Long-range projection neurons of the mouse ventral tegmental area: A single-cell axon tracing analysis

Pathways arising from the ventral tegmental area (VTA) release dopamine and other neurotransmitters during the expectation and achievement of reward, and are regarded as central links of the brain networks that create drive, pleasure, and addiction. While the global pattern of VTA projections is well-known, the actual axonal wiring of individual VTA neurons had never been investigated. Here, we labeled and analyzed the axons of 30 VTA single neurons by means of single-cell transfection with the Sindbis-pal-eGFP vector in mice. These observations were complemented with those obtained by labeling the axons of small populations of VTA cells with iontophoretic microdeposits of biotinylated dextran amine. In the single-cell labeling experiments, each entire axonal tree was reconstructed from serial sections, the length of terminal axonal arbors was estimated by stereology, and the dopaminergic phenotype was tested by double-labeling for tyrosine hydroxylase immunofluorescence. We observed two main, markedly different VTA cell morphologies: neurons with a single main axon targeting only forebrain structures (FPN cells), and neurons with multibranched axons targeting both the forebrain and the brainstem (F + BSPN cells). Dopaminergic phenotype was observed in FPN cells. Moreover, four “subtypes” could be distinguished among the FPN cells based on their projection targets: (1) “Mesocorticolimbic” FPN projecting to both neocortex and basal forebrain; (2) “Mesocortical” FPN innervating the neocortex almost exclusively; (3) “Mesolimbic” FPN projecting to the basal forebrain, accumbens and caudateputamen; and (4) “Mesostriatal” FPN targeting only the caudateputamen. While the F + BSPN cells were scattered within VTA, the mesolimbic neurons were abundant in the paranigral nucleus. The observed diversity in wiring architectures is consistent with the notion that different VTA cell subpopulations modulate the activity of specific sets of prosencephalic and brainstem structuresThe project was supported by Grants from the Fundación Eugenio Rodríguez Pascual, the Spanish MINECO (BFU2010-19695) and the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 604102 (Human Brain Project

Regulation of the transcriptional program by DNA methylation during human αβ T-cell development

© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. Thymocyte differentiation is a complex process involving well-defined sequential developmental stages that ultimately result in the generation of mature T-cells. In this study, we analyzed DNA methylation and gene expression profiles at successive human thymus developmental stages. Gain and loss of methylation occurred during thymocyte differentiation, but DNA demethylation was much more frequent than de novo methylation and more strongly correlated with gene expression. These changes took place in CpG-poor regions and were closely associated with T-cell differentiation and TCR function. Up to 88 genes that encode transcriptional regulators, some of whose functions in T-cell development are as yet unknown, were differentially methylated during differentiation. Interestingly, no reversion of accumulated DNA methylation changes was observed as differentiation progressed, except in a very small subset of key genes (RAG1, RAG2, CD8A, PTCRA, etc.), indicating that methylation changes are mostly unique and irreversible events. Our study explores the contribution of DNA methylation to T-cell lymphopoiesis and provides a fine-scale map of differentially methylated regions associated with gene expression changes. These can lay the molecular foundations for a better interpretation of the regulatory networks driving human thymopoiesis.Plan Nacional de [I+D+I 2008–2011]; Instituto de Salud Carlos III [grant number PI12/02587]; Red Española de Investigación Renal (REDinREN) [grant number RD12/0021/0018 and RD12/0021/0021]; Spanish Ministry of Science and Innovation [grant number SAF2010- 15106 and PLE2009-0110]; European Union [Fondos FEDER]Peer Reviewe

Genetic association study of dyslexia and ADHD candidate genes in a Spanish cohort: Implications of comorbid samples

