Subsynaptic Distribution, Lipid Raft Targeting and G Protein-Dependent Signalling of the Type 1 Cannabinoid Receptor in Synaptosomes from the Mouse Hippocampus and Frontal Cortex

Numerous studies have investigated the roles of the type 1 cannabinoid receptor (CB1) in glutamatergic and GABAergic neurons. Here, we used the cell-type-specific CB1 rescue model in mice to gain insight into the organizational principles of plasma membrane targeting and Gαi/o protein signalling of the CB1 receptor at excitatory and inhibitory terminals of the frontal cortex and hippocampus. By applying biochemical fractionation techniques and Western blot analyses to synaptosomal membranes, we explored the subsynaptic distribution (pre-, post-, and extra-synaptic) and CB1 receptor compartmentalization into lipid and non-lipid raft plasma membrane microdomains and the signalling properties. These data infer that the plasma membrane partitioning of the CB1 receptor and its functional coupling to Gαi/o proteins are not biased towards the cell type of CB1 receptor rescue. The extent of the canonical Gαi/o protein-dependent CB1 receptor signalling correlated with the abundance of CB1 receptor in the respective cell type (glutamatergic versus GABAergic neurons) both in frontal cortical and hippocampal synaptosomes. In summary, our results provide an updated view of the functional coupling of the CB1 receptor to Gαi/o proteins at excitatory and inhibitory terminals and substantiate the utility of the CB1 rescue model in studying endocannabinoid physiology at the subcellular level.This research was funded by the Basque Government (IT1230-19), MINECO, Spanish Ministry of Science, Innovation and Universities (CTQ2017-85686-R)

Endocannabinoid 2-Arachidonoylglycerol Synthesis and Metabolism at Neuronal Nuclear Matrix Fractions Derived from Adult Rat Brain Cortex

In this report, we describe the kinetics characteristics of the diacylglycerol lipase-α (DGLα) located at the nuclear matrix of nuclei derived from adult cortical neurons. Thus, using high-resolution fluorescence microscopy, classical biochemical subcellular fractionation, and Western blot techniques, we demonstrate that the DGLα enzyme is located in the matrix of neuronal nuclei. Furthermore, by quantifying the 2-arachidonoylglycerol (2-AG) level by liquid chromatography and mass spectrometry when 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) was exogenously added as substrate, we describe the presence of a mechanism for 2-AG production through DGLα dependent biosynthesis with an apparent Km (Kmapp) of 180 µM and a Vmax of 1.3 pmol min−1 µg−1 protein. We also examined the presence of enzymes with hydrolytic and oxygenase activities that are able to use 2-AG as substrate, and described the localization and compartmentalization of the major 2-AG degradation enzymes, namely monoacylglycerol lipase (MGL), fatty acid amide hydrolase (FAAH), α/β-hydrolase domain 12 protein (ABHD12) and cyclooxygenase-2 (COX2). Of these, only ABHD12 exhibited the same distribution with respect to chromatin, lamin B1, SC-35 and NeuN as that described for DGLα. When 2-AG was exogenously added, we observed the production of arachidonic acid (AA), which was prevented by inhibitors (but not specific MGL or ABHD6 inhibitors) of the ABHD family. Overall, our results expand knowledge about the subcellular distribution of neuronal DGLα, and provide biochemical and morphological evidence to ensure that 2-AG is produced in the neuronal nuclear matrix. Thus, this work paves the way for proposing a working hypothesis about the role of 2-AG produced in neuronal nuclei.This research was funded by the Spanish Ministry of Science and Innovation (Grant ID, CTQ2017-85686-R), Basque Government (Research Groups of the Basque University System, Grant IDs, IT1492-22 and IT1620-22) and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM). Miquel Saumell-Esnaola is in receipt of a PhD contract awarded by the Department of Education of the Basque Government

