Lipid metabolism of leukocytes in the unstimulated and activated states.

Lipidomics has emerged as a powerful technique to study cellular lipid metabolism. As the lipidome contains numerous isomeric and isobaric species resulting in a significant overlap between different lipid classes, cutting-edge analytical technology is necessary for a comprehensive analysis of lipid metabolism. Just recently, differential mobility spectrometry (DMS) has evolved as such a technology, helping to overcome several analytical challenges. We here set out to apply DMS and the Lipidyzer™ platform to obtain a comprehensive overview of leukocyte-related lipid metabolism in the resting and activated states. First, we tested the linearity and repeatability of the platform by using HL60 cells. We obtained good linearities for most of the thirteen analyzed lipid classes (correlation coefficient > 0.95), and good repeatability (%CV < 15). By comparing the lipidome of neutrophils (PMNs), monocytes (CD14+), and lymphocytes (CD4+), we shed light on leukocyte-specific lipid patterns as well as lipidomic changes occurring through differential stimulation. For example, at the resting state, PMNs proved to contain higher amounts of triacylglycerides compared to CD4+ and CD14+ cells. On the other hand, CD4+ and CD14+ cells contained higher levels of phospholipids and ceramides. Upon stimulation, diacylglycerides, hexosylceramides, phosphatidylcholines, phosphoethanolamines, and lysophosphoethanolamines were upregulated in CD4+ cells and PMNs, whereas CD14+ cells did not show significant changes. By exploring the fatty acid content of the significantly upregulated lipid classes, we mainly found increased concentrations of very long and polyunsaturated fatty acids. Our results indicate the usefulness of the Lipidyzer™ platform for studying cellular lipid metabolism. Its application allowed us to explore the lipidome of leukocytes. Graphical abstract.

Cholesterol Metabolism Is a Druggable Axis that Independently Regulates Tau and Amyloid-ß in iPSC-Derived Alzheimer's Disease Neurons.

Genetic, epidemiologic, and biochemical evidence suggests that predisposition to Alzheimer's disease (AD) may arise from altered cholesterol metabolism, although the molecular pathways that may link cholesterol to AD phenotypes are only partially understood. Here, we perform a phenotypic screen for pTau accumulation in AD-patient iPSC-derived neurons and identify cholesteryl esters (CE), the storage product of excess cholesterol, as upstream regulators of Tau early during AD development. Using isogenic induced pluripotent stem cell (iPSC) lines carrying mutations in the cholesterol-binding domain of APP or APP null alleles, we found that while CE also regulate Aß secretion, the effects of CE on Tau and Aß are mediated by independent pathways. Efficacy and toxicity screening in iPSC-derived astrocytes and neurons showed that allosteric activation of CYP46A1 lowers CE specifically in neurons and is well tolerated by astrocytes. These data reveal that CE independently regulate Tau and Aß and identify a druggable CYP46A1-CE-Tau axis in AD.

Zebrafish Larvae Are a Suitable Model to Investigate the Metabolic Phenotype of Drug-Induced Renal Tubular Injury.

Prevention and treatment of drug-induced renal injury (DIRI) rely on the availability of sensitive and specific biomarkers of early kidney injury and predictive animal models of human pathophysiology. This study aimed to evaluate the potential of zebrafish larvae as translational model in metabolic profiling of DIRI. Zebrafish larvae were exposed to the lethal concentration for 10% of the larvae (LC10) or ½ LC10 of gentamicin, paracetamol and tenofovir as tenofovir disoproxil fumarate (TDF) and tenofovir (TFV). Metabolites were extracted from whole larvae and analyzed by liquid chromatography-mass spectrometry. Principal component analysis showed that drug exposition to the LC10 of paracetamol, TFV, and TDF was the main source of the variance of the data. To identify the metabolites responsible for the toxic effects of the drugs, partial least squares discriminant analyses were built between the LC10 and ½ LC10 for each drug. Features with variable importance in projection> 1.0 were selected and Venn diagrams were built to differentiate between the common and drug specific metabolites of DIRI. Creatine, tyrosine, glutamine, guanosine, hypoxanthine were identified as common metabolites, adenosine and tryptophan as paracetamol-specific and xanthine and oxidized glutathione as tenofovir-specific. Those metabolic changes can be associated with alterations in energy metabolism, xenobiotic detoxification and protein catabolism, all described in the human pathophysiology of DIRI. Thus, zebrafish proved to be a suitable model to characterize the metabolic changes associated with DIRI. This information can be useful to early diagnose DIRI and to improve our knowledge on the mechanisms of DIRI.

