ABSTRACT
Glioblastoma multiforme is the most common and fatal brain tumor among human cancers. Ceramide (Cer) and Sphingosine 1-phosphate (S1P) have emerged as bioeffector molecules that control several biological processes involved in both cancer development and resistance. Cer acts as a tumor suppressor, inhibiting cancer progression, promoting apoptosis, enhancing immunotherapy and sensitizing cells to chemotherapy. In contrast, S1P functions as an onco-promoter molecule, increasing proliferation, survival, invasiveness, and resistance to drug-induced apoptosis. The pro-survival PI3K/Akt pathway is a recognized downstream target of S1P, and we have previously demonstrated that in glioma cells it also improves Cer transport and metabolism towards complex sphingolipids in glioma cells. Here, we first examined the possibility that, in T98G glioma cells, S1P may regulate Cer metabolism through PI3K/Akt signaling. Our research showed that exogenous S1P increases the rate of vesicular trafficking of Cer from the endoplasmic reticulum (ER) to the Golgi apparatus through S1P receptor-mediated activation of the PI3K/Akt pathway. Interestingly, the effect of S1P results in cell protection against toxicity arising from Cer accumulation in the ER, highlighting the role of S1P as a survival factor to escape from the Cer-generating cell death response.
Subject(s)
Cell Survival , Ceramides , Endoplasmic Reticulum , Glioma , Golgi Apparatus , Lysophospholipids , Proto-Oncogene Proteins c-akt , Signal Transduction , Sphingosine , Humans , Lysophospholipids/metabolism , Lysophospholipids/pharmacology , Ceramides/metabolism , Ceramides/pharmacology , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Golgi Apparatus/metabolism , Golgi Apparatus/drug effects , Cell Line, Tumor , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/drug effects , Cell Survival/drug effects , Glioma/metabolism , Glioma/pathology , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Apoptosis/drug effectsABSTRACT
HT-2 toxin is a type of mycotoxin which is shown to affect gastric and intestinal lesions, hematopoietic and immunosuppressive effects, anorexia, lethargy, nausea. Recently, emerging evidences indicate that HT-2 also disturbs the reproductive system. In this study, we investigated the impact of HT-2 toxin exposure on the organelles of porcine oocytes. Our results found that the abnormal distribution of endoplasmic reticulum increased after HT-2 treatment, with the perturbation of ribosome protein RPS3 and GRP78 expression; Golgi apparatus showed diffused localization pattern and GM130 localization was also impaired, thereby affecting the Rab10-based vesicular transport; Due to the impairment of ribosomes, ER, and Golgi apparatus, the protein supply to lysosomes was hindered, resulting in lysosomal damage, which further disrupted the LC3-based autophagy. Moreover, the results indicated that the function and distribution of mitochondria were also affected by HT-2 toxin, showing with fragments of mitochondria, decreased TMRE and ATP level. Taken together, our study suggested that HT-2 toxin exposure induces damage to the organelles for endomembrane system, which further inhibited the meiotic maturation of porcine oocytes.
Subject(s)
In Vitro Oocyte Maturation Techniques , Oocytes , Animals , Swine , Oocytes/drug effects , In Vitro Oocyte Maturation Techniques/veterinary , T-2 Toxin/toxicity , T-2 Toxin/analogs & derivatives , Female , Golgi Apparatus/drug effects , Endoplasmic Reticulum/drug effects , Mitochondria/drug effectsABSTRACT
Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon compound that is generated during combustion processes, and is present in various substances such as foods, tobacco smoke, and burning emissions. BaP is extensively acknowledged as a highly carcinogenic substance to induce multiple forms of cancer, such as lung cancer, skin cancer, and stomach cancer. Recently it is shown to adversely affect the reproductive system. Nevertheless, the potential toxicity of BaP on oocyte quality remains unclear. In this study, we established a BaP exposure model via mouse oral gavage and found that BaP exposure resulted in a notable decrease in the ovarian weight, number of GV oocytes in ovarian, and oocyte maturation competence. BaP exposure caused ribosomal dysfunction, characterized by a decrease in the expression of RPS3 and HPG in oocytes. BaP exposure also caused abnormal distribution of the endoplasmic reticulum (ER) and induced ER stress, as indicated by increased expression of GRP78. Besides, the Golgi apparatus exhibited an abnormal localization pattern, which was confirmed by the GM130 localization. Disruption of vesicle transport processes was observed by the abnormal expression and localization of Rab10. Additionally, an enhanced lysosome and LC3 fluorescence intensity indicated the occurrence of protein degradation in oocytes. In summary, our results suggested that BaP exposure disrupted the distribution and functioning of organelles, consequently affecting the developmental competence of mouse oocytes.
