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

Sistema endokannabinoidea: etorkizun handiko itu terapeutikoa gaixotasun psikiatrikoetan

Sistema endokannabinoideak banaketa zabala dauka giza garunean, eta sistema neuromodulatzailerik garrantzitsuenetako bat izan litekeela proposatu da. Epidemiologi ikasketetan zein animali ereduetan, sistema endokannabinoidearen seinaleztapenak emozio eta kognizio funtzioak erregulatzen dituela ikusi da. Testuinguru honetan, azkeneko urteotan lan ugari bideratu dira sistema endokannabinoideak gaixotasun neuropsikiatrikoen fisiopatologian eta tratamenduan eduki dezakeen garrantzia finkatzera. Artikulu honetan, depresioa eta antsietatea bezalako gogo-aldartearen asalduretan eta eskizofrenia gaixotasunean ikusitako sistema endokannabinoidearen parte-hartzeari buruzko ebidentziak azaltzen saiatuko gara. Gaur egungo ebidentziek agerian uzten dute sistema endokannabinoideak gaixotasun psikiatrikoen azpiko zelula-prozesuetan betetzen duen funtzioa. Halere, arlo honetan asko dago ezagutzeke oraindik, eta etorkizunean sistema endokannabinoidearen gaineko ikerkuntza oso interesgarria izango da gaixotasun hauen prozesu patologikoen oinarriak ezagutzeko, edota terapiarako tresna terapeutiko berrien garapenerako

New Insights into Gene Delivery to Human Neuronal Precursor NT2 Cells: A Comparative Study between Lipoplexes, Nioplexes, and Polyplexes

The transfection of human NTera2/D1 teratocarcinoma-derived cell line (or NT2 cells) represents a promising strategy for the delivery of exogenous proteins or biological agents into the central nervous system (CNS). The development of suitable nonviral vectors with high transfection efficiencies requires a profound knowledge of the whole transfection process. In this work, we elaborated and characterized in terms of size and zeta potential three different nonviral vectors: lipoplexes (144 nm; -29.13 mV), nioplexes (142.5 nm; +35.4 mV), and polyplexes (294.8 nm; +15.1 mV). We compared the transfection efficiency, cellular uptake, and intracellular trafficking of the three vectors in NT2 cell line. Lipoplexes exhibited the highest percentages of EGFP positive cells. The values obtained with polyplexes were lower compared to lipoplexes but higher than the percentages obtained with nioplexes. Cellular uptake results had a clear correlation with respect to the corresponding transfection efficiencies. Regarding the endocytosis mechanism, lipoplexes enter in the cell, mainly, via clathrin-mediated endocytosis (CME) while polyplexes via caveolae-mediated endocytosis (CvME). Nioplexes were discarded for this experiment due to their low cellular uptake. By simulating an artificial endosome, we demonstrated that the vectors were able to release the DNA cargo once inside the late endosome. The data collected from this assay showed that at 6 h the genetic material carried by polyplexes was still located in the late endosome, while DNA carried by lipoplexes was already in the nucleus. This result indicates a faster intracellular traffic of the lipid-based vectors. Overall, our work gives new insights into the transfection process of NT2 cells by different nonviral vectors as a first step in the development of ex vivo gene therapy platform.This project was partially supported by the University of the Basque Country UPV/EHU (UFI 11/32 and UFI11/35), the Basque Government (Department of Education, GIC-12/150 to J.S, University and Research, for the predoctoral BFI-2011-2226 fellowship, and Department of Industry SAIOTEK S-PE13UN193) and the National Council of Science and Technology (CONACYT, Mexico, Reg. 217101). Technical and human support provided by SGIker (UPV/EHU) is gratefully acknowledged. Authors also wish to thank the intellectual and technical assistance from the ICTS “NANBIOSIS”, more specifically, by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/EHU).Peer reviewe

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

Isolation of Platelet-Derived Exosomes from Human Platelet-Rich Plasma: Biochemical and Morphological Characterization

