Vaccinia virus subverts xenophagy through phosphorylation and nuclear targeting of p62.

Autophagy is an essential degradation program required for cell homeostasis. Among its functions is the engulfment and destruction of cytosolic pathogens, termed xenophagy. Not surprisingly, many pathogens use various strategies to circumvent or co-opt autophagic degradation. For poxviruses, it is known that infection activates autophagy, which however is not required for successful replication. Even though these complex viruses replicate exclusively in the cytoplasm, autophagy-mediated control of poxvirus infection has not been extensively explored. Using the prototypic poxvirus, vaccinia virus (VACV), we show that overexpression of the xenophagy receptors p62, NDP52, and Tax1Bp1 restricts poxvirus infection. While NDP52 and Tax1Bp1 were degraded, p62 initially targeted cytoplasmic virions before being shunted to the nucleus. Nuclear translocation of p62 was dependent upon p62 NLS2 and correlated with VACV kinase mediated phosphorylation of p62 T269/S272. This suggests that VACV targets p62 during the early stages of infection to avoid destruction and further implies that poxviruses exhibit multi-layered control of autophagy to facilitate cytoplasmic replication.

Active growth signaling promotes senescence and cancer cell sensitivity to CDK7 inhibition.

Tumor growth is driven by continued cellular growth and proliferation. Cyclin-dependent kinase 7's (CDK7) role in activating mitotic CDKs and global gene expression makes it therefore an attractive target for cancer therapies. However, what makes cancer cells particularly sensitive to CDK7 inhibition (CDK7i) remains unclear. Here, we address this question. We show that CDK7i, by samuraciclib, induces a permanent cell-cycle exit, known as senescence, without promoting DNA damage signaling or cell death. A chemogenetic genome-wide CRISPR knockout screen identified that active mTOR (mammalian target of rapamycin) signaling promotes samuraciclib-induced senescence. mTOR inhibition decreases samuraciclib sensitivity, and increased mTOR-dependent growth signaling correlates with sensitivity in cancer cell lines. Reverting a growth-promoting mutation in PIK3CA to wild type decreases sensitivity to CDK7i. Our work establishes that enhanced growth alone promotes CDK7i sensitivity, providing an explanation for why some cancers are more sensitive to CDK inhibition than normally growing cells.

Cardiac glycosides restore autophagy flux in an iPSC-derived neuronal model of WDR45 deficiency.

Beta-Propeller Protein-Associated Neurodegeneration (BPAN) is one of the commonest forms of Neurodegeneration with Brain Iron Accumulation, caused by mutations in the gene encoding the autophagy-related protein, WDR45. The mechanisms linking autophagy, iron overload and neurodegeneration in BPAN are poorly understood and, as a result, there are currently no disease-modifying treatments for this progressive disorder. We have developed a patient-derived, induced pluripotent stem cell (iPSC)-based midbrain dopaminergic neuronal cell model of BPAN (3 patient, 2 age-matched controls and 2 isogenic control lines) which shows defective autophagy and aberrant gene expression in key neurodegenerative, neurodevelopmental and collagen pathways. A high content imaging-based medium-throughput blinded drug screen using the FDA-approved Prestwick library identified 5 cardiac glycosides that both corrected disease-related defective autophagosome formation and restored BPAN-specific gene expression profiles. Our findings have clear translational potential and emphasise the utility of iPSC-based modelling in elucidating disease pathophysiology and identifying targeted therapeutics for early-onset monogenic disorders.

Automated high-content imaging in iPSC-derived neuronal progenitors.

Induced pluripotent stem cells (iPSCs) have great potential as physiological disease models for human disorders where access to primary cells is difficult, such as neurons. In recent years, many protocols have been developed for the generation of iPSCs and the differentiation into specialised cell subtypes of interest. More recently, these models have been modified to allow large-scale phenotyping and high-content screening of small molecule compounds in iPSC-derived neuronal cells. Here, we describe the automated seeding of day 11 ventral midbrain progenitor cells into 96-well plates, administration of compounds, automated staining for immunofluorescence, the acquisition of images on a high-content screening platform and workflows for image analysis.

Salmonella Exhibit Altered Cellular Localization in the Presence of HLA-B27 and Codistribute with Endo-Reticular Membrane.