Subject(s)
Benzo(a)pyrene , Endoplasmic Reticulum Chaperone BiP , Oocytes , Animals , Benzo(a)pyrene/toxicity , Oocytes/drug effects , Female , Mice , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , Organelles/drug effects , Mice, Inbred ICRABSTRACT
Thermoregulation and thermal homeostasis at the cellular and subcellular organelle level are poorly understood events. In this work, we used BV2, a microglial cell line, and a series of thermo-sensitive subcellular organelle-specific probes to analyze the relative changes in the spatio-temporal temperatures of different subcellular organelles, both qualitatively and quantitatively. These methodologies allowed us to understand the thermal relationship of different subcellular organelles also. We modulated BV2 cells by pharmacological application of activator or inhibitor of TRPM8 ion channel (a cold-sensitive ion channel) and/or by treating the cells with LPS, a molecule that induces pathogen-associated molecular patterns (PAMPs) signaling. We demonstrate that the temperatures of individual organelles remain variable within a physiological range, yet vary in different conditions. We also demonstrate that treating BV2 cells by TRPM8 modulators and/or LPS alters the organelle temperatures in a specific and context-dependent manner. We show that TRPM8 modulation and/or LPS can alter the relationship of mitochondrial membrane potential to mitochondrial temperature. Our work suggests that mitochondrial temperature positively influences ER temperature and negatively influences Golgi temperature. Golgi temperature positively influences membrane temperature. This understanding of thermal relationships may be crucial for dissecting cellular structures, function, and stress signaling and may be relevant for different diseases.
Subject(s)
Microglia , TRPM Cation Channels , TRPM Cation Channels/metabolism , Microglia/metabolism , Microglia/drug effects , Microglia/cytology , Animals , Mice , Cell Line , Mitochondria/metabolism , Mitochondria/drug effects , Lipopolysaccharides/pharmacology , Membrane Potential, Mitochondrial/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/drug effects , Organelles/metabolism , Organelles/drug effects , Golgi Apparatus/metabolism , Golgi Apparatus/drug effectsABSTRACT
Chemotherapy is a crucial treatment for colorectal tumors. However, its efficacy is restricted by chemoresistance. Recently, Golgi dispersal has been suggested to be a potential response to chemotherapy, particularly to drugs that induce DNA damage. However, the underlying mechanisms by which Golgi dispersal enhances the capacity to resist DNA-damaging agents remain unclear. Here, we demonstrated that DNA-damaging agents triggered Golgi dispersal in colorectal cancer (CRC), and cancer stem cells (CSCs) possessed a greater degree of Golgi dispersal compared with differentiated cancer cells (non-CSCs). We further revealed that Golgi dispersal conferred resistance against the lethal effects of DNA-damaging agents. Momentously, Golgi dispersal activated the Golgi stress response via the PKCα/GSK3α/TFE3 axis, resulting in enhanced protein and vesicle trafficking, which facilitated drug efflux through ABCG2. Identification of Golgi dispersal indicated an unexpected pathway regulating chemoresistance in CRC.
Subject(s)
Colorectal Neoplasms , Drug Resistance, Neoplasm , Golgi Apparatus , Neoplastic Stem Cells , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Humans , Golgi Apparatus/metabolism , Golgi Apparatus/drug effects , Colorectal Neoplasms/pathology , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Drug Resistance, Neoplasm/drug effects , Animals , Cell Line, Tumor , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics , DNA Damage , Mice , Mice, Nude , Neoplasm Proteins/metabolism , Neoplasm Proteins/genetics , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic useABSTRACT
Nutrient or energy deprivation, especially glucose restriction, is a promising anticancer therapeutic approach. However, establishing a precise and potent deprivation strategy remains a formidable task. The Golgi morphology is crucial in maintaining the function of transport proteins (such as GLUT1) driving glycolysis. Thus, in this study, we present a "Golgi-customized Trojan horse" based on tellurium loaded with apigenin (4',5,7-trihydroxyflavone) and human serum albumin, which was able to induce GLUT1 plasma membrane localization disturbance via Golgi dispersal leading to the inhibition of tumor glycolysis. Diamond-shaped delivery system can efficiently penetrate into cells as a gift like Trojan horse, which decomposes into tellurite induced by intrinsically high H2O2 and GSH levels. Consequently, tellurite acts as released warriors causing up to 3.8-fold increase in Golgi apparatus area due to the down-regulation of GOLPH3. Further, this affects GLUT1 membrane localization and glucose transport disturbance. Simultaneously, apigenin hinders ongoing glycolysis and causes significant decrease in ATP level. Collectively, our "Golgi-customized Trojan horse" demonstrates a potent antitumor activity because of its capability to deprive energy resources of cancer cells. This study not only expands the applications of tellurium-based nanomaterials in the biomedicine but also provides insights into glycolysis restriction for anticancer therapy.
