Reactive or transgenic increase in microglial TYROBP reveals a TREM2-independent TYROBP-APOE link in wild-type and Alzheimer's-related mice.

INTRODUCTION: Microglial TYROBP (DAP12) is a network hub and driver in sporadic late-onset Alzheimer's disease (AD). TYROBP is a cytoplasmic adaptor for TREM2 and other receptors, but little is known about its roles and actions in AD. Herein, we demonstrate that endogenous Tyrobp transcription is specifically increased in recruited microglia. METHODS: Using a novel transgenic mouse overexpressing TYROBP in microglia, we observed a decrease of the amyloid burden and an increase of TAU phosphorylation stoichiometry when crossed with APP/PSEN1 or MAPTP301S mice, respectively. Characterization of these mice revealed Tyrobp-related modulation of apolipoprotein E (Apoe) transcription. We also showed that Tyrobp and Apoe mRNAs were increased in Trem2-null microglia recruited around either amyloid beta deposits or a cortical stab injury. Conversely, microglial Apoe transcription was dramatically diminished when Tyrobp was absent. CONCLUSIONS: Our results provide evidence that TYROBP-APOE signaling does not require TREM2 and could be an initiating step in establishment of the disease-associated microglia (DAM) phenotype.

Viral expression of a SERCA2a-activating PLB mutant improves calcium cycling and synchronicity in dilated cardiomyopathic hiPSC-CMs.

There is increasing momentum toward the development of gene therapy for heart failure (HF) that is defined by impaired calcium (Ca2+) transport and reduced contractility. We have used FRET (fluorescence resonance energy transfer) between fluorescently-tagged SERCA2a (the cardiac Ca2+ pump) and PLB (phospholamban, ventricular peptide inhibitor of SERCA) to test directly the effectiveness of loss-of-inhibition/gain-of-binding (LOI/GOB) PLB mutants (PLBM) that were engineered to compete with the binding of inhibitory wild-type PLB (PLBWT). Our therapeutic strategy is to relieve PLBWT inhibition of SERCA2a by using the reserve adrenergic capacity mediated by PLB to enhance cardiac contractility. Using a FRET assay, we determined that the combination of a LOI PLB mutation (L31A) and a GOB PLB mutation (I40A) results in a novel engineered LOI/GOB PLBM (L31A/I40A) that effectively competes with PLBWT binding to cardiac SERCA2a in HEK293-6E cells. We demonstrated that co-expression of PLBM enhances SERCA Ca-ATPase activity by increasing enzyme Ca2+ affinity (1/KCa) in PLBWT-inhibited HEK293 cell homogenates. For an initial assessment of PLBM physiological effectiveness, we used human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from a healthy individual. In this system, we observed that adeno-associated virus 2 (rAAV2)-driven expression of PLBM enhances the amplitude of SR Ca2+ release and the rate of SR Ca2+ re-uptake. To assess therapeutic potential, we used a hiPSC-CM model of dilated cardiomyopathy (DCM) containing PLB mutation R14del, where we observed that rAAV2-driven expression of PLBM rescues arrhythmic Ca2+ transients and alleviates decreased Ca2+ transport. Thus, we propose that PLBM transgene expression is a promising gene therapy strategy that directly targets the underlying pathophysiology of abnormal Ca2+ transport and thus contractility in underlying systolic heart failure.

Lipidomics and biodistribution of extracellular vesicles-secreted by hepatocytes from Zucker lean and fatty rats.

