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/ß-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.

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-CB1414-472 and GST-CB1414-442 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.

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.

Fit-for-purpose based testing and validation of antibodies to amino- and carboxy-terminal domains of cannabinoid receptor 1.

Specific and selective anti-CB1 antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB1 receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five commercial antibodies against different regions of CB1 receptor as the best choice for specific end-use purposes. Specifically, an antibody against a long fragment of the extracellular amino tail of CB1 receptor (but not one against a short sequence of the extreme amino-terminus) detected strong surface staining when applied to live cells, whereas two different antibodies against an identical fragment of the extreme carboxy-terminus of CB1 receptor (but not one against an upstream peptide) showed acceptable performance on all platforms, although they behaved differently in immunohistochemical assays depending on the tissue fixation procedure used and showed different specificity in Western blot assays, which made each of them particularly suitable for one of those techniques. Our results provide a framework to interpret past and future results derived from the use of different anti-CB1 antibodies in the context of current knowledge about the CB1 receptor at the molecular level, and highlight the need for an adequate validation for specific purposes, not only before antibodies are placed on the market, but also before the decision to discontinue them is made.

Effects of Platelet-Rich Plasma on Cellular Populations of the Central Nervous System: The Influence of Donor Age.

Platelet-rich plasma (PRP) is a biologic therapy that promotes healing responses across multiple medical fields, including the central nervous system (CNS). The efficacy of this therapy depends on several factors such as the donor's health status and age. This work aims to prove the effect of PRP on cellular models of the CNS, considering the differences between PRP from young and elderly donors. Two different PRP pools were prepared from donors 65‒85 and 20‒25 years old. The cellular and molecular composition of both PRPs were analyzed. Subsequently, the cellular response was evaluated in CNS in vitro models, studying proliferation, neurogenesis, synaptogenesis, and inflammation. While no differences in the cellular composition of PRPs were found, the molecular composition of the Young PRP showed lower levels of inflammatory molecules such as CCL-11, as well as the presence of other factors not found in Aged PRP (GDF-11). Although both PRPs had effects in terms of reducing neural progenitor cell apoptosis, stabilizing neuronal synapses, and decreasing inflammation in the microglia, the effect of the Young PRP was more pronounced. In conclusion, the molecular composition of the PRP, conditioned by the age of the donors, affects the magnitude of the biological response.

Nuclear diacylglycerol lipase-α in rat brain cortical neurons: evidence of 2-arachidonoylglycerol production in concert with phospholipase C-ß activity.

In this report, we describe the localization of diacylglycerol lipase-α (DAGLα) in nuclei from adult cortical neurons, as assessed by double-immunofluorescence staining of rat brain cortical sections and purified intact nuclei and by western blot analysis of subnuclear fractions. Double-labeling assays using the anti-DAGLα antibody and NeuN combined with Hoechst staining showed that only nuclei of neuronal origin were DAGLα positive. At high resolution, DAGLα-signal displayed a punctate pattern in nuclear subdomains poor in Hoechst's chromatin and lamin B1 staining. In contrast, SC-35- and NeuN-signals (markers of the nuclear speckles) showed a high overlap with DAGLα within specific subdomains of the nuclear matrix. Among the members of the phospholipase C-ß (PLCß) family, PLCß1, PLCß2, and PLCß4 exhibited the same distribution with respect to chromatin, lamin B1, SC-35, and NeuN as that described for DAGLα. Furthermore, by quantifying the basal levels of 2-arachidonoylglycerol (2-AG) by liquid chromatography and mass spectrometry (LC-MS), and by characterizing the pharmacology of its accumulation, we describe the presence of a mechanism for 2-AG production, and its PLCß/DAGLα-dependent biosynthesis in isolated nuclei. These results extend our knowledge about subcellular distribution of neuronal DAGLα, providing biochemical grounds to hypothesize a role for 2-AG locally produced within the neuronal nucleus.

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.

PLCß1 by-passes early growth response -1 to induce the differentiation of neuronal cells.

The Gαq/phospholipase C-ß (PLCß) signaling system mediates calcium responses to a variety of hormones and neurotransmitters. Recent studies suggest that PLCß1 expression plays a role in the differentiation of two types of cultured neuronal cells (PC12 and SK-N-SH) through a mechanism independent of Gαq. Here, we show that, similar to that observed in PC12 and SK-N-SH cells, PLCß1 expression increases when human NT2 cells are induced to differentiate either through cytosine-ß-D-arabinofuranoside or retinoic acid. Preventing this increase, abolishes differentiation, and down-regulating PLCß1 in rat primary astrocytes causes cells to adapt an undifferentiated morphology. Surprisingly, transfecting PLCß1 into undifferentiated PC12 or NT2 cells induces differentiation without the need for differentiating agents. Studies to uncover the underlying mechanism focused on the transcription factor early growth response 1 (Egr-1) which mediates PLCß1 expression early in differentiation. Over-expressing PLCß1 in HEK293 cells enhances Egr-1 expression and induces morphological changes. We show that increased levels of cytosolic PLCß1 in undifferentiated PC12 cells disrupts the association between Egr-1 and its cytosolic binding partner (Tar RNA binding protein), promoting relocalization of Egr-1 to the nucleus, which promotes transcription of proteins needed for differentiation. These studies show a novel mechanism through which differentiation can be modulated.

