Nicoletta Pasquariello

Selective CB2 Receptor Agonism Protects Central Neurons from Remote Axotomy-Induced Apoptosis through the PI3K/Akt Pathway

Endocannabinoids are neuroprotective in vivo and in vitro, but the mechanisms by which they act are largely unknown. The present study addressed the role of cannabinoid receptors during remote cell death of central neurons in a model that is based on cerebellar lesions. A lesion in one cerebellar hemisphere induced remote cell death and type 2 cannabinoid receptor (CB2R) expression in contralateral precerebellar neurons. Of the selective agonists and antagonists that modulated cannabinoid receptor activity, we found that the CB2R agonist JWH-015 reduced neuronal loss and cytochrome-c release, leading to neurological recovery; these effects were reversed by the selective CB2R antagonist SR144528. Analysis of CB2R-triggered signal transduction demonstrated that in axotomized neurons, CB2R regulated Akt and JNK phosphorylation through a PI3K-dependent pathway, whereas other major signaling routes that are dependent on CB2R, such as ERK1/2 and p38, were not involved. This result was corroborated by the observation that the selective PI3K inhibitor LY294002 blocked the CB2R stimulation effects on neuronal survival as well as Akt and JNK phosphorylation levels. Together, these data demonstrate that axonal damage induces CB2R expression in central neurons and that stimulation of this receptor has a neuroprotective effect that is achieved through PI3K/Akt signaling.

Endocannabinoids in adipocytes during differentiation and their role in glucose uptake.

Abstract.The molecular basis for the control of energy balance by the endocannabinoid anandamide (AEA) is still unclear. Here, we show that murine 3T3-L1 fibroblasts have the machinery to bind, synthesize and degrade AEA, and that their differentiation into adipocytes increases by approximately twofold the binding efficiency of cannabinoid receptors (CBR), and by approximately twofold and approximately threefold, respectively, the catalytic efficiency of the AEA transporter and AEA hydrolase. In contrast, the activity of the AEA synthetase and the binding efficiency of vanilloid receptor were not affected by the differentiation process. In addition, we demonstrate that AEA increases by approximately twofold insulin-stimulated glucose uptake in differentiated adipocytes, according to a CB1R-dependent mechanism that involves nitric oxide synthase, but not lipoxygenase or cyclooxygenase. We also show that AEA binding to peroxisome proliferator-activated receptor-γ, known to induce differentiation of 3T3-L1 fibroblasts into adipocytes, is not involved in the stimulation of glucose uptake.

Molecular identification of albumin and Hsp70 as cytosolic anandamide-binding proteins.

The cellular uptake and the intracellular synthesis/degradation of anandamide are crucial steps for controlling its extracellular level and the duration of its activity. Although the biosynthesis and breakdown of anandamide are well understood, little is known about the mechanisms underlying its intracellular transport. Here, we investigated the presence of a potential carrier-mediated trafficking of anandamide within the cytosol, using a biotinylated analog as a tool to catch by affinity chromatography anandamide-interacting proteins. The identity of two of these anandamide-binding proteins, Hsp70 and serum albumin, was determined by mass spectrometry, confirmed by western blotting and confocal microscopy, and further validated through an anandamide-binding assay. These findings suggest that the trafficking of anandamide from the plasma membrane to the internal compartments of a cell occur via a nonvesicular mechanism mediated by cytosolic carriers.

Characterization of the Endocannabinoid System in Human Spermatozoa and Involvement of Transient Receptor Potential Vanilloid 1 Receptor in Their Fertilizing Ability

Human spermatozoa express type-1 cannabinoid receptor (CB1), whose activation by anandamide (AEA) affects motility and acrosome reaction (AR). In this study, we extended the characterization of the AEA-related endocannabinoid system in human spermatozoa, and we focused on the involvement of the AEA-binding vanilloid receptor (TRPV1) in their fertilizing ability. Protein expression was revealed for CB1 ( approximately 56 kDa), TRPV1 ( approximately 95 kDa), AEA-synthesizing phospholipase D (NAPE-PLD) ( approximately 46 kDa), and AEA-hydrolyzing enzyme [fatty acid amide hydrolase (FAAH), approximately 66 kDa]. Both AEA-binding receptors (CB1 and TRPV1) exhibited a functional binding activity; enzymatic activity was demonstrated for NAPE-PLD, FAAH, and the purported endocannabinoid membrane transporter (EMT). Immunoreactivity for CB1, NAPE-PLD, and FAAH was localized in the postacrosomal region and in the midpiece