Extracellular vesicles (EVs) have been involved in metabolic syndrome, although their specific role in the development of the pathology is still unknown. To further study the role of EVs, we have analysed by Raman tweezers microspectroscopy and mass spectrometry-based lipidomics the small EVs population secreted by fatty (ZF) and lean (ZL) hepatocytes obtained from Zucker rats. We have also explored in vivo and ex vivo biodistribution of these EVs through fluorine-18-radiolabelling using a positron emission tomography imaging. Based on the proportion of proteins to lipids and the types of lipids, our results indicate that within the range of small EVs, primary hepatocytes secrete different subpopulations of particles. These differences were observed in the enrichment of triglyceride species in EVs secreted by ZF hepatocytes. Biodistribution experiments showed accumulation in the brain, heart, lungs, kidney and specially in bladder after intravenous administration. In summary, we show that EVs released by a fatty hepatocytes carry a different lipid signature compared to their lean counterpart. Biodistribution experiment has shown no difference in the distribution of EVs secreted by ZF and ZL hepatocytes but has given us a first view of possible target organs for these particles. Our results might open a door to both pathology studies and therapeutic interventions.

Unbiased identification of novel transcription factors in striatal compartmentation and striosome maturation.

Many diseases are linked to dysregulation of the striatum. Striatal function depends on neuronal compartmentation into striosomes and matrix. Striatal projection neurons are GABAergic medium spiny neurons (MSNs), subtyped by selective expression of receptors, neuropeptides, and other gene families. Neurogenesis of the striosome and matrix occurs in separate waves, but the factors regulating compartmentation and neuronal differentiation are largely unidentified. We performed RNA- and ATAC-seq on sorted striosome and matrix cells at postnatal day 3, using the Nr4a1-EGFP striosome reporter mouse. Focusing on the striosome, we validated the localization and/or role of Irx1, Foxf2, Olig2, and Stat1/2 in the developing striosome and the in vivo enhancer function of a striosome-specific open chromatin region 4.4 Kb downstream of Olig2. These data provide novel tools to dissect and manipulate the networks regulating MSN compartmentation and differentiation, including in human iPSC-derived striatal neurons for disease modeling and drug discovery.

Differences in the metabolite composition and mechanical properties of extracellular vesicles secreted by hepatic cellular models.

Liver constitutes the major metabolic factory in the organism and is involved in the synthesis, secretion and clearance of many blood-circulating molecules. Previously, we have characterised the protein and RNA cargo of extracellular vesicles (EVs) secreted by two hepatic cellular models, a mouse hepatocyte progenitor cell line (MLP29) and primary rat hepatocytes (RHs). Here, we report the metabolome profile of these vesicles by performing a targeted UHPLC-MS metabolomics analysis of these two cellular models and their respective secreted EVs. Visual inspection of the data through principal component analysis allows clear separation of the metabolic profile of cells and EVs, and also of both cellular models. Correlation matrix supported that lipid composition of EVs is mainly determined by parent cell composition. EVs derived from MLP29 and RHs showed a negative correlation in their percentage composition of ceramides, glycerophospholipids, sphingomyelins and triglycerides. Metabolites enriched in EVs were also different depending on the cellular model. EVs secreted by MLP29 were enriched in different species of sphingomyelins and ceramides underrepresented in EVs secreted by RHs. Remarkably, triglycerides constitute an important percentage of the composition of EVs derived from RHs. We further investigate if the differences in lipid composition were also accompanied by differences in mechanical behaviour, by using atomic force microscopy complemented with nanoindentation-based methodology. To compare the stiffness and brittleness of EVs derived from MLP29 cell line and RH primary cells, FZ curves were performed in the centre of single vesicles and the differences found in their force-vs.-indentation curves suggest that RHs EVs are softer (less stiff) and less resistance to mechanical failure than MLP29 EVs. Therefore, we can conclude that EVs from different origin carry a characteristic lipid composition related to their parental cell composition, and exhibit different mechanical properties. Abbreviations: For the identification of the different species of lipids, the following abbreviations has been employed: Cer, ceramide; ChoE, Cholesteryl Ester; CMH, monohexosylceramide; DAG, diglycerid; LPC, lysophosphatidylcholin; LPI, lysophosphatidyinositol; PC, phosphocoline; PE, phoethanolamine; PI, phosphoinositol; SM, sphingomyelin; TAG, triglycerid.

Extracellular Vesicles from Hypoxic Adipocytes and Obese Subjects Reduce Insulin-Stimulated Glucose Uptake.