Laser bidezko ablazioaren egungo egoera eta aplikazioak

80ko hamarkadaz geroztik, laser bidezko ablazioa eta akoplamendu induktibozko plasma-masa espektrometria (LA-ICPMS) konbinatzen dituen teknika analitikoa sakonki ikertu eta garatu da, eta, gaur egun, lagin solidoen analisi elemental zein isotopiko zuzena egiteko erreferentziazko teknika bilakatu da. Ezaugarri bereizgarrienak hauexek dira: erabilpen erraza, analisi-denbora laburra, laginari eragindako kalte urriak, sentsibilitate handia eta elementu nagusi zein minoritarioen eta ratio isotopikoen aldibereko neurketa ahalbidetzen duen tarte dinamiko zabala. Lan honetan, LA-ICPMS teknikaren oinarriak eta konfigurazio orokorra azaldu eta femtosegundo/ nanosegundo laser bidezko ablazioaren erabileraren arteko desberdintasun nagusiak nabarmentzen dira. Azkenik, teknika honen egungo egoeraz jabetzeko, jatorri askotariko laginen analisi elementala laburbildu eta berrikusten da biologian eta ingurumenean, osasunean, geokimikan zein auzitegi zientzien alorretan.; Since the early 80s, laser-ablation inductively-coupled-plasma mass spectrometry (LA-ICPMS) has been widely explored and nowadays is considered to be one of the most versatile analytical techniques for the direct trace elemental and isotopic analyses of solid samples. The most remarkable features are ease of use, fast sample throughput, limited sample damages, high sensitivity and wide dynamic range which allows the simultaneous acquisition of major and trace elements, as well as the measurement of isotope ratios. Throughout this manuscript, the principles and general setup of LA-ICPMS will be extensively explained and the performance of femtosecondLA in comparison to nanosecond-LA discussed. In order to show the state of the art in this field a variety of examples for elemental analysis of solid samples in biological/environmental science, geochemistry and forensic science applications will be presented

Hormones and bile acids as biomarkers for the characterization of animal management in prehistoric sheepfold caves: El Mirador case (Sierra de Atapuerca, Burgos, Spain

Early husbandry practices that include herd management and the use of livestock areas such as sheepfold caves can be analysed in the context of different disciplines (e.g. zooarchaeology, micromorphology, and archaeobotany). In this study, a new and standard method for the determination of bile acids and steroidal hormones that incorporates microwave extraction-liquid chromatography-mass spectrometry was used. This method has been applied successfully to analyse Neolithic fumier deposit facies from the El Mirador cave, a location that was used as a prehistoric sheepfold and is located in the Atapuerca range (Burgos, Spain). The results obtained demonstrated that the analysis of bile acids can be useful for the identification of remains of ruminant residues in the facies studied. In addition, the progesterone/deoxycholic acid ratio has been used as a possible biomarker to improve our understanding of flock management, including the separation of pregnant and nursing ewes from the rest of the herd to avoid the rejection of the lamb and keep them safe and healthy.The authors thank the technical and human support provided by the Alava Central Service of Analysis of SGIker (UPV/EHU, MINECO, GV/ EJ, ERDF, and ESF) and Paula Rivero for the elaboration of the graphical abstract. Patricia Martín is grateful for her postdoctoral fellowship to Juan de la Cierva Subprogramme (FJCI-2016-29045) with financial sponsorship from the Spanish Ministry of Economy, Industry, and Competitiveness and for her recent "Maria de Maeztu" excellenceaccreditation from the Spanish Minstry of Science and Innovation (CEX2019-000945-M), and to Ane Gorostizu-Orkaiztegi for her pre- doctoral fellowships to the University of the Basque Country. This work was funded by the Department of Industry, Innovation, Commerce, and Tourism of the Basque Government (SAI12/25 Project), the Spanish Ministry of Science, Innovation and University (PGC 2018-093925-B- C32 project) and by the Basque Government, Research Groups of the Basque University System (Project No. IT925-16)

Solid-phase synthesis of imprinted nanoparticles as artificial antibodies against the C-terminus of the cannabinoid CB1 receptor: exploring a viable alternative for bioanalysis

[EN]The production of artificial anti-CB1 antibodies in nanoparticle format is described using the solid-phase imprinting approach. Instead of whole protein imprinting, a linear C-terminus sequence of the receptor comprising 15 amino acids (458-KVTMSVSTDTSAEAL-472) has been used as template, in accordance with the epitope imprinting approach. This sequence is located intracellularly, and it is involved in coupling to G(i/o) proteins, being responsible for CB1 receptor desensitisation and internalisation. Developed molecularly imprinted materials were found to be in the nanometre scale, with a particle size of 126.4 +/- 10.5 nm at pH 3 (25 oC) and spherical shape. It was also observed that the size was sensible to temperature changes being reduced to 106.3 +/- 15.2 nm at 35 degrees C. Lower critical solution temperature of this polymer was found to be approximate to 33.4 degrees C. The affinity and selectivity of the artificial antibody were assessed through dot blot and Western blot experiments. For the latter, recombinant fusion proteins GST-CB1(414-472) and GST-CB1(414-442) were produced to work respectively as target and negative control proteins. The control protein did not carry the target epitope for being devoid of last 30 amino acids at the C-terminus. The results demonstrated that the anti-CB1 material recognised selectively the target protein, thanks to the presence of the 15-amino acid sequence selected as epitope, which revealed that binding occurred at the C-terminus of the receptor itself. The methodology presented may pave the way for the development of novel imprinted nanomaterials for other proteins included in the superfamily of the G-protein-coupled receptors (GPCR).Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Funding for this research was provided by the Spanish Ministry of Science, Innovation and Universities (project CTQ2017-85686-R) and by the Basque Government (Research Groups of the Basque University System, Project No IT 1186-19

Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods

Background: Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. Results: Here we generated highly soluble and stable recombinant protein constructs GST-CB1(41)(4-)(472 )and GST-CB1(414-442) containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443-473 of the mouse CB1 receptor that corresponds to residues 442-472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. Conclusions: Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints.This work was funded by Spanish Ministry of Science, Innovation and Universities (Grant ID, CTQ2017-85686-R) and Basque Government (Research Groups of the Basque University System, Grant IDs, IT1492-22 and IT1620-22)

Ikerketa metabolomikoak haurretan gertatzen den giltzurrun gutxiegitasun kronikoaren diagnostikorako biomarkatzaile berrien identifikazioan

The assessment of chronic kidney disease (CKD) is performed by means of glomerular filtration rate (GFR), which is calculated from equations using serum creatinine concentration. However, serum creatinine concentration changes according to several factors, and this might endanger CKD diagnosis, especially in early stages of the disease. In addition to metabolic and cardiovascular complications, CKD is related to growth complications and malnutrition. All of these complications lead to a 30 times higher mortality rate in paediatrics suffering from advanced CKD compared to healthy counterparts. Overall, the existence of biomarkers for a defined disease allows earlier diagnosis as well as better response. For that reason, comprehensive research has been performed in adults suffering from CKD aimed at finding new biomarkers, however only a few studies are available in paediatrics with CKD so far, and there is a need for new biomarkers in this population. For that purpose, following targeted and untargeted metabolomics approaches seven potential biomarkers have been found which could be useful for paediatric CKD by comparing metabolic profiles. The use of these metabolites in addition to creatinine enables better differentiation of paediatrics with CKD and control ones, unlike just creatinine itself.; Giltzurrun gutxiegitasun kronikoaren (GGK) ebaluazioa egiten da iragazpen glomerularraren tasaren (IGT) bidez, eta kalkulatzen da serumeko kreatininaren kontzentrazioan oinarritutako ekuazio ezberdinen bidez. Hala ere, serumeko kreatinina kontzentrazioa hainbat faktoreren arabera alda daiteke, eta honek GGKaren diagnostikoa arriskuan jar dezake, bereziki gaixotasunaren fase goiztiarrenetan. Haurretan GGKak arazo metaboliko eta kardiobaskularrez gain, garapen-arazoak eta malnutrizioa eragiten ditu. Haur osasuntsuekin alderatuta, zailtasun horiek 30 aldiz handitzen dute heriotza-tasa GGKaren fase aurreratuenetan dauden haurretan. Orokorrean, gaixotasun jakin baterako biomarkatzaileen presentziak diagnostiko goiztiarragoa eta erantzun hobea ahalbidetzen dute. Hori dela eta, GGK pairatzen duten helduetan ikerketa integrala egin da biomarkatzaile berriak topatzeko, baina populazio pediatrikoan ikerlanak oraindik oso mugatuak dira eta biomarkatzaile berrien beharra dago. Helburu horrekin, haur osasuntsuen eta GGK pairatzen duten haur gaixoen arteko profil metabolikoen konparaketa egin da, metabolomika bideratu zein ez-bideratuak aplikatuz. Ikerketaren ondorioz, GGK pediatrikoaren detekzio goiztiarrean erabilgarri izan daitezkeen zazpi metabolito berri aurkitu dira, kreatinina biomarkatzaile klasikoarekin batera. Metabolito horiek, kreatininarekin batera erabiliz, kreatinina soilaren erabilerarekin alderatuz haur gaixoen eta osasuntsuen artean bereizketa egitea ahalbidetzen dute