Published: October 31, 2018Dyslexia and attention deficit hyperactivity disorder (ADHD) are two complex neuro-behaviorally disorders that co-occur more often than expected, so that reading disability has been linked to inattention symptoms. We examined 4 SNPs located on genes previously associated to dyslexia (KIAA0319, DCDC2, DYX1C1 and FOXP2) and 3 SNPs within genes related to ADHD (COMT, MAOA and DBH) in a cohort of Spanish children (N = 2078) that met the criteria of having one, both or none of these disorders (dyslexia and ADHD). We used a case-control approach comparing different groups of samples based on each individual diagnosis. In addition, we also performed a quantitative trait analysis with psychometric measures on the general population (N = 3357). The results indicated that the significance values for some markers change depending on the phenotypic groups compared and/or when considering pair-wise marker interactions. Furthermore, our quantitative trait study showed significant genetic associations with specific cognitive processes. These outcomes advocate the importance of establishing rigorous and homogeneous criteria for the diagnosis of cognitive disorders, as well as the relevance of considering cognitive endophenotypes.The work of MSM and MC was supported by CONSOLIDER-Ingenio- 2010_COEDUCA (CSD2008-00048). AMA, LB and AG-L’s work was supported by the Basque Department of Industry, Tourism and Trade (Etortek Program), Innovation Technology Department of Bizkaia and CIBERehd Network. MC was also supported by grants (PSI2015-67353-R), and Ayuda Centro de Excelencia Severo Ochoa SEV-2015-0490 from the MINECO, and by grant (ERC-2011-ADG-295362) from the European Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Identification of proximal SUMO-dependent interactors using SUMO-ID

[EN]The fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We develop an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allow us to identify interactors of SUMOylated SALL1, a less characterized SUMO substrate. Furthermore, using TP53 as a substrate, we identify SUMO1, SUMO2 and Ubiquitin preferential interactors. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code.We are thankful to Iraide Escobes for her work in the Proteomics Platform at CIC bioGUNE and Arnoud de Ru in the Center for Proteomics and Metabolomics at the LUMC. O.B.-G., F.T., R.B. and A.C.O.V. acknowledge funding by the grant 765445-EU (UbiCODE Program). R.B. acknowledges funding by grants BFU2017-84653-P and PID2020-114178GB-I00 (MINECO/FEDER, EU), SEV-2016-0644 (Severo Ochoa Excellence Program), SAF2017-90900-REDT (UBIRed Program) and IT1165-19 (Basque Country Government). Additional support was provided by the Department of Industry, Tourism, and Trade of the Basque Country Government (Elkartek Research Programs) and by the Innovation Technology Department of the Bizkaia County. VM acknowledges FPI grant PRE2018-086230 (MINECO/FEDER, EU). F.E. is at Proteomics Platform, member of ProteoRed-ISCIII (PT13/0001/0027). F.E. and A.M.A. acknowledge CIBERehd. A.C. acknowledges the Basque Department of education (IKERTALDE IT1106-16), the MCIU (PID2019-108787RB-I00 (FEDER/EU), Severo Ochoa Excellence Accreditation SEV-2016-0644, Excellence Networks RED2018-102769-T), the AECC (GCTRA18006CARR), La Caixa Foundation (ID 100010434), under the agreement LCF/PR/HR17, and the European Research Council (Starting Grant 336343, PoC 754627, Consolidator grant 819242). CIBERONC was co-funded with FEDER funds. U.M. acknowledges the Basque Government Department of Education (IT1165-19) and the Spanish MCIU (SAF2016-76898-P (FEDER/EU))

Genetic Diversity of Toscana Virus

Distribution of Toscana virus (TOSV) is evolving with climate change, and pathogenicity may be higher in nonexposed populations outside areas of current prevalence (Mediterranean Basin). To characterize genetic diversity of TOSV, we determined the coding sequences of isolates from Spain and France. TOSV is more diverse than other well-studied phleboviruses (e.g.,Rift Valley fever virus)

A Comprehensive Study of Vesicular and Non-Vesicular miRNAs from a Volume of Cerebrospinal Fluid Compatible with Clinical Practice