SCOPE: We investigate the effects of extracellular vesicles (EVs) obtained from in vitro adipocyte cell models and from obese subjects on glucose transport and insulin responsiveness. METHODS AND RESULTS: EVs are isolated from the culture supernatant of adipocytes cultured under normoxia, hypoxia (1% oxygen), or exposed to macrophage conditioned media (15% v/v). EVs are isolated from the plasma of lean individuals and subjects with obesity. Cultured adipocytes are incubated with EVs and activation of insulin signalling cascades and insulin-stimulated glucose transport are measured. EVs released from hypoxic adipocytes impair insulin-stimulated 2-deoxyglucose uptake and reduce insulin mediated phosphorylation of AKT. Insulin-mediated phosphorylation of extracellular regulated kinases (ERK1/2) is not affected. EVs from individuals with obesity decrease insulin stimulated 2-deoxyglucose uptake in adipocytes (p = 0.0159). CONCLUSION: EVs released by stressed adipocytes impair insulin action in neighboring adipocytes.

Abundance of Cytochromes in Hepatic Extracellular Vesicles Is Altered by Drugs Related With Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a serious worldwide health problem that accounts for more than 50% of acute liver failure. There is a great interest in clinical diagnosis and pharmaceutical industry to elucidate underlying molecular mechanisms and find noninvasive biomarkers for this pathology. Cell-secreted extracellular vesicles (EVs) have provided a new biological source to identify low disease invasive markers. Despite the intense research developed on these vesicles, there is currently a gap on their patho-physiological effects. Here, we study EVs secreted by primary rat hepatocytes challenged with galactatosamine (GalN), acetaminophen, or diclofenac as DILI in vitromodels. Proteomics analysis of these EVs revealed an increase in enzymes already associated with liver damage, such as catecholamine-methyl transferase and arginase 1. An increase in translation-related proteins and a decrease in regulators of apoptosis were also observed. In addition, we show the presence of enzymatic activity of P450 cytochrome 2d1 in EVs. The activity specifically is decreased in EVs secreted by hepatocytes after acetaminophen treatment and increased in EVs derived from GalN-treated hepatocytes. By using in vivo preclinical models, we demonstrate the presence of this cytochrome activity in circulation under normal conditions and an increased activity after GalN-induced injury. Conclusion: Hepatocyte-secreted EVs carry active xenobiotic-metabolizing enzymes that might be relevant in extracellular metabolism of drugs and be associated with DILI. (Hepatology Communications 2018;0:00-00).

Transcriptome-wide isoform-level dysregulation in ASD, schizophrenia, and bipolar disorder.

Most genetic risk for psychiatric disease lies in regulatory regions, implicating pathogenic dysregulation of gene expression and splicing. However, comprehensive assessments of transcriptomic organization in diseased brains are limited. In this work, we integrated genotypes and RNA sequencing in brain samples from 1695 individuals with autism spectrum disorder (ASD), schizophrenia, and bipolar disorder, as well as controls. More than 25% of the transcriptome exhibits differential splicing or expression, with isoform-level changes capturing the largest disease effects and genetic enrichments. Coexpression networks isolate disease-specific neuronal alterations, as well as microglial, astrocyte, and interferon-response modules defining previously unidentified neural-immune mechanisms. We integrated genetic and genomic data to perform a transcriptome-wide association study, prioritizing disease loci likely mediated by cis effects on brain expression. This transcriptome-wide characterization of the molecular pathology across three major psychiatric disorders provides a comprehensive resource for mechanistic insight and therapeutic development.

Metabolically active extracellular vesicles released from hepatocytes under drug-induced liver-damaging conditions modify serum metabolome and might affect different pathophysiological processes.