The Schistosoma mansoni lipidome: Leads for immunomodulation.

Schistosoma mansoni is a parasitic helminth that infects millions of people mostly in tropical parts of the world. Different life cycle stages of S.mansoni, that infect or develop in the human host, promote distinct immune responses and are known for their ability to modulate host immune responses. However, the molecular mechanisms through which the parasites interact with, and modulate the host immune system remain incompletely understood. Despite the well-known ability of various lipids to modulate immune responses, a comprehensive analysis of the lipidome of the different life cycle stages has not been performed. Using three complementary MS-based platforms to detect and quantify around 350 lipid species, we here characterized the lipid profiles of S. mansoni cercariae, worms and eggs, as well as extracts and excretory/secretory (ES) products of different life cycle stages of S. mansoni. We identified life cycle stage specific signatures of lipid classes of which cercariae were found to have the most distinct profile. Moreover, we detected several immunolomodulatory oxylipids in the different life cycle stages. Specifically, prostaglandins were found to be most highly enriched in egg preparations, while resolvins were specifically detected in cercariae. Together, the generation of this detailed lipid database of the different life cycle stages of S. mansoni will not only be important for a better understanding of the biology of the parasite itself but also of host-parasite interactions and how that could result in immunomodulation.

Usefulness of zebrafish larvae to evaluate drug-induced functional and morphological renal tubular alterations.

Prediction and management of drug-induced renal injury (DIRI) rely on the knowledge of the mechanisms of drug insult and on the availability of appropriate animal models to explore it. Zebrafish (Danio rerio) offers unique advantages for assessing DIRI because the larval pronephric kidney has a high homology with its human counterpart and it is fully mature at 3.5 days post-fertilization. Herein, we aimed to evaluate the usefulness of zebrafish larvae as a model of renal tubular toxicity through a comprehensive analysis of the renal alterations induced by the lethal concentrations for 10% of the larvae for gentamicin, paracetamol and tenofovir. We evaluated drug metabolic profile by mass spectrometry, renal function with the inulin clearance assay, the 3D morphology of the proximal convoluted tubule by two-photon microscopy and the ultrastructure of proximal convoluted tubule mitochondria by transmission electron microscopy. Paracetamol was metabolized by conjugation and oxidation with further detoxification with glutathione. Renal clearance was reduced with gentamicin and paracetamol. Proximal tubules were enlarged with paracetamol and tenofovir. All drugs induced mitochondrial alterations including dysmorphic shapes ("donuts", "pancakes" and "rods"), mitochondrial swelling, cristae disruption and/or loss of matrix granules. These results are in agreement with the tubular effects of gentamicin, paracetamol and tenofovir in man and demonstrate that zebrafish larvae might be a good model to assess functional and structural damage associated with DIRI.

2-picoline-borane: a non-toxic reducing agent for oligosaccharide labeling by reductive amination.

Analysis of N-glycans is often performed by LC coupled to fluorescence detection. The N-glycans are usually labeled by reductive amination with a fluorophore containing a primary amine to allow fluorescence detection. Moreover, many of the commonly applied labels also allow improved mass spectrometric detection of oligosaccharides. For reductive amination, the amine group of the label reacts with the reducing-end aldehyde group of the oligosaccharide to form a Schiff base, which is reduced to a secondary amine. Here, we propose the use of 2-picoline-borane as the reducing agent, as a non-toxic alternative to the extensively used, but toxic sodium cyanoborohydride. Using dextran oligosaccharides and plasma N-glycans, we demonstrate similar labeling efficacies for 2-picoline-borane and sodium cyanoborohydride. Therefore, 2-picoline-borane is a non-toxic alternative to sodium cyanoborohydride for the labeling of oligosaccharides.