Subject(s)
Apigenin , Cell Membrane , Glucose Transporter Type 1 , Glycolysis , Golgi Apparatus , Tellurium , Humans , Glycolysis/drug effects , Golgi Apparatus/metabolism , Golgi Apparatus/drug effects , Cell Membrane/metabolism , Cell Membrane/drug effects , Glucose Transporter Type 1/metabolism , Apigenin/administration & dosage , Apigenin/pharmacology , Tellurium/administration & dosage , Cell Line, Tumor , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Glucose/metabolismABSTRACT
The Golgi apparatus (GA) is a vital target for anticancer therapy due to its sensitivity against reactive oxygen species (ROS)-induced oxidative stress that could lead to cell death. In this study, we designed a series of aggregation-induced emission (AIE)-based photosensitizers (TPAPyTZ, TPAPyTC, TPAPyTM, and TPAPyTI) carrying different ROS with selective GA-targeted ability. The in vitro study showed that TPAPyTZ and TPAPyTC displayed strong AIE characteristics, robust type-I/II ROS production capabilities, specific GA-targeted, high photostability, and high imaging quality. The cell-uptake of TPAPyTZ was found primarily through an energy-dependent caveolae/raft-mediated endocytosis pathway. Remarkably, TPAPyTZ induced GA-oxidative stress, leading to GA fragmentation, downregulation of GM130 expression, and activation of mitochondria caspase-related apoptosis during photodynamic therapy (PDT). In vivo experiments revealed that TPAPyTZ significantly inhibited tumor proliferation under lower-intensity white light irradiation with minimal side effects. Overall, our work presents a promising strategy for designing AIEgens for fluorescence imaging-guided PDT. Additionally, it enriched the collection of GA-targeted leads for the development of cancer theranostics capable of visualizing dynamic changes in the GA during cancer cell apoptosis, which could potentially enable early diagnosis applications in the future. STATEMENT OF SIGNIFICANCE: AIE luminogens (AIEgens) are potent phototheranostic agents that can exhibit strong fluorescence emission and enhance ROS production in the aggregate states. In this study, through the precise design of photosensitizers with four different electron-acceptors, we constructed a series of potent AIEgens (TPAPyTZ, TPAPyTC, TPAPyTM, and TPAPyTI) with strong fluorescence intensity and ROS generation capacity. Among them, TPAPyTZ with an extended π-conjugation displayed the strongest ROS generation ability and anti-tumor activity, resulting in an 88 % reduction in tumor weight. Our studies revealed that the enhanced activity of TPAPyTZ may be due to its unique Golgi apparatus (GA)-targeted ability, which causes GA oxidative stress followed by effective cancer cell apoptosis. This unique GA-targeted feature of TPAPyTZ remains rare in the reported AIEgens, which mainly target organelles such as lysosome, mitochondria, and cell membrane. The successful design of a GA-targeted and potent AIEgen could enrich the collection of GA-targeted luminogens, providing a lead theranostic for the further development of fluorescence imaging-guided PDT, and serving as a tool to explore the potential mechanism and discover new GA-specific drug targets.