Hepatocytes are involved in the endogenous and drug metabolism; many of the enzymes involved in those processes are incorporated into extracellular vesicles and secreted into the bloodstream. Liver-damaging conditions modify the molecular cargo of those vesicles significantly. However, no information about the effect of these hepatic vesicles on the extracellular environment is available. Drug-induced liver damage increases the number of circulating extracellular vesicles and affects the release and content of hepatocyte-derived vesicles. In this work, we evaluated the metabolic effect of these vesicles on the composition of the serum. We performed a targeted ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics analysis of serum samples. The samples had been first incubated with hepatic extracellular vesicles from hepatocytes challenged with acetaminophen or diclofenac. The incubation affected the serum levels of 67 metabolites, such as amino acids and different species of lipids. The metabolites included various species of phosphatidylcholines and phosphatidylethanolamines. These compounds are the components of biological membranes; our observations suggest that the vesicles might take part in remodelling and maintenance of the membranes. Alterations in the levels of some other serum metabolites might have deleterious consequences, for example, the tetracosanoic acid with its cardiovascular effects. However, some of the metabolites whose levels were increased, including alpha-linoleic and tauroursodeoxycholic acids, have been reported to have a protective effect. Our targeted metabolomics analysis indicated that the hepatic extracellular vesicles act as nano-metabolic machines supplying the extracellular environment with the means to integrate diverse tissue responses. In conclusion, we show that the hepatic extracellular vesicles are metabolically active and might play a role in the physiopathological response to hepatic insults, including drug-induced liver injury.

Hepatocyte-secreted extracellular vesicles modify blood metabolome and endothelial function by an arginase-dependent mechanism.

Hepatocytes release extracellular vesicles (EVs) loaded with signaling molecules and enzymes into the bloodstream. Although the importance of EVs in the intercellular communication is already recognized, the metabolic impact of the enzymes carried by these vesicles is still unclear. We evaluated the global effect of the enzymatic activities of EVs by performing untargeted metabolomic profiling of serum samples after their exposure to EVs. This approach revealed a significant change in the abundance of 94 serum metabolic signals. Our study shows that these vesicles modify the concentration of metabolites of different chemical nature including metabolites related to arginine metabolism, which regulates vascular function. To assess the functional relevance of this finding, we examined the levels of arginase-1 protein and its activity in the hepatic EVs carrying the exosomal markers CD81 and CD63. Remarkably, the arginase activity was also detected in EVs isolated from the serum in vivo, and this vesicular activity significantly increased under liver-damaging conditions. Finally, we demonstrated that EVs secreted by hepatocytes inhibited the acetylcholine-induced relaxation in isolated pulmonary arteries, via an arginase-dependent mechanism. In summary, our study demonstrates that the hepatocyte-released EVs are metabolically active, affecting a number of serum metabolites involved in oxidative stress metabolism and the endothelial function.

Identification of the metabolic alterations associated with the multidrug resistant phenotype in cancer and their intercellular transfer mediated by extracellular vesicles.

Multidrug resistance (MDR) is a serious obstacle to efficient cancer treatment. Overexpression of P-glycoprotein (P-gp) plays a significant role in MDR. Recent studies proved that targeting cellular metabolism could sensitize MDR cells. In addition, metabolic alterations could affect the extracellular vesicles (EVs) cargo and release. This study aimed to: i) identify metabolic alterations in P-gp overexpressing cells that could be involved in the development of MDR and, ii) identify a potential role for the EVs in the acquisition of the MDR. Two different pairs of MDR and their drug-sensitive counterpart cancer cell lines were used. Our results showed that MDR (P-gp overexpressing) cells have a different metabolic profile from their drug-sensitive counterparts, demonstrating decreases in the pentose phosphate pathway and oxidative phosphorylation rate; increases in glutathione metabolism and glycolysis; and alterations in the methionine/S-adenosylmethionine pathway. Remarkably, EVs from MDR cells were capable of stimulating a metabolic switch in the drug-sensitive cancer cells, towards a MDR phenotype. In conclusion, obtained results contribute to the growing knowledge about metabolic alterations in MDR cells and the role of EVs in the intercellular transfer of MDR. The specific metabolic alterations identified in this study may be further developed as targets for overcoming MDR.