Salmonella enteritica (S. enteritica) induce and require unfolded protein response (UPR) pathways for intracellular replication. Salmonella infections can lead to reactive arthritis (ReA), which can exhibit associations with Human Leucocyte Antigen (HLA)-B∗27 : 05. S. enteritica normally reside in a juxtanuclear position to the Golgi apparatus, representing the formation and residence within the Salmonella-containing vacuole (SCV). Changes in cellular localization of infecting Salmonella can alter their ability to replicate. We therefore used isogenic epithelial cell lines expressing physiological levels of HLA-B∗27 : 05 heavy chain (HC) and a control HLA-B allele, HLA-B∗35 : 01.HC to determine any changes in Salmonella localization within epithelial cells. Expression of HLA-B∗27 : 05 but not HLA-B∗35 : 01 was associated with a quantifiable change in S. enteritica cellular distribution away from the Golgi apparatus. Furthermore, the Salmonella requirements for UPR induction and the consequences of the concomitant endoplasmic reticulum (ER) membrane expansion were determined. Using confocal imaging, S. enteritica bacteria exhibited a significant and quantifiable codistribution with endo-reticular membrane as determined by ER tracker staining. Isogenic S. enterica Typhimurium mutant strains, which can infect but exhibit impaired intracellular growth, demonstrated that the activation of the UPR was dependent on an integral intracellular niche. Therefore, these data identify cellular changes accompanying Salmonella induction of the UPR and in the presence of HLA-B27.

Hydralazine protects the heart against acute ischaemia/reperfusion injury by inhibiting Drp1-mediated mitochondrial fission.

AIMS: Genetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission. METHODS AND RESULTS: Pre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6±1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated IRI, hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7±2.5% vs. control 34.1±1.5%, P=0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6±6.5% vs. vehicle control 54.1±4.9%, P=0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9±3.0% vs. vehicle control 58.2±3.8%, P<0.001). CONCLUSION: We show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.

Vaccinia virus hijacks ESCRT-mediated multivesicular body formation for virus egress.

Poxvirus egress is a complex process whereby cytoplasmic single membrane-bound virions are wrapped in a cell-derived double membrane. These triple-membrane particles, termed intracellular enveloped virions (IEVs), are released from infected cells by fusion. Whereas the wrapping double membrane is thought to be derived from virus-modified trans-Golgi or early endosomal cisternae, the cellular factors that regulate virus wrapping remain largely undefined. To identify cell factors required for this process the prototypic poxvirus, vaccinia virus (VACV), was subjected to an RNAi screen directed against cellular membrane-trafficking proteins. Focusing on the endosomal sorting complexes required for transport (ESCRT), we demonstrate that ESCRT-III and VPS4 are required for packaging of virus into multivesicular bodies (MVBs). EM-based characterization of MVB-IEVs showed that they account for half of IEV production indicating that MVBs are a second major source of VACV wrapping membrane. These data support a model whereby, in addition to cisternae-based wrapping, VACV hijacks ESCRT-mediated MVB formation to facilitate virus egress and spread.

Identification and functional validation of FDA-approved positive and negative modulators of the mitochondrial calcium uniporter.

The mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ entry, plays important roles in physiology and pathology. However, only few pharmacological compounds directly and selectively modulate its activity. Here, we perform high-throughput screening on a US Food and Drug Administration (FDA)-approved drug library comprising 1,600 compounds to identify molecules modulating mitochondrial Ca2+ uptake. We find amorolfine and benzethonium to be positive and negative MCU modulators, respectively. In agreement with the positive effect of MCU in muscle trophism, amorolfine increases muscle size, and MCU silencing is sufficient to blunt amorolfine-induced hypertrophy. Conversely, in the triple-negative breast cancer cell line MDA-MB-231, benzethonium delays cell growth and migration in an MCU-dependent manner and protects from ceramide-induced apoptosis, in line with the role of mitochondrial Ca2+ uptake in cancer progression. Overall, we identify amorolfine and benzethonium as effective MCU-targeting drugs applicable to a wide array of experimental and disease conditions.

New Modalities of 3D Pluripotent Stem Cell-Based Assays in Cardiovascular Toxicity.

The substantial progress of the human induced pluripotent stem cell (hiPSC) technologies over the last decade has provided us with new opportunities for cardiovascular drug discovery, regenerative medicine, and disease modeling. The combination of hiPSC with 3D culture techniques offers numerous advantages for generating and studying physiological and pathophysiological cardiac models. Cells grown in 3D can overcome many limitations of 2D cell cultures and animal models. Furthermore, it enables the investigation in an architecturally appropriate, complex cellular environment in vitro. Yet, generation and study of cardiac organoids-which may contain versatile cardiovascular cell types differentiated from hiPSC-remain a challenge. The large-scale and high-throughput applications require accurate and standardised models with highly automated processes in culturing, imaging and data collection. Besides the compound spatial structure of organoids, their biological processes also possess different temporal dynamics which require other methods and technologies to detect them. In this review, we summarise the possibilities and challenges of acquiring relevant information from 3D cardiovascular models. We focus on the opportunities during different time-scale processes in dynamic pharmacological experiments and discuss the putative steps toward one-size-fits-all assays.