Subject(s)
Golgi Apparatus , Photochemotherapy , Photosensitizing Agents , Reactive Oxygen Species , Photochemotherapy/methods , Golgi Apparatus/metabolism , Golgi Apparatus/drug effects , Photosensitizing Agents/pharmacology , Photosensitizing Agents/chemistry , Humans , Animals , Reactive Oxygen Species/metabolism , Mice , Mice, Nude , Mice, Inbred BALB C , Cell Line, Tumor , Apoptosis/drug effects , FemaleABSTRACT
Cellular stress is a crucial factor in regulating and maintaining both organismal and microenvironmental homeostasis. It induces a response that also affects the micropolarity of specific cellular compartments, which is essential for early disease diagnosis. In this contribution, we present a quantitative study of micropolarity changes inside the endoplasmic reticulum (ER) during the G1/S and G2/M phases, using a biocompatible small-molecule fluorophore called ER-Oct. This probe is selectively driven to the ER by its hydrophobicity, and it has the fastest diffusion properties among a series of analogous probes. We found that induced ER stress caused cell cycle arrests leading to an increase in ER micropolarity which is well supported by lambda scanning experiments and fluorescence lifetime imaging microscopy (FLIM) as well. ER-Oct is a versatile staining agent that could effectively stain the ER in various living/fixed mammalian cells, isolated ER, Caenorhabditis elegans, and mice tissues. Furthermore, we used this probe to visualize a well-known biological event, ER to Golgi transport, by live-cell fluorescence microscopy. Our exhaustive investigation of micropolarity using ER-staining dye provides a new way to study ER stress, which could provide a deeper understanding of proteostasis in model systems and even in fixed patient samples.
Subject(s)
Caenorhabditis elegans , Endoplasmic Reticulum Stress , Endoplasmic Reticulum , Golgi Apparatus , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/drug effects , Animals , Golgi Apparatus/metabolism , Golgi Apparatus/drug effects , Humans , Endoplasmic Reticulum Stress/drug effects , Mice , Caenorhabditis elegans/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Microscopy, FluorescenceABSTRACT
BACKGROUND: Sepsis is often accompanied by multiple organ dysfunction, in which the incidence of cardiac injury is about 60%, and is closely related to high mortality. Recent studies have shown that Golgi stress is involved in liver injury, kidney injury, and lung injury in sepsis. However, whether it is one of the key mechanisms of sepsis-induced cardiomyopathy (SIC) is still unclear. The aim of this study is to investigate whether Golgi stress mediates SIC and the specific mechanism. METHODS: Sepsis model of male C57BL/6J mice was established by cecal ligation and puncture. To observe the effect of Golgi stress on SIC, mice were injected with Golgi stimulant (Brefeldin A) or Golgi inhibitor (Glutathione), respectively. The 7-day survival rate of mice were recorded, and myocardial injury indicators including cardiac function, myocardial enzymes, myocardial pathological tissue score, myocardial inflammatory factors, and apoptosis were detected. The morphology of Golgi was observed by immunofluorescence, and the Golgi stress indices including GM-130, GOLPH3 and Goligin97 were detected by WB and qPCR. RESULTS: After CLP, the cardiac function of mice was impaired and the levels of myocardial enzymes were significantly increased. Golgi stress was accompanied by increased myocardial inflammation and apoptosis. Moreover, the expressions of morphological proteins GM-130 and Golgin97 were decreased, and the expression of stress protein GOLPH3 was increased. In addition, Brefeldin A increased 7-day mortality and the above indicators in mice. The use of glutathione improves all of the above indicators. CONCLUSION: Golgi stress mediates SIC, and the inhibition of Golgi stress can improve SIC by inhibiting apoptosis and inflammation.
Subject(s)
Apoptosis , Brefeldin A , Cardiomyopathies , Golgi Apparatus , Mice, Inbred C57BL , Sepsis , Animals , Apoptosis/drug effects , Male , Sepsis/complications , Sepsis/drug therapy , Golgi Apparatus/metabolism , Golgi Apparatus/drug effects , Cardiomyopathies/etiology , Cardiomyopathies/drug therapy , Mice , Brefeldin A/pharmacology , Inflammation/drug therapy , Disease Models, Animal , Glutathione/metabolism , Myocardium/pathology , Myocardium/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , HumansABSTRACT
Prodiginines have been studied extensively for their anticancer activity, however, the majority of the research has focused on prodigiosin. In this study, cycloheptylprodigiosin (S-1) is extracted from marine bacterium Spartinivicinus ruber MCCC 1K03745T, and its anticancer property was investigated. It exhibits remarkable cytotoxicity against a panel of human lung cancer cell lines, with the IC50 values ranging from 84.89 nM to 661.2 nM. After 6 h of treatment, S-1 gradually accumulates on mitochondria and lysosomes. While lower doses of S-1 induce cell cycle arrest, treatment with higher doses results in cell death in apoptotic independent manner in both NCI-H1299 and NCI-H460 cell lines. Interestingly, treatment with S-1 leads to the accumulation of LC3B-II via pathways that vary among different cell lines. In addition to its role as an autophagy inhibitor, S-1 also promotes autophagy initiation as demonstrated by the increment of EGFP fragment in the EGFP-LC3 degradation assay, however, inhibition of autophagy does not rescue cells from death induced by S-1. Mechanistically, S-1 impairs autophagic flux through disrupting acidic lysosomal pH and blocking the maturation of cathepsin D. Moreover, treatment with S-1 enhanced secretion of both pro- and mature forms of cathepsin D, coincident with disintegration of trans-Golgi network. Interestingly, S-1 does not induce ferroptosis, pyroptosis or necroptosis in NCI-H1299 cells. However, treatment of NCI-H460 cells with S-1 induces methuosis, which can be suppressed by Rac1 inhibitor EHT 1864. Our data demonstrate that S-1 is an effective anticancer agent with potential therapeutic application.
