Data_Sheet_2_Overlapping Distribution of Orexin and Endocannabinoid Receptors and Their Functional Interaction in the Brain of Adult Zebrafish.docx

<p>Hypocretins/Orexins neuropeptides are known to regulate numerous physiological functions, such as energy homeostasis, food intake, sleep/wake cycle, arousal and wakefulness, in vertebrates. Previous studies on mice have revealed an intriguing orexins/endocannabinoids (ECs) signaling interaction at both structural and functional levels, with OX-A behaving as a strong enhancer of 2-arachydonoyl-glycerol (2-AG) biosynthesis. In this study, we describe, for the first time in the brain of zebrafish, the anatomical distribution and co-expression of orexin (OX-2R) and endocannabinoid (CB1R) receptors, suggesting a functional interaction. The immunohistochemical colocalization of these receptors by confocal imaging in the dorsal and ventral telencephalon, suprachiasmatic nucleus (SC), thalamus, hypothalamus, preoptic area (PO) and cerebellum, is reported. Moreover, biochemical quantification of 2-AG levels by LC-MS supports the occurrence of OX-A-induced 2-AG biosynthesis in the zebrafish brain after 3 h of OX-A intraperitoneal (i.p.; 3 pmol/g) or intracerebroventricular (i.c.v.; 0.3 pmol/g) injection. This effect is likely mediated by OX-2R as it is counteracted by i.p./i.c.v administration of OX-2R antagonist (SB334867, 10 pmol/g). This study provides compelling morphological and functional evidence of an OX-2R/CB1R signaling interaction in the brain of adult zebrafish, suggesting the use of this well-established vertebrate animal model for the study of complex and phylogenetically conserved physiological functions.</p

The Cannabinoid Receptor <i>Type 2</i> as Mediator of Mesenchymal Stromal Cell Immunosuppressive Properties

<div><p>Mesenchymal stromal cells are non-hematopoietic, multipotent progenitor cells producing cytokines, chemokines, and extracellular matrix proteins that support hematopoietic stem cell survival and engraftment, influence immune effector cell development, maturation, and function, and inhibit alloreactive T-cell responses. The immunosuppressive properties of human mesenchymal stromal cells have attracted much attention from immunologists, stem cell biologists and clinicians.</p><p>Recently, the presence of the endocannabinoid system in hematopoietic and neural stem cells has been demonstrated. Endocannabinoids, mainly acting through the cannabinoid receptor <i>subtype 2</i>, are able to modulate cytokine release and to act as immunosuppressant when added to activated T lymphocytes.</p><p>In the present study, we have investigated, through a multidisciplinary approach, the involvement of the endocannabinoids in migration, viability and cytokine release of human mesenchymal stromal cells.</p><p>We show, for the first time, that cultures of human mesenchymal stromal cells express all of the components of the endocannabinoid system, suggesting a potential role for the cannabinoid CB2 receptor as a mediator of anti-inflammatory properties of human mesenchymal stromal cells, as well as of their survival pathways and their capability to home and migrate towards endocannabinoid sources.</p></div

mTOR/S6K pathway is involved in the hMSCs migration 2AG-mediated.

<p>Immunocytochemical expression of the phosphorylated S6K1thr389 in the cytoplasm of hMSCs after 1h of exposure in absence (<b>A</b>) or presence (<b>B</b>) of 2-AG [10 µM] in the medium. The pS6K1 thr389 expression is strongly reduced by pretreatment of hMSCs with CB2 antagonist AM630 [2 µM] 15 min before the 2AG addition (<b>C</b>) whereas is unaffected by similar pretreatment with CB1 antagonist AM251(<b>D</b>). The Mammalian Target of Rapamycin (mTOR)-dependent action on 2-AG-induced phosporylation of pS6K1 thr389 in hMSCs is prevented by addition of rapamycin in medium 15 min before the 2AG addition (<b>E</b>). Scale bar = 10 microns.</p

Stimulation of the CB2 receptor partially reverses the LPS-induced modulation of pro- and anti- inflammatory cytokines in hMSCs.

<p>The release of the anti-inflammatory IL-10 (A) and of the pro-inflammatory IL-1β (B), IL-8 (C) and IL-17 (D), as well as of IL-6 (E), TNF-α (F) and INF-γ (G) from MSCs have been investigated through a multi-ELISA assay. IL-10 secretion by MSC cells was significantly reduced in response to 500 ng/ml LPS, while IL-1β, IL-8 and IL-17 were significantly increased. The treatment with the CB2 agonist JWH.-133 1 µM was able to revert the LPS-induced effect. The CB2 stimulation decreased the release of all the cytokines, even of the anti-inflammatory IL-10, thus was conversely shown to be up-regulated in total cellular lysates both at P6 and after CB2 stimulation, as revealed by the western blot included in the A1 inset. The release of IL-6, TNF-α and INF-γ by MSC cells, undetectable at basal condition, was significantly increased in response to 500 ng/ml LPS. The treatment with the CB2 agonist JWH-133 1 µM was able to fully revert the LPS-induced effect. Mean concentration ± SD (pg/ml) for all the cytokines from triplicate cultures is shown. A t-test has been used for statistical analysis. <i>p</i><0.05 has been considered statistically significant.</p

Stimulation of CB2 directly stimulates the ERK2 pathway.

