Characterization of an influenza virus pseudotyped with Ebolavirus glycoprotein

We have produced a new Ebola virus pseudotype: E-S-FLU, which can be handled in biosafety level-1/2 containment for laboratory analysis. E-S-FLU is a single cycle influenza virus coated with Ebolavirus glycoprotein, and it encodes enhanced green fluorescence protein as a reporter that replaces the influenza haemagglutinin. MDCK-SIAT1 cells were transduced to express Ebolavirus glycoprotein as a stable transmembrane protein for E-S-FLU production. Infection of cells by E-S-FLU was dependent on Niemann-Pick C1 protein, which is the well-characterized receptor for Ebola virus entry at the late endosome/lysosome membrane. E-S-FLU was neutralized specifically by anti-Ebola glycoprotein antibody and a variety of small drug molecules that are known to inhibit entry of wild-type Ebola virus. To demonstrate the application of this new Ebola virus pseudotype, we show that a single laboratory batch was sufficient to screen a library (LOPAC®1280 Sigma) of 1280 pharmacologically active compounds for inhibition of virus entry. 215 compounds inhibited E-S-FLU infection, while only 22 inhibited the control H5-S-FLU virus coated in an H5 haemagglutinin. These inhibitory compounds have very dispersed targets and mechanisms of action e.g. calcium channel blockers, estrogen receptor antagonists, anti-histamines, serotonin uptake inhibitors etc. and this correlates with inhibitor screening results with other pseudotypes or wild-type Ebola virus in the literature. E-S-FLU is a new tool for Ebola virus cell entry studies and is easily applied to high throughput screening assays for small molecule inhibitors or antibodies.Importance Ebola virus is from the Filoviridae family and is a biosafety level 4 pathogen. There are no FDA-approved therapeutics for Ebola virus. These characteristics warrant the development of surrogates of Ebola virus that can be handled in more convenient laboratory containment to study the biology of the virus, and screen for inhibitors. Here we characterized a new surrogate named E-S-FLU, that is based on a disabled influenza virus core coated with the Ebola virus surface protein, but does not contain any genetic information from the Ebola virus itself. We show that E-S-FLU uses the same cell entry pathway as wild-type Ebola virus. As an example of the ease of use of E-S-FLU in biosafety level-1/2 containment, we showed that a single production batch could provide enough surrogate virus to screen a standard small molecule library of 1280 candidates for inhibitors of viral entry

A novel strategy for the comprehensive analysis of the biomolecular composition of isolated plasma membranes

A methodology for rapid, high-purity isolation of plasma membranes using superparamagnetic nanoparticles is described. The method is illustrated with high-resolution proteomic, glycomic and lipidomic analyses of presenilin-deficient cells

A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics : the case of NPC1 deficiency

Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions

CRISPR/Cas9 screen in human iPSC‐derived cortical neurons identifies NEK6 as a novel disease modifier of C9orf72 poly(PR) toxicity

Introduction The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. Methods We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. Results Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. Discussion We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS

Superparamagnetic nanoparticles based isolation of subcellular compartments as a method to identify spatial alterations in protein and lipid composition

Summary The more than ~25,000 human genes give rise to many more proteins, a.o. by alternative splicing. Genome-wide approaches using database annotations attempt to reveal patterns of protein interactions, however generally ignore the protein locations. Mass spectrometry handles a huge number of proteins ranging widely in abundance, yet its resolution can be enhanced by analyzing purified organellar fractions. This proposal aims to generate proteome inventories of plasma membranes (PM) and endosomes in a disease-related context. Classical purification schemes cannot be used as disease-related mutations often alter the physical parameters used for organellar isolation. This caveat is circumvented by administering surface-coated superparamagnetic iron oxide nanoparticles (SPIONs). We have developed lipid-coated SPIONs which target the cell surface and allow purification of PM. By exploring the PM proteome we will search for stage-specific or unique expression of channels, receptors or adhesion molecules that may become novel targets or early biomarkers in disease. SPION variants will further be optimized to load endosomes. Coupling biomolecules (ligands, toxins, drugs) combined with pulse-chase experiments will allow isolating distinct endosome populations. Many diseases including neurodegenerative and metabolic diseases originate from a block in endosomal trafficking and degradation although the causal genes are not always identified. By comparing the endosomal proteomes in patient cell lines with control, we aim to identify aberrant protein expression patterns from which causal genes and/or novel biomarkers could be identified. Production and QC of SPIONs is done at IMEC and VIB11. Mass spectrometry driven proteomics to catalogue organellar proteomes is performed at VIB9.nrpages: 203status: publishe