Subject(s)
Antineoplastic Agents , Carcinoma, Non-Small-Cell Lung , Cathepsin D , Golgi Apparatus , Lung Neoplasms , Prodigiosin , Humans , Cell Line, Tumor , Prodigiosin/pharmacology , Prodigiosin/analogs & derivatives , Cathepsin D/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Antineoplastic Agents/pharmacology , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Autophagy/drug effects , Apoptosis/drug effects , Lysosomes/drug effects , Lysosomes/metabolism , Cell Death/drug effects , Mitochondria/drug effects , Mitochondria/metabolismABSTRACT
The development of alcohol-associated liver disease (ALD) is associated with disorganized Golgi apparatus and accelerated phagophore formation. While Golgi membranes may contribute to phagophores, association between Golgi alterations and macroautophagy/autophagy remains unclear. GOLGA4/p230 (golgin A4), a dimeric Golgi matrix protein, participates in phagophore formation, but the underlying mechanism is elusive. Our prior research identified ethanol (EtOH)-induced Golgi scattering, disrupting intra-Golgi trafficking and depleting RAB3D GTPase from the trans-Golgi. Employing various techniques, we analyzed diverse cellular and animal models representing chronic and chronic/binge alcohol consumption. In trans-Golgi of non-treated hepatocytes, we found a triple complex formed between RAB3D, GOLGA4, and MYH10/NMIIB (myosin, heavy polypeptide 10, non-muscle). However, EtOH-induced RAB3D downregulation led to MYH10 segregation from the Golgi, accompanied by Golgi fragmentation and tethering of the MYH10 isoform, MYH9/NMIIA, to dispersed Golgi membranes. EtOH-activated autophagic flux is evident through increased WIPI2 recruitment to the Golgi, phagophore formation, enhanced LC3B lipidation, and reduced SQSTM1/p62. Although GOLGA4 dimerization and intra-Golgi localization are unaffected, loss of RAB3D leads to an extension of the cytoplasmic N terminal domain of GOLGA4, forming GOLGA4-positive phagophores. Autophagy inhibition by hydroxychloroquine (HCQ) prevents alcohol-mediated Golgi disorganization, restores distribution of ASGR (asialoglycoprotein receptor), and mitigates COL (collagen) deposition and steatosis. In contrast to short-term exposure to HCQ, extended co-treatment with both EtOH and HCQ results in the depletion of LC3B protein via proteasomal degradation. Thus, (a) RAB3D deficiency and GOLGA4 conformational changes are pivotal in MYH9-driven, EtOH-mediated Golgiphagy, and (b) HCQ treatment holds promise as a therapeutic approach for alcohol-induced liver injury.Abbreviation: ACTB: actin, beta; ALD: alcohol-associated liver disease; ASGR: asialoglycoprotein receptor; AV: autophagic vacuoles; EM: electron microscopy; ER: endoplasmic reticulum; EtOH: ethanol; HCQ: hydroxychloroquine; IP: immunoprecipitation; KD: knockdown; KO: knockout; MYH10/NMIIB: myosin, heavy polypeptide 10, non-muscle; MYH9/NMIIA: myosin, heavy polypeptide 9, non-muscle; PLA: proximity ligation assay; ORO: Oil Red O staining; PM: plasma membrane; TGN: trans-Golgi network; SIM: structured illumination super-resolution microscopy.
