Subcellular activation of ß-adrenergic receptors using a spatially restricted antagonist.

Gprotein-coupled receptors (GPCRs) regulate several physiological and pathological processes and represent the target of approximately 30% of Food and Drug Administration-approved drugs. GPCR-mediated signaling was thought to occur exclusively at the plasma membrane. However, recent studies have unveiled their presence and function at subcellular membrane compartments. There is a growing interest in studying compartmentalized signaling of GPCRs. This requires development of tools to separate GPCR signaling at the plasma membrane from the ones initiated at intracellular compartments. We leveraged the structural and pharmacological information available for ß-adrenergic receptors (ßARs) and focused on ß1AR as exemplary GPCR that functions at subcellular compartments, and rationally designed spatially restricted antagonists. We generated a cell-impermeable ßAR antagonist by conjugating a suitable pharmacophore to a sulfonate-containing fluorophore. This cell-impermeable antagonist only inhibited ß1AR on the plasma membrane. In contrast, a cell-permeable ßAR antagonist containing a nonsulfonated fluorophore efficiently inhibited both the plasma membrane and Golgi pools of ß1ARs. Furthermore, the cell-impermeable antagonist selectively inhibited the phosphorylation of PKA downstream effectors near the plasma membrane, which regulate sarcoplasmic reticulum (SR) Ca2+ release in adult cardiomyocytes, while the ß1AR Golgi pool remained active. Our tools offer promising avenues for investigating compartmentalized ßAR signaling in various contexts, potentially advancing our understanding of ßAR-mediated cellular responses in health and disease. They also offer a general strategy to study compartmentalized signaling for other GPCRs in various biological systems.

Sphingosine 1-Phosphate Stimulates ER to Golgi Ceramide Traffic to Promote Survival in T98G Glioma Cells.

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.

General toxicity and screening of reproductive and developmental toxicity following bioaccumulation of oral-dosed perfluorooctanoic acid: Loss of the Golgi apparatus.

Despite its widespread use as a stabilizer across various industries over the past several decades, the health effects of chronic exposure to PFOA are still unclear. We administered PFOA by oral gavage (0, 12.5, 50, and 200 µg/day/mouse, eight groups) to male and female mice for six months. Body weight gain decreased with dose accompanied by increased liver weight, and PFOA altered liver damage-related-blood biochemical indicators and induced pathological lesions, including hepatocellular hypertrophy, cholangiofibrosis, and centrilobular hepatocellular vacuolation. Loss of the Golgi apparatus, formation of lamellar body-like structures, and lipid accumulation were observed in the liver of PFOA-treated mice. We also cohabited five pairs of male and female mice for the last ten days of administration, dosed PFOA to dam up to 28 days after birth, and investigated effects on reproduction and development. The survival rate of pups and the sex ratio of surviving mice decreased significantly at the highest dose. PFOA tissue concentration increased with the dose in the parent mice's liver and the pups' blood and brain. Taken together, we suggest that PFOA primarily affects the liver and reproduction system and that disturbance in lipid metabolism and Golgi's structural stability may be involved in PFOA-induced toxicity.

HT-2 toxin impairs porcine oocyte in vitro maturation through disruption of endomembrane system.

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.

TRPM8 affects relative "cooling and heating" of subcellular organelles in microglia in a context-dependent manner.

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.

Benzo[a]pyrene exposure disrupts the organelle distribution and function of mouse oocytes.

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.

Golgi dispersal in cancer stem cells promotes chemoresistance of colorectal cancer via the Golgi stress response.

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.

A small-molecule probe to decipher stress-induced ER microenvironments and ER-Golgi communication.

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.

"Golgi-customized Trojan horse" nanodiamonds impair GLUT1 plasma membrane localization and inhibit tumor glycolysis.

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.

A structure-guided strategy to design Golgi apparatus-targeted type-I/II aggregation-induced emission photosensitizers for efficient photodynamic therapy.

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.

Inhibition of Golgi stress alleviates sepsis-induced cardiomyopathy by reducing inflammation and apoptosis.

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.

Cycloheptylprodigiosin from marine bacterium Spartinivicinus ruber MCCC 1K03745T induces a novel form of cell death characterized by Golgi disruption and enhanced secretion of cathepsin D in non-small cell lung cancer cell lines.

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.

Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

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.

A Golgi Apparatus-Targeted Photothermal Agent with Protein Anchoring for Enhanced Cancer Photothermal Therapy.

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.

Fluorescence Lifetime Imaging of pH along the Secretory Pathway.

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.

Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection.

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.

Rottlerin inhibits La Crosse virus-induced encephalitis in mice and blocks release of replicating virus from the Golgi body in neurons.

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.

Single-cell analysis reveals androgen receptor regulates the ER-to-Golgi trafficking pathway with CREB3L2 to drive prostate cancer progression.

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.

Glucocerebrosidase 1 and leucine-rich repeat kinase 2 in Parkinson disease and interplay between the two genes.

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.

GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis.

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.