Cerebrospinal fluid (CSF) microRNAs (miRNAs) have emerged as potential biomarkers for minimally invasive diagnosis of central nervous system malignancies. However, despite significant advances in recent years, this field still suffers from poor data reproducibility. This is especially true in cases of infants, considered a new subject group. Implementing efficient methods to study miRNAs from clinically realistic CSF volumes is necessary for the identification of new biomarkers. Methods: We compared six protocols for characterizing miRNAs, using 200-mu L CSF from infants (aged 0-7). Four of the methods employed extracellular vesicle (EV) enrichment step and the other two obtained the miRNAs directly from cleared CSF. The efficiency of each method was assessed using real-time PCR and small RNA sequencing. We also determined the distribution of miRNAs among different CSF shuttles, using size-exclusion chromatography. Results: We identified 281 CSF miRNAs from infants. We demonstrated that the miRNAs could be efficiently detected using only 200 mu L of biofluid in case of at least two of the six methods. In the exosomal fraction, we found 12 miRNAs that might be involved in neurodevelopment. Conclusion: The Norgen and Invitrogen protocols appear suitable for the analysis of a large number of miRNAs using small CSF samples.This work was supported by the Basque Government [IT989-16], the Spanish Ministry of Economy and Competitiveness MINECO [SAF2015066312], and the Ramon Areces Foundation [FRA-17-JMF]. We thank MINECO for the REDIEX (Spanish Excellence Network in Exosomes) and the Severo Ochoa Excellence Accreditation (SEV-2016-0644). Funding for open access charge: Severo Ochoa Excellence Accreditation (SEV-2016-0644)

microRNA-based signatures obtained from endometrial fluid identify implantative endometrium

STUDY QUESTION Is it possible to use free and extracellular vesicle-associated microRNAs (miRNAs) from human endometrial fluid (EF) samples as non-invasive biomarkers for implantative endometrium? SUMMARY ANSWER The free and extracellular vesicle-associated miRNAs can be used to detect implantative endometrium in a non-invasive manner. WHAT IS KNOWN ALREADY miRNAs and extracellular vesicles (EVs) from EF have been described as mediators of the embryo-endometrium crosstalk. Therefore, the analysis of miRNA from this fluid could become a non-invasive technique for recognizing implantative endometrium. This analysis could potentially help improve the implantation rates in ART. STUDY DESIGN, SIZE, DURATION In this prospective study, we first optimized different protocols for EVs and miRNA analyses using the EF of a setup cohort (n = 72). Then, we examined differentially expressed miRNAs in the EF of women with successful embryo implantation (discovery cohort n = 15/validation cohort n = 30) in comparison with those for whom the implantation had failed (discovery cohort n = 15/validation cohort n = 30). Successful embryo implantation was considered when pregnancy was confirmed by vaginal ultrasound showing a gestational sac 4 weeks after embryo transfer (ET). PARTICIPANTS/MATERIALS, SETTING, METHODS The EF of the setup cohort was obtained before starting fertility treatment during the natural cycle, 16-21 days after the beginning of menstruation. For the discovery and validation cohorts, the EF was collected from women undergoing frozen ET on Day 5, and the samples were collected immediately before ET. In this study, we compared five different methods; two of them based on direct extraction of RNA and the other three with an EV enrichment step before the RNA extraction. Small RNA sequencing was performed to determine the most efficient method and find a predictive model differentiating between implantative and non-implantative endometrium. The models were confirmed using quantitative PCR in two sets of samples (discovery and validation cohorts) with different implantation outcomes. MAIN RESULTS AND THE ROLE OF CHANCE The protocols using EV enrichment detected more miRNAs than the methods based on direct RNA extraction. The two most efficient protocols (using polymer-based precipitation (PBP): PBP-M and PBP-N) were used to obtain two predictive models (based on three miRNAs) allowing us to distinguish between an implantative and non-implantative endometrium. The first Model 1 (PBP-M) (discovery: AUC = 0.93; P-value = 0.003; validation: AUC = 0.69; P-value = 0.019) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-148b-3p. Model 2 (PBP-N) (discovery: AUC = 0.92; P-value = 0.0002; validation: AUC = 0.78; P-value = 0.0002) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-99b-5p. Functional analysis of these miRNAs showed strong association with key implantation processes such as in utero embryonic development or transforming growth factor-beta signaling. LARGE SCALE DATA The FASTQ data are available in the GEO database (access number GSE178917). LIMITATIONS, REASONS FOR CAUTION One important factor to consider is the inherent variability among the women involved in the trial and among the transferred embryos. The embryos were pre-selected based on morphology, but neither genetic nor molecular studies were conducted, which would have improved the accuracy of our tests. In addition, a limitation in miRNA library construction is the low amount of input RNA. WIDER IMPLICATIONS OF THE FINDINGS We describe new non-invasive protocols to analyze miRNAs from small volumes of EF. These protocols could be implemented in clinical practice to assess the status of the endometrium before attempting ET. Such evaluation could help to avoid the loss of embryos transferred to a non-implantative endometrium. STUDY FUNDING/COMPETING INTEREST(S) J.I.-P. was supported by a predoctoral grant from the Basque Government (PRE_2017_0204). This study was partially funded by the Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). It was also supported by the Spanish Ministry of Economy and Competitiveness MINECO within the National Plan RTI2018-094969-B-I00, the European Union's Horizon 2020 research and innovation program (860303), the Severo Ochoa Centre of Excellence Innovative Research Grant (SEV-2016-0644) and the Instituto de Salud Carlos III (PI20/01131). The funding entities did not play any role in the study design, collection, analysis and interpretation of data, writing of the report or the decision to submit the article for publication. The authors declare no competing interests.J.I.-P. was supported by a predoctoral grant from the Basque Government (PRE_2017_0204). This study was partially funded by the Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). The project was also supported by the Spanish Ministry of Economy and Competitiveness MINECO within the national plan RTI2018-094969-B-I00, the European Union's Horizon 2020 research and innovation program (860303), the Severo Ochoa Centre of Excellence Innovative Research Grant (SEV-2016-0644) and the Instituto de Salud Carlos III (PI20/01131). The funding entities did not have any role in study design, sample collection, analysis and interpretation of data, report writing or decision to submit the article for publication