Subject(s)
Autophagy , Down-Regulation , Ethanol , Golgi Apparatus , Golgi Apparatus/metabolism , Golgi Apparatus/drug effects , Golgi Apparatus/ultrastructure , Humans , Animals , Down-Regulation/drug effects , Autophagy/drug effects , Autophagy/physiology , Ethanol/pharmacology , rab3 GTP-Binding Proteins/metabolism , Mice , Hepatocytes/metabolism , Hepatocytes/drug effects , Mice, Inbred C57BLABSTRACT
The Golgi apparatus (GA) is central in shuttling proteins from the endoplasmic reticulum to different cellular areas. Therefore, targeting the GA to precisely destroy its proteins through local heat could induce apoptosis, offering a potential avenue for effective cancer therapy. Herein, a GA-targeted photothermal agent based on protein anchoring is introduced for enhanced photothermal therapy of tumor through the modification of near-infrared molecular dye with maleimide derivative and benzene sulfonamide. The photothermal agent can actively target the GA and covalently anchor to its sulfhydryl proteins, thereby increasing its retention within the GA. Under laser irradiation, the heat generated by the photothermal agent efficiently disrupts sulfhydryl proteins in situ, leading to GA dysfunction and ultimately inducing cell apoptosis. In vivo experiments demonstrate that the photothermal agent can precisely treat tumors and significantly reduce side effects.
Subject(s)
Golgi Apparatus , Photothermal Therapy , Golgi Apparatus/metabolism , Golgi Apparatus/drug effects , Photothermal Therapy/methods , Animals , Humans , Mice , Apoptosis/drug effects , Cell Line, Tumor , Neoplasms/therapy , Neoplasms/metabolism , Neoplasms/drug therapy , Neoplasms/pathology , Mice, Nude , Mice, Inbred BALB C , Maleimides/chemistry , Maleimides/pharmacologyABSTRACT
Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein, pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution is unsuitable to follow the rapid transit of cargo between organelles. Therefore, we applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway and compared the pH values obtained by the FLIM-based measurements with those obtained by conventional ratiometric imaging. Then, we analyzed the dynamic pH changes within cells treated with Bafilomycin A1, to block the vesicular ATPase, and Brefeldin A, to block endoplasmic reticulum (ER)-Golgi trafficking. Finally, we followed the pH changes of newly synthesized molecules of the inflammatory cytokine tumor necrosis factor-α while they were in transit from the ER via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution and can be used to assess organellar pH in disease models.
Subject(s)
Hydrogen-Ion Concentration , Optical Imaging/methods , Secretory Pathway , Adenosine Triphosphatases/antagonists & inhibitors , Brefeldin A/pharmacology , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum/metabolism , Golgi Apparatus/drug effects , Golgi Apparatus/enzymology , Golgi Apparatus/metabolism , Humans , Macrolides/pharmacology , Microscopy, Fluorescence/methods , Protein TransportABSTRACT
Chronic kidney disease (CKD) is one of the top ten leading causes of death in the USA. Acute kidney injury (AKI), while often recoverable, predisposes patients to CKD later in life. Kidney epithelial cells have been identified as key signaling nodes in both AKI and CKD, whereby the cells can determine the course of the disease through the secretion of cytokines and other proteins. In CKD especially, several lines of evidence have demonstrated that maladaptively repaired tubular cells drive disease progression through the secretion of transforming growth factor-beta (TGF-ß), connective tissue growth factor (CTGF), and other profibrotic cytokines. However, identifying the source and the relative number of secreted proteins from different cell types in vivo remains challenging. This paper describes a technique using brefeldin A (BFA) to prevent the secretion of cytokines, enabling the staining of cytokines in kidney tissue using standard immunofluorescent techniques. BFA inhibits endoplasmic reticulum (ER)-to-Golgi apparatus transport, which is necessary for the secretion of cytokines and other proteins. Injection of BFA 6 h before sacrifice leads to a build-up of TGF-ß, PDGF, and CTGF inside the proximal tubule cells (PTCs) in a mouse cisplatin model of AKI and TGF-ß in a mouse aristolochic acid (AA) model of CKD. Analysis revealed that BFA + cisplatin or BFA + AA increased TGF-ß-positive signal significantly compared to BFA + saline, cisplatin, or AA alone. These data suggest that BFA can be used to identify the cell type producing specific cytokines and quantify the relative amounts and/or different types of cytokines produced.
