER due to factors like protein folding chaperone dysfunction, toxicity, oxidative stress, or inflammation, it triggers the Unfolded protein response (UPR). The UPR involves the activation of chaperones like calnexin, calreticulin, glucose-regulating protein 78 (GRP78), and Glucose-regulating protein 94 (GRP94), along with oxidoreductases like protein disulphide isomerases (PDIs). Cells employ the Endoplasmic reticulum-associated degradation (ERAD) mechanism to counteract protein misfolding. ERAD disruption causes the detachment of GRP78 from transmembrane proteins, initiating a cascade involving Inositol-requiring kinase/endoribonuclease 1 (IRE1), Activating transcription factor 6 (ATF6), and Protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathways. The accumulation and deposition of misfolded proteins within the cell are hallmarks of numerous neurodegenerative diseases. These aberrant proteins disrupt normal neuronal signalling and contribute to impaired cellular homeostasis, including oxidative stress and compromised protein degradation pathways. In essence, ER stress is defined as the cellular response to the accumulation of misfolded proteins in the endoplasmic reticulum, encompassing a series of signalling pathways and molecular events that aim to restore cellular homeostasis. This comprehensive review explores ER stress and its profound implications for the pathogenesis and progression of neurodegenerative diseases.