Organellar OmicsA Reviving Strategy to Untangle the Biomolecular Complexity of the Cell

A eukaryotic cell encompasses many membrane-enclosed organelles, each of these holding several types of biomolecules that exhibit tremendous diversity in terms of their localization and expression. Despite the development of increasingly sensitive analytical tools, the enormous biomolecular complexity that exists within a cell cannot yet be fully resolved as low abundant molecules often remain unrecognized. Moreover, a drawback of whole cell analysis is that it does not provide spatial information and therefore it is not capable of assigning distinct biomolecules to specific compartments or analyzing changes in the composition of these compartments. Reduction of the biomolecular complexity of a sample helps to identify low abundant molecules, but such a reductionist approach requires methods that enable proper isolation and purification of individual cellular organelles. Decades of research have led to the development of a plethora of isolation methods for a broad range of subcellular organelles; yet, in particular, intrinsically dynamic compartments belonging to the endocytic machinery, including the plasma membrane, remain difficult to isolate in a sufficiently pure fraction. In this review, we discuss various methods that are commonly used to isolate subcellular organelles from cells and evaluate their advantages and disadvantages.status: publishe

Organellar omics : a reviving strategy to untangle the biomolecular complexity of the cell

A eukaryotic cell encompasses many membrane-enclosed organelles, each of these holding several types of biomolecules that exhibit tremendous diversity in terms of their localization and expression. Despite the development of increasingly sensitive analytical tools, the enormous biomolecular complexity that exists within a cell cannot yet be fully resolved as low abundant molecules often remain unrecognized. Moreover, a drawback of whole cell analysis is that it does not provide spatial information and therefore it is not capable of assigning distinct biomolecules to specific compartments or analyzing changes in the composition of these compartments. Reduction of the biomolecular complexity of a sample helps to identify low abundant molecules, but such a reductionist approach requires methods that enable proper isolation and purification of individual cellular organelles. Decades of research have led to the development of a plethora of isolation methods for a broad range of subcellular organelles; yet, in particular, intrinsically dynamic compartments belonging to the endocytic machinery, including the plasma membrane, remain difficult to isolate in a sufficiently pure fraction. In this review, we discuss various methods that are commonly used to isolate subcellular organelles from cells and evaluate their advantages and disadvantages

A toxic gain-of-function mechanism in C9orf72 ALS impairs the autophagy-lysosome pathway in neurons

Abstract Background Motor neurons (MNs), which are primarily affected in amyotrophic lateral sclerosis (ALS), are a specialized type of neurons that are long and non-dividing. Given their unique structure, these cells heavily rely on transport of organelles along their axons and the process of autophagy to maintain their cellular homeostasis. It has been shown that disruption of the autophagy pathway is sufficient to cause progressive neurodegeneration and defects in autophagy have been associated with various subtypes of ALS, including those caused by hexanucleotide repeat expansions in the C9orf72 gene. A more comprehensive understanding of the dysfunctional cellular mechanisms will help rationalize the design of potent and selective therapies for C9orf72-ALS. Methods In this study, we used induced pluripotent stem cell (iPSC)-derived MNs from C9orf72-ALS patients and isogenic control lines to identify the underlying mechanisms causing dysregulations of the autophagy-lysosome pathway. Additionally, to ascertain the potential impact of C9orf72 loss-of-function on autophagic defects, we characterized the observed phenotypes in a C9orf72 knockout iPSC line (C9-KO). Results Despite the evident presence of dysfunctions in several aspects of the autophagy-lysosome pathway, such as disrupted lysosomal homeostasis, abnormal lysosome morphology, inhibition of autophagic flux, and accumulation of p62 in C9orf72-ALS MNs, we were surprised to find that C9orf72 loss-of-function had minimal influence on these phenotypes. Instead, we primarily observed impairment in endosome maturation as a result of C9orf72 loss-of-function. Additionally, our study shed light on the pathological mechanisms underlying C9orf72-ALS, as we detected an increased TBK1 phosphorylation at S172 in MNs derived from C9orf72 ALS patients. Conclusions Our data provides further insight into the involvement of defects in the autophagy-lysosome pathway in C9orf72-ALS and strongly indicate that those defects are mainly due to the toxic gain-of-function mechanisms underlying C9orf72-ALS