Platelet-Rich Plasma (PRP) is enriched in molecular messengers with restorative effects on altered tissue environments. Upon activation, platelets release a plethora of growth factors and cytokines, either in free form or encapsulated in exosomes, which have been proven to promote tissue repair and regeneration. Translational research on the potential of exosomes as a safe nanosystem for therapeutic cargo delivery requires standardizing exosome isolation methods along with their molecular and morphological characterization. With this aim, we isolated and characterized the exosomes released by human PRP platelets. Western blot analysis revealed that CaCl2-activated platelets (PLT-Exos-Ca2+) released more exosomes than non-activated ones (PLT-Exos). Moreover, PLT-Exos-Ca2+ exhibited a molecular signature that meets the most up-to-date biochemical criteria for platelet-derived exosomes and possessed morphological features typical of exosomes as assessed by transmission electron microscopy. Array analysis of 105 analytes including growth factors and cytokines showed that PLT-Exos-Ca2+ exhibited lower levels of most analytes compared to PLT-Exos, but relatively higher levels of those consistently validated as components of the protein cargo of platelet exosomes. In summary, the present study provides new insights into the molecular composition of human platelet-derived exosomes and validates a method for isolating highly pure platelet exosomes as a basis for future preclinical studies in regenerative medicine and drug delivery.This work was funded by the Provincial Council of Alava through the Alava Innova Program, Basque Government (IT1230-19), MINECO CTQ2017-85686-R (Spanish Ministry of Economy and Competitiveness and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM)

Teratokartzinometatik eratorritako zelula-lerro neuronalen (NT2N) trasplantea: istripu zerebrobaskularra izandako gaixo kronikoentzako estrategia terapeutiko berria

Currently, Cerebrovascular accident (CVA) also known as stroke, is the third leading cause of disability and death worldwide. CVA is a sudden alteration of cerebral circulation, which results in neural cell death and consequently a temporary or permanent loss in certain neuronal functions. Therefore, in addition to working on prevention, in clinical practice is essential to have effective treatments aimed at avoiding neuronal dead. Unfortunately, the efficacy of the currently used therapeutic strategies is limited and, in most cases, patients suffering from stroke do not recover an adequate quality of life. This article presents cell therapy as a future alternative of interest in the treatment of chronic patients of CVA. This new therapy aims to replace the lost neuronal cells that are the main cause of disability in patients. Specifically, the review article presents recent progress on the use of teratocarcinoma-derived Ntera2/D1 neuronlike cells (NT2N cells), as graft source for cell transplantation in stroke, discussing the safety and efficacy demonstrated by preclinical and clinical trials made to date.; Gaur egun, istripu zerebrobaskularra (IZB) mundu osoan bigarren heriotza-kausa eta ezgaitasunen hirugarren eragilea da. IZB, garuneko zirkulazioaren bat-bateko asaldura da, non, neuronen galera baten ondorioz, aldi baterako edo betirako garunaren eskualde jakin baten funtzioa eraldatzen den. Hortaz, praktika klinikoan, prebentzioan lan egiteaz gain, ezinbestekoa da funtzio neurologikoen galeraren oinarrian dagoen neuronen heriotzaren kontra zuzendutako tratamendu eraginkorrak izatea. Tamalez, gaur egun erabiltzen diren estrategia terapeutikoen eraginkortasuna mugatua da, eta, kasu gehienetan, IZB izandako pazienteek ez dute bizi-kalitate egokia berreskuratzen. Artikulu honetan, gaixoen ezgaitasunaren arrazoia den neuronen galerari aurre egiteko zelula hauen ordezkapena itu duen terapia zelularra aurkezten da. Zehazki, Ntera2/D1 edo NT2 deituriko zelula-lerrotik eratorritako zelula neuronalen (NT2N) transplantean sakontzen dugu, IZB izandako gaixoetan estrategia berri bezala eskainiko lituzkeen abantailak eta gaur egun arte egindako entsegu pre-klinikoetan zein klinikoetan frogatutako segurtasuna eta eraginkortasuna aztertuz

Lack of the transient receptor potential vanilloid 1 shifts cannabinoid-dependent excitatory synaptic plasticity in the dentate gyrus of the mouse brain hippocampus