Up-regulation of CB1 cannabinoid receptors located at glutamatergic terminals in the medial prefrontal cortex of the obese Zucker rat.

The present study describes a detailed neuroanatomical distribution map of the cannabinoid type 1 (CB1) receptor, along with the biochemical characterization of the expression and functional coupling to their cognate G i/o proteins in the medial prefrontal cortex (mPCx) of the obese Zucker rats. The CB1 receptor density was higher in the prelimbic (PL) and infralimbic (IL) subregions of the mPCx of obese Zucker rats relative to their lean littermates which was associated with a higher percentage of CB1 receptor immunopositive excitatory presynaptic terminals in PL and IL. Also, a higher expression of CB1 receptors and WIN55,212-2-stimulated [35S]GTPγS binding was observed in the mPCx but not in the neocortex (NCx) and hippocampus of obese rats. Low-frequency stimulation in layers II/III of the mPCx induced CB1 receptor-dependent long-term synaptic plasticity in IL of area obese Zucker but not lean rats. Overall, the elevated 2-AG levels, up-regulation of CB1 receptors, and increased agonist-stimulated [35S]GTPγS binding strongly suggest that hyperactivity of the endocannabinoid signaling takes place at the glutamatergic terminals of the mPCx in the obese Zucker rat. These findings could endorse the importance of the CB1 receptors located in the mPCx in the development of obesity in Zucker rats.

Highly efficient generation of glutamatergic/cholinergic NT2-derived postmitotic human neurons by short-term treatment with the nucleoside analogue cytosine ß-D-arabinofuranoside.

The human NTERA2/D1 (NT2) cells generate postmitotic neurons (NT2N cells) upon retinoic acid (RA) treatment and are functionally integrated in the host tissue following grafting into the rodent and human brain, thus representing a promising source for neuronal replacement therapy. Yet the major limitations of this model are the lengthy differentiation procedure and its low efficiency, although recent studies suggest that the differentiation process can be shortened to less than 1 week using nucleoside analogues. To explore whether short-term exposure of NT2 cells to the nucleoside analogue cytosine ß-d-arabinofuranoside (AraC) could be a suitable method to efficiently generate mature neurons, we conducted a neurochemical and morphometric characterization of AraC-differentiated NT2N (AraC/NT2N) neurons and improved the differentiation efficiency by modifying the cell culture schedule. Moreover, we analyzed the neurotransmitter phenotypes of AraC/NT2N neurons. Cultures obtained by treatment with AraC were highly enriched in postmitotic neurons and essentially composed of dual glutamatergic/cholinergic neurons, which contrasts with the preferential GABAergic phenotype that we found after RA differentiation. Taken together, our results further reinforce the notion NT2 cells are a versatile source of neuronal phenotypes and provide a new encouraging platform for studying mechanisms of neuronal differentiation and for exploring neuronal replacement strategies.

Data for the morphometric characterization of NT2-derived postmitotic neurons.

NTERA2/D1 human teratocarcinoma progenitors induced to differentiate into postmitotic neurons by either long-term treatment with retinoic acid or short-term treatment with the nucleoside analog cytosine ß-D-arabinofuranoside were subjected to morphometric analysis and compared. Our data provide a methodological and conceptual framework for future investigations aiming at distinguishing neuronal phenotypes on the basis of morphometric analysis. Data presented here are related to research concurrently published in "Highly Efficient Generation of Glutamatergic/Cholinergic NT2-Derived Postmitotic Human Neurons by Short-Term treatment with the Nucleoside Analogue Cytosine ß-D-Arabinofuranoside" [1].

Nuclear phospholipase C-ß1 and diacylglycerol LIPASE-α in brain cortical neurons.

Phosphoinositide (PtdIns) signaling involves the generation of lipid second messengers in response to stimuli in a receptor-mediated manner at the plasma membrane. In neuronal cells of adult brain, the standard model proposes that activation of metabotropic receptors coupled to Phospholipase C-ß1 (PLC-ß1) is linked to endocannabinoid signaling through the production of diacylglycerol (DAG), which could be systematically metabolized by 1,2-diacylglycerol Lipases (DAGL) to produce an increase of 2-arachidonoyl-glycerol (2-AG), the most abundant endocannabinoid in the brain. However, the existence of a nuclear PtdIns metabolism independent from that occurring elsewhere in the cell is now widely accepted, suggesting that the nucleus constitutes both a functional and a distinct compartment for PtdIns metabolism. In this review, we shall highlight the main achievements in the field of neuronal nuclear inositol lipid metabolism with particular attention to progress made linked to the 2-AG biosynthesis. Our aim has been to identify potential sites of 2-AG synthesis other than the neuronal cytoplasmic compartment by determining the subcellular localization of PLC-ß1 and DAGL-α, which is much more abundant than DAGL-ß in brain. Our data show that PLC-ß1 and DAGL-α are detected in discrete brain regions, with a marked predominance of pyramidal morphologies of positive cortical cells, consistent with their role in the biosynthesis and release of 2-AG by pyramidal neurons to control their synaptic inputs. However, as novelty, we showed here an integrated description of the localization of PLC-ß1 and DAGL-α in the neuronal nuclear compartment. We discuss our comparative analysis of the expression patterns of PLC-ß1 and DAGL-α, providing some insight into the potential autocrine role of 2-AG production in the neuronal nuclear compartment that probably subserve additional roles to the recognized activation of the CB1 cannabinoid receptor.