Subject(s)
Brefeldin A , Endoplasmic Reticulum , Golgi Apparatus , Kidney , Animals , Brefeldin A/pharmacology , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , Humans , Kidney/drug effects , Kidney/metabolism , Mice , Transforming Growth Factor beta/metabolismABSTRACT
The glucocerebrosidase 1 gene (GBA1), bi-allelic variants of which cause Gaucher disease (GD), encodes the lysosomal enzyme glucocerebrosidase (GCase) and is a risk factor for Parkinson Disease (PD). GBA1 variants are linked to a reduction in GCase activity in the brain. Variants in Leucine-Rich Repeat Kinase 2 (LRRK2), such as the gain-of-kinase-function variant G2019S, cause the most common familial form of PD. In patients without GBA1 and LRRK2 mutations, GCase and LRRK2 activity are also altered, suggesting that these two genes are implicated in all forms of PD and that they may play a broader role in PD pathogenesis. In this review, we review the proposed roles of GBA1 and LRRK2 in PD, focussing on the endolysosomal pathway. In particular, we highlight the discovery of Ras-related in brain (Rab) guanosine triphosphatases (GTPases) as LRRK2 kinase substrates and explore the links between increased LRRK2 activity and Rab protein function, lysosomal dysfunction, alpha-synuclein accumulation and GCase activity. We also discuss the discovery of RAB10 as a potential mediator of LRRK2 and GBA1 interaction in PD. Finally, we discuss the therapeutic implications of these findings, including current approaches and future perspectives related to novel drugs targeting LRRK2 and GBA1.
Subject(s)
Epistasis, Genetic/genetics , Glucosylceramidase/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Parkinson Disease/genetics , Animals , Glucosylceramidase/antagonists & inhibitors , Glucosylceramidase/metabolism , Golgi Apparatus/drug effects , Golgi Apparatus/genetics , Golgi Apparatus/metabolism , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/antagonists & inhibitors , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Parkinson Disease/drug therapy , Parkinson Disease/metabolism , rab GTP-Binding Proteins/antagonists & inhibitors , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
Androgen receptor (AR) plays a central role in driving prostate cancer (PCa) progression. How AR promotes this process is still not completely clear. Herein, we used single-cell transcriptome analysis to reconstruct the transcriptional network of AR in PCa. Our work shows AR directly regulates a set of signature genes in the ER-to-Golgi protein vesicle-mediated transport pathway. The expression of these genes is required for maximum androgen-dependent ER-to-Golgi trafficking, cell growth, and survival. Our analyses also reveal the signature genes are associated with PCa progression and prognosis. Moreover, we find inhibition of the ER-to-Golgi transport process with a small molecule enhanced antiandrogen-mediated tumor suppression of hormone-sensitive and insensitive PCa. Finally, we demonstrate AR collaborates with CREB3L2 in mediating ER-to-Golgi trafficking in PCa. In summary, our findings uncover a critical role for dysregulation of ER-to-Golgi trafficking expression and function in PCa progression, provide detailed mechanistic insights for how AR tightly controls this process, and highlight the prospect of targeting the ER-to-Golgi pathway as a therapeutic strategy for advanced PCa.
Subject(s)
Androgens/pharmacology , Basic-Leucine Zipper Transcription Factors/metabolism , Endoplasmic Reticulum/pathology , Gene Expression Regulation, Neoplastic/drug effects , Golgi Apparatus/pathology , Prostatic Neoplasms/pathology , Receptors, Androgen/metabolism , Animals , Apoptosis , Basic-Leucine Zipper Transcription Factors/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Proliferation , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Gene Regulatory Networks , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , Humans , Male , Mice , Prognosis , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Receptors, Androgen/genetics , Single-Cell Analysis/methods , Survival Rate , Transcriptome , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
La Crosse virus (LACV) is a mosquito-borne orthobunyavirus that causes approximately 60 to 80 hospitalized pediatric encephalitis cases in the United States yearly. The primary treatment for most viral encephalitis, including LACV, is palliative care, and specific antiviral therapeutics are needed. We screened the National Center for Advancing Translational Sciences library of 3,833 FDA-approved and bioactive small molecules for the ability to inhibit LACV-induced death in SH-SY5Y neuronal cells. The top three hits from the initial screen were validated by examining their ability to inhibit virus-induced cell death in multiple neuronal cell lines. Rottlerin consistently reduced LACV-induced death by 50% in multiple human and mouse neuronal cell lines with an effective concentration of 0.16-0.69 µg ml-1 depending on cell line. Rottlerin was effective up to 12 hours post-infection in vitro and inhibited virus particle trafficking from the Golgi apparatus to trans-Golgi vesicles. In human inducible pluripotent stem cell-derived cerebral organoids, rottlerin reduced virus production by one log and cell death by 35% compared with dimethyl sulfoxide-treated controls. Administration of rottlerin in mice by intraperitoneal or intracranial routes starting at 3 days post-infection decreased disease development by 30-50%. Furthermore, rottlerin also inhibited virus replication of other pathogenic California serogroup orthobunyaviruses (Jamestown Canyon and Tahyna virus) in neuronal cell lines.