[EN] The transient receptor potential vanilloid 1 (TRPV1) participates in synaptic functions in the brain. In the dentate gyrus, post-synaptic TRPV1 in the granule cell (GC) dendritic spines mediates a type of long-term depression (LTD) of the excitatory medial perforant path (MPP) synapses independent of pre-synaptic cannabinoid CB1 receptors. As CB1 receptors also mediate LTD at these synapses, both CB1 and TRPV1 might be influencing the activity of each other acting from opposite synaptic sites. We tested this hypothesis in the MPP–GC synapses of mice lacking TRPV1 (TRPV1-/-). Unlike wild-type (WT) mice, low-frequency stimulation (10min at 10Hz) of TRPV1-/- MPP fibers elicited a form of long-term potentiation (LTP) that was dependent on (1) CB1 receptors, (2) the endocannabinoid 2-arachidonoylglycerol (2-AG), (3) rearrangement of actin filaments, and (4) nitric oxide signaling. These functional changes were associated with an increase in the maximum binding efficacy of guanosine-5′-O-(3-[35S]thiotriphosphate) ([35S]GTPgS) stimulated by the CB1 receptor agonist CP 55,940, and a significant decrease in receptor basal activation in the TRPV1-/- hippocampus. Finally, TRPV1-/- hippocampal synaptosomes showed an augmented level of the guanine nucleotide-binding (G) Gai1, Gai2, and Gai3 protein alpha subunits. Altogether, the lack of TRPV1 modifies CB1 receptor signaling in the dentate gyrus and causes the shift from CB1 receptor-mediated LTD to LTP at the MPP–GC synapses.This work was supported by the Basque Government (IT1230- 19, to PG); MINECO/FEDER, UE (SAF2015-65034-R, to PG); Ministry of Science and Innovation (PID2019-107548RBI00, to PG); Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III); and European Regional Development Funds-European Union (ERDF-EU, Investing in your future; RD16/0017/0012, to PG); MINECO CTQ2017-85686-R (Spanish Ministry of Economy and Competitiveness, to JS); JE-H is a Postdoctoral Researcher contracted with funds of Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III), and European Regional Development Funds-European Union (ERDF-EU, Investing in your future; RD16/0017/0012), and the Basque Government (IT1230-19). IB-D holds a Postdoctoral Orientation Period contract (BES-2016-076766, BES-C-2016-0051). SA has a Ph.D. contract granted by University of the Basque Country (PIF 16/251). ES-G is funded by Ikerbasque and MINECO (PGC2018- 093990-A-I00; MICIU/AEI/FEDER, UE)

The Absence of the Transient Receptor Potential Vanilloid 1 Directly Impacts on the Expression and Localization of the Endocannabinoid System in the Mouse Hippocampus

The transient receptor potential vanilloid 1 (TRPV1) is a non-selective ligand-gated cation channel involved in synaptic transmission, plasticity, and brain pathology. In the hippocampal dentate gyrus, TRPV1 localizes to dendritic spines and dendrites postsynaptic to excitatory synapses in the molecular layer (ML). At these same synapses, the cannabinoid CB1 receptor (CB1R) activated by exogenous and endogenous cannabinoids localizes to the presynaptic terminals. Hence, as both receptors are activated by endogenous anandamide, co-localize, and mediate long-term depression of the excitatory synaptic transmission at the medial perforant path (MPP) excitatory synapses though by different mechanisms, it is plausible that they might be exerting a reciprocal influence from their opposite synaptic sites. In this anatomical scenario, we tested whether the absence of TRPV1 affects the endocannabinoid system. The results obtained using biochemical techniques and immunoelectron microscopy in a mouse with the genetic deletion of TRPV1 show that the expression and localization of components of the endocannabinoid system, included CB1R, change upon the constitutive absence of TRPV1. Thus, the expression of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) drastically increased in TRPV1(-/-) whole homogenates. Furthermore, CB1R and MAGL decreased and the cannabinoid receptor interacting protein 1a (CRIP1a) increased in TRPV1(-/-) synaptosomes. Also, CB1R positive excitatory terminals increased, the number of excitatory terminals decreased, and CB1R particles dropped significantly in inhibitory terminals in the dentate ML of TRPV1(-/-) mice. In the outer 2/3 ML of the TRPV1(-/-) mutants, the proportion of CB1R particles decreased in dendrites, and increased in excitatory terminals and astrocytes. In the inner 1/3 ML, the proportion of labeling increased in excitatory terminals, neuronal mitochondria, and dendrites. Altogether, these observations indicate the existence of compensatory changes in the endocannabinoid system upon TRPV1 removal, and endorse the importance of the potential functional adaptations derived from the lack of TRPV1 in the mouse brain.This work was supported by the Basque Government (IT123019, to PG); MINECO/FEDER, UE (SAF2015-65034-R, to PG); Ministry of Science and Innovation (PID2019-107548RBI00, to PG); Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III) and European Regional Development Funds-European Union (ERDF-EU, Investing in your future; RD16/0017/0012, to PG); MINECO CTQ2017-85686-R (Spanish Ministry of Economy and Competitiviness, to JS); JE-H is a Postdoctoral Researcher contracted with funds of Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III) and European Regional Development Funds-European Union (ERDF-EU, Investing in your future; RD16/0017/0012), and the Basque Government (IT1230-19); IB-DR holds a Postdoctoral contract (BES2016-076766, BES-C-2016-0051); AM is the recipient of a PhD contract granted by the Department of Education of the Basque Governmen

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)