Modulation of endothelial organelle size as an antithrombotic strategy.

BACKGROUND: It is long established that von Willebrand factor (VWF) is central to hemostasis and thrombosis. Endothelial VWF is stored in cell-specific secretory granules, Weibel-Palade bodies (WPBs), organelles generated in a wide range of lengths (0.5-5.0 µm). WPB size responds to physiological cues and pharmacological treatment, and VWF secretion from shortened WPBs dramatically reduces platelet and plasma VWF adhesion to an endothelial surface. OBJECTIVE: We hypothesized that WPB-shortening represented a novel target for antithrombotic therapy. Our objective was to determine whether compounds exhibiting this activity do exist. METHODS: Using a microscopy approach coupled to automated image analysis, we measured the size of WPB bodies in primary human endothelial cells treated with licensed compounds for 24 hours. RESULTS AND CONCLUSIONS: A novel approach to identification of antithrombotic compounds generated a significant number of candidates with the ability to shorten WPBs. In vitro assays of two selected compounds confirm that they inhibit the pro-hemostatic activity of secreted VWF. This set of compounds acting at a very early stage of the hemostatic process could well prove to be a useful adjunct to current antithrombotic therapeutics. Further, in the current SARS-CoV-2 pandemic, with a considerable fraction of critically ill COVID-19 patients affected by hypercoagulability, these WPB size-reducing drugs might also provide welcome therapeutic leads for frontline clinicians and researchers.

Expression of mutant exon 1 huntingtin fragments in human neural stem cells and neurons causes inclusion formation and mitochondrial dysfunction.

Robust cellular models are key in determining pathological mechanisms that lead to neurotoxicity in Huntington's disease (HD) and for high throughput pre-clinical screening of potential therapeutic compounds. Such models exist but mostly comprise non-human or non-neuronal cells that may not recapitulate the correct biochemical milieu involved in pathology. We have developed a new human neuronal cell model of HD, using neural stem cells (ReNcell VM NSCs) stably transduced to express exon 1 huntingtin (HTT) fragments with variable length polyglutamine (polyQ) tracts. Using a system with matched expression levels of exon 1 HTT fragments, we investigated the effect of increasing polyQ repeat length on HTT inclusion formation, location, neuronal survival, and mitochondrial function with a view to creating an in vitro screening platform for therapeutic screening. We found that expression of exon 1 HTT fragments with longer polyQ tracts led to the formation of intra-nuclear inclusions in a polyQ length-dependent manner during neurogenesis. There was no overt effect on neuronal viability, but defects of mitochondrial function were found in the pathogenic lines. Thus, we have a human neuronal cell model of HD that may recapitulate some of the earliest stages of HD pathogenesis, namely inclusion formation and mitochondrial dysfunction.

High-content screening image dataset and quantitative image analysis of Salmonella infected human cells.

OBJECTIVES: Salmonella bacteria can induce the unfolded protein response, a cellular stress response to misfolding proteins within the endoplasmic reticulum. Salmonella can exploit the host unfolded protein response leading to enhanced bacterial replication which was in part mediated by the induction and/or enhanced endo-reticular membrane synthesis. We therefore wanted to establish a quantitative confocal imaging assay to measure endo-reticular membrane expansion following Salmonella infections of host cells. DATA DESCRIPTION: High-content screening confocal fluorescence microscopic image set of Salmonella infected HeLa cells is presented. The images were collected with a PerkinElmer Opera LX high-content screening system in seven 96-well plates, 50 field-of-views and DAPI, endoplasmic reticulum tracker channels and Salmonella mCherry protein in each well. Totally 93,300 confocal fluorescence microscopic images were published in this dataset. An ImageJ high-content image analysis workflow was used to extract features. Cells were classified as infected and non-infected, the mean intensity of endoplasmic reticulum tracker under Salmonella bacteria was calculated. Statistical analysis was performed by an R script, quantifying infected and non-infected cells for wild-type and ΔsifA mutant cells. The dataset can be further used by researchers working with big data of endoplasmic reticulum fluorescence microscopic images, Salmonella bacterial infection images and human cancer cells.

Fibroblastic Reticular Cells Control Conduit Matrix Deposition during Lymph Node Expansion.