<p>(<b>A</b>) Western Blot experiment and the relative quantification for p-ERK2 expression normalized with respect to β-tubulin in total MSC lysates from different passages and (<b>B</b>) after CB2 selective stimulation at multiple time-points. Data show the maximum increase of the protein levels at P1 and after 20-40 minutes and 24 hours from CB2 selective stimulation with the synthetic agonist JWH-133. The minimal p-ERK2 expression is revealed at P6. The CB2 antagonist AM630 significantly counteracts the JWH-induced increase of p-ERK2. Data are represented as a mean ± SD from three different assays. A t-test has been used for statistical analysis. <i>p</i><0.05 was considered statistically significant.</p

2-AG is Chemoattractant for hMSCs through CB2 receptor.

<p>(A) Image-based detection of 2-AG chemoattractive effect on cultured hMSCs. Representative morphological data of hMSCs time-lapse migration recorded at starting point (0 min), 90 min and 120 min from the 2-AG (filled gray arrows) or vehicle (white arrows) exposure. The cells were maintained in the relative medium or pretreated with the CB2 antagonist AM630 (lower panel) 15 min before the exposure to 2-AG during the chemotaxic assay. The arrowheads indicate static reference points in the cell port chamber; the black arrows point to soma or cellular processes without noticeable movements. The numbers indicate the hMSCs or their processes affected by 2-AG chemoattractive movements. Scale bar = 30 microns. A source of HGF has been used as positive control. (B) Quantative data of percentage of hMSCs effected by chemotaxic movements. n = 300 cells per each treatment. *** <i>P</i><0.0001 of hMSCs migration toward 2-AG at 90 min and 120 min <i>versus</i> hMSCs migration at each different treatment and time course.</p

CB2 selective stimulation is associated with mTOR pathway activation.

<p>(<b>A</b>) Western Blot experiment and the relative quantification for pS6K1 expression normalized with respect to β-tubulin in total MSC lysates from different passages and (<b>B</b>) after CB2 selective stimulation at multiple time-points. Data show the maximum increase of the protein levels at P1 and P9 and after 24 hours from CB2 selective stimulation with the synthetic agonist JWH-133. The minimal expression is revealed at P6. The CB2 antagonist AM630 reduces the JWH-induced increase of p-AKT. Data are represented as a mean ± SD from three different assays. A t-test has been used for statistical analysis. <i>p</i><0.05 was considered statistically significant.</p

CB2 expression level in hMSCs increases with their maturation.

<p>(<b>A</b>) Western Blot experiment and (<b>B</b>) the relative quantification for CB2 receptor expression in total MSC lysates from different passages normalized with respect to β-tubulin reveal a significant increase of the protein levels at P5 with a maximal expression at P6. Data are represented as a mean ± SD from three different assays. A t-test has been used for statistical analysis. <i>p</i><0.05 was considered statistically significant. (<b>C</b>) A positive staining for the specific mesenchymal CD105 marker (in red) and an increased CB2 receptor expression from passage P1 to P7 (in green) is highlighted by immunocytochemistry analysis. Scale bar 50 = 50 microns.</p

Endocannabinoid levels.

<p>Human mesenchymal stem cells (MSCs) contained measurable amounts of AEA, 2-AG (A), PEA and OEA (B). A significantly decrease of all the endocannabinoids was detected during expansion passages from P1 to P9 with respect to P0. The minimum amount of AEA, 2-AG and PEA was detected at P6 (AEA 1.26±0.27 pmol/mg extract; 2-AG 4.79±0.81 pmol/mg extract; PEA 20.10±4.76 pmol/mg extract). The minimum amount of OEA was detected at P5 (AEA 6.13±1.11 pmol/mg extract). Data are represented as a mean ± SD from N = 6 experiments. A t-test has been used for statistical analysis. <i>p</i><0.05 has been considered statistically significant.</p

Expression of CB1 and CB2 receptors in human MSCs.

<p>(<b>A</b>) Significant increase of Cannabinoid Receptor type 2 (CB2) expression levels in human Mesenchymal Stromal Cells from passage 3, P3. (<b>B</b>) Cannabinoid Receptor type 1 (CB1) expression levels showed a significant decrease starting from passage 1. Data are revealed from human in vitro MSCs by real time PCR by using three serial dilution 2X starting from 250 ng of total mRNA for the RT reaction and normalized for the housekeeping gene β-actin. Assays are at least in triplicate. Data are represented as a mean ± SD. A t-test has been used for statistical analysis. <i>p</i><0.05 was considered statistically significant.</p