Subject(s)
Acetophenones/administration & dosage , Antiviral Agents/administration & dosage , Benzopyrans/administration & dosage , Encephalitis, California/virology , Golgi Apparatus/virology , La Crosse virus/drug effects , La Crosse virus/physiology , Neurons/virology , Animals , Encephalitis, California/drug therapy , Female , Golgi Apparatus/drug effects , Humans , La Crosse virus/genetics , Male , Mice , Mice, Inbred C57BL , Neurons/drug effects , Virus Release/drug effects , Virus Replication/drug effectsABSTRACT
The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.
Subject(s)
Glycosphingolipids/metabolism , Golgi Apparatus/metabolism , Golgi Matrix Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Autoantigens/genetics , Autoantigens/metabolism , Brefeldin A/pharmacology , Ceramides/metabolism , Cholera Toxin/pharmacology , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Gene Expression , Glycosylation/drug effects , Golgi Apparatus/drug effects , Golgi Apparatus/genetics , Golgi Matrix Proteins/genetics , HeLa Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Shiga Toxin/pharmacologyABSTRACT
Inducing the formation of new oligodendrocytes from oligodendrocyte progenitor cells (OPCs) represents a potential approach to repairing the loss of myelin observed in multiple sclerosis and other diseases. Recently, we demonstrated that accumulation of specific cholesterol precursors, 8,9-unsaturated sterols, is a dominant mechanism by which dozens of small molecules enhance oligodendrocyte formation. Here, we evaluated a library of 56 sterols and steroids to evaluate whether other classes of bioactive sterol derivatives may also influence mouse oligodendrocyte precursor cell (OPC) differentiation or survival. From this library, we identified U-73343 as a potent enhancer of oligodendrocyte formation that induces 8,9-unsaturated sterol accumulation by inhibition of the cholesterol biosynthesis enzyme sterol 14-reductase. In contrast, we found that mouse OPCs are remarkably vulnerable to treatment with the glycosterol OSW-1, an oxysterol-binding protein (OSBP) modulator that induces Golgi stress and OPC death in the low picomolar range. A subsequent small-molecule suppressor screen identified mTOR signaling as a key effector pathway mediating OSW-1's cytotoxic effects in mouse OPCs. Finally, evaluation of a panel of ER and Golgi stress-inducing small molecules revealed that mouse OPCs are highly sensitive to these perturbations, more so than closely related neural progenitor cells. Together, these studies highlight the wide-ranging influence of sterols and steroids on OPC cell fate, with 8,9-unsaturated sterols positively enhancing differentiation to oligodendrocytes and OSW-1 able to induce lethal Golgi stress with remarkable potency.
Subject(s)
Cell Differentiation/drug effects , Oligodendrocyte Precursor Cells/drug effects , Sterols/pharmacology , Animals , Cell Survival/drug effects , Cholestenones/pharmacology , Cholestenones/toxicity , Drug Evaluation, Preclinical , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum Stress/drug effects , Estrenes/pharmacology , Golgi Apparatus/drug effects , HeLa Cells , Humans , Mice , Oligodendrocyte Precursor Cells/metabolism , Oligodendroglia/metabolism , Pyrrolidinones/pharmacology , Saponins/pharmacology , Saponins/toxicity , Small Molecule Libraries/pharmacology , Small Molecule Libraries/toxicity , Sterols/toxicityABSTRACT
We studied the prolonged action of kainic acid on glutamatergic neurons in the dorsal hippocampus and the endocannabinoid-dependent protection against neurodegeneration. The pyramidal neurons of the CA3 field of the hippocampus, as well as granular and mossy cells of the dentate gyrus were examined. Light and electron microscopy revealed substantial damage to the components of the protein-synthesizing (rough endoplasmic reticulum, Golgi apparatus, and polyribosomes) and catabolic (lysosomes, autophagosomes, multivesicular structures, and lipofuscin formations) systems in all cells. Pyramidal and mossy neurons die mainly by the necrotic pathway. The death of granular cells occurred through both apoptosis and necrosis. The most vulnerable cells are mossy neurons located in the hilus. Activation of the endocannabinoid system induced by intracerebral injection of URB597, an inhibitor of degradation of endocannabinoid anandamide, protected the normal structure of the hippocampus and prevented neuronal damage and death induced by KA.