Peripheral Blood Mononuclear Cells (PBMC) Microbiome is Not Affected by Colon Microbiota in Healthy Goats

BACKGROUND: The knowledge about blood circulating microbiome and its functional relevance in healthy individuals remains limited. An assessment of changes in the circulating microbiome was performed by sequencing peripheral blood mononuclear cells (PBMC) bacterial DNA from goats supplemented or not in early life with rumen liquid transplantation. RESULTS: Most of the bacterial DNA associated to PBMC was identified predominantly as Proteobacteria (55%) followed by Firmicutes (24%), Bacteroidetes (11%) and Actinobacteria (8%). The predominant genera found in PBMC samples were Pseudomonas, Prevotella, Sphingomonas, Acinetobacter, Corynebacterium and Ruminococcus. Other genera such as Butyrivibrivio, Bifidobacterium, Dorea and Coprococcus were also present in lower proportions. Several species known as blood pathogens or others involved in gut homeostasis such as Faecalibacterium prausnitzii were also identified. However, the PBMC microbiome phylum composition differed from that in the colon of goats (P≤0.001), where Firmicutes was the predominant phylum (83%). Although, rumen liquid administration in early-life altered bacterial community structure and increased Tlr5 expression (P=0.020) in colon pointing to higher bacterial translocation, less than 8% of OTUs in colon were also observed in PBMCs. CONCLUSIONS: Data suggest that in physiological conditions, PBMC microbiome differs from and is not affected by colon gut microbiota in small ruminants. Although, further studies with larger number of animals and covering other animal tissues are required, results point to a common circulating bacterial profile on mammals being phylum Proteobacteria, and genera Pseudomonas and Prevotella the most abundants. All suggest that PBMC microbiome in healthy ruminants could be implicated in homeostatic condition. This study expands our knowledge about PBMC microbiome contribution to health in farm animals.This work was supported by grants from the Spanish Ministry of Science and Innovation (MCI) co-financed with FEDER funds [AGL2017-86757- to LA, AGL2017-86938-R to DRY]. Other contributions were SAF2015-65327-R to JA and SAF2015-73549-JIN to HR. LA is a Ramón y Cajal fellow [RYC-2013-13666] from the Spanish Ministry of Science and Innovation. APC is a recipient of a fellowship from the University of the Basque Country. We thank the MCI for the Severo Ochoa Excellence accreditation (SEV-2016-0644) and the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs

S-adenosylmethionine Levels Regulate the Schwann Cell DNA Methylome

SummaryAxonal myelination is essential for rapid saltatory impulse conduction in the nervous system, and malformation or destruction of myelin sheaths leads to motor and sensory disabilities. DNA methylation is an essential epigenetic modification during mammalian development, yet its role in myelination remains obscure. Here, using high-resolution methylome maps, we show that DNA methylation could play a key gene regulatory role in peripheral nerve myelination and that S-adenosylmethionine (SAMe), the principal methyl donor in cytosine methylation, regulates the methylome dynamics during this process. Our studies also point to a possible role of SAMe in establishing the aberrant DNA methylation patterns in a mouse model of diabetic neuropathy, implicating SAMe in the pathogenesis of this disease. These critical observations establish a link between SAMe and DNA methylation status in a defined biological system, providing a mechanism that could direct methylation changes during cellular differentiation and in diverse pathological situations

Mitochondrial dysfunction governs immunometabolism in leukocytes of patients with acute-on-chronic liver failure.

Background & aims: Patients with acute-on-chronic liver failure (ACLF) present a systemic hyperinflammatory response associated with increased circulating levels of small-molecule metabolites. To investigate whether these alterations reflect inadequate cell energy output, we assessed mitochondrial morphology and central metabolic pathways with emphasis on the tricarboxylic acid (TCA) cycle in peripheral leukocytes from patients with acutely decompensated (AD) cirrhosis, with and without ACLF. Methods: The study included samples from patients with AD cirrhosis (108 without and 128 with ACLF) and 41 healthy individuals. Leukocyte mitochondrial ultrastructure was visualized by transmission electron microscopy and cytosolic and mitochondrial metabolic fluxes were determined by assessing NADH/FADH2 production from various substrates. Plasma GDF15 and FGF21 were determined by Luminex and acylcarnitines by LC-MS/MS. Gene expression was analyzed by RNA-sequencing and PCR-based glucose metabolism profiler array. Results: Mitochondrial ultrastructure in patients with advanced cirrhosis was distinguished by cristae rarefication and swelling. The number of mitochondria per leukocyte was higher in patients, accompanied by a reduction in their size. Increased FGF21 and C6:0- and C8:0-carnitine predicted mortality whereas GDF15 strongly correlated with a gene set signature related to leukocyte activation. Metabolic flux analyses revealed increased energy production in mononuclear leukocytes from patients with preferential involvement of extra-mitochondrial pathways, supported by upregulated expression of genes encoding enzymes of the glycolytic and pentose phosphate pathways. In patients with ACLF, mitochondrial function analysis uncovered break-points in the TCA cycle at the isocitrate dehydrogenase and succinate dehydrogenase level, which were bridged by anaplerotic reactions involving glutaminolysis and nucleoside metabolism. Conclusions: Our findings provide evidence at the cellular, organelle and biochemical levels that severe mitochondrial dysfunction governs immunometabolism in leukocytes from patients with AD cirrhosis and ACLF. Lay summary: Patients at advanced stages of liver disease have dismal prognosis due to vital organ failures and the lack of treatment options. In this study, we report that the functioning of mitochondria, which are known as the cell powerhouse, is severely impaired in leukocytes of these patients, probably as a consequence of intense inflammation. Mitochondrial dysfunction is therefore a hallmark of advanced liver disease