Lymph nodes (LNs) act as filters, constantly sampling peripheral cues. This is facilitated by the conduit network, a tubular structure of aligned extracellular matrix (ECM) fibrils ensheathed by fibroblastic reticular cells (FRCs). LNs undergo rapid 3- to 5-fold expansion during adaptive immune responses, but these ECM-rich structures are not permanently damaged. Whether conduit flow or filtering function is affected during LN expansion is unknown. Here, we show that conduits are partially disrupted during acute LN expansion, but FRC-FRC contacts remain connected. We reveal that polarized FRCs deposit ECM basolaterally using LL5-ß and that ECM production is regulated at transcriptional and secretory levels by the C-type lectin CLEC-2, expressed by dendritic cells. Inflamed LNs maintain conduit size exclusion, and flow is disrupted but persists, indicating the robustness of this structure despite rapid tissue expansion. We show how dynamic communication between peripheral tissues and LNs provides a mechanism to prevent inflammation-induced fibrosis in lymphoid tissue.

Salmonella exploits HLA-B27 and host unfolded protein responses to promote intracellular replication.

OBJECTIVE: Salmonella enterica infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the Salmonella life-cycle within epithelial cells. METHODS: Isogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S.enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane. RESULTS: S.enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism. CONCLUSIONS: HLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention.

Identification of Kinases and Phosphatases That Regulate ATG4B Activity by siRNA and Small Molecule Screening in Cells.

Autophagy protease ATG4B is a key regulator of the LC3/GABARAP conjugation system required for autophagosome formation, maturation and closure. Members of the ATG4 and the LC3/GABARAP family have been implicated in various diseases including cancer, and targeting the ATG4B protease has been suggested as a potential therapeutic anti-cancer strategy. Recently, it has been demonstrated that ATG4B is regulated by multiple post-translational modifications, including phosphorylation and de-phosphorylation. In order to identify regulators of ATG4B activity, we optimized a cell-based luciferase assay based on ATG4B-dependent release of Gaussia luciferase. We applied this assay in a proof-of-concept small molecule compound screen and identified activating compounds that increase cellular ATG4B activity. Next, we performed a high-throughput screen to identify kinases and phosphatases that regulate cellular ATG4B activity using siRNA mediated knockdown and cDNA overexpression. Of these, we provide preliminary evidence that the kinase AKT2 enhances ATG4B activity in cells. We provide all raw and processed data from the screens as a resource for further analysis. Overall, our findings provide novel insights into the regulation of ATG4B and highlight the importance of post-translational modifications of ATG4B.

A single cell high content assay detects mitochondrial dysfunction in iPSC-derived neurons with mutations in SNCA.

Mitochondrial dysfunction is implicated in many neurodegenerative diseases including Parkinson's disease (PD). Induced pluripotent stem cells (iPSCs) provide a unique cell model for studying neurological diseases. We have established a high-content assay that can simultaneously measure mitochondrial function, morphology and cell viability in iPSC-derived dopaminergic neurons. iPSCs from PD patients with mutations in SNCA and unaffected controls were differentiated into dopaminergic neurons, seeded in 384-well plates and stained with the mitochondrial membrane potential dependent dye TMRM, alongside Hoechst-33342 and Calcein-AM. Images were acquired using an automated confocal screening microscope and single cells were analysed using automated image analysis software. PD neurons displayed reduced mitochondrial membrane potential and altered mitochondrial morphology compared to control neurons. This assay demonstrates that high content screening techniques can be applied to the analysis of mitochondria in iPSC-derived neurons. This technique could form part of a drug discovery platform to test potential new therapeutics for PD and other neurodegenerative diseases.

Molecular Signatures of Regression of the Canine Transmissible Venereal Tumor.

The canine transmissible venereal tumor (CTVT) is a clonally transmissible cancer that regresses spontaneously or after treatment with vincristine, but we know little about the regression mechanisms. We performed global transcriptional, methylation, and functional pathway analyses on serial biopsies of vincristine-treated CTVTs and found that regression occurs in sequential steps; activation of the innate immune system and host epithelial tissue remodeling followed by immune infiltration of the tumor, arrest in the cell cycle, and repair of tissue damage. We identified CCL5 as a possible driver of CTVT regression. Changes in gene expression are associated with methylation changes at specific intragenic sites. Our results underscore the critical role of host innate immunity in triggering cancer regression.

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

Corrigendum: Image-based siRNA screen to identify kinases regulating Weibel-Palade body size control using electroporation.

This corrects the article DOI: 10.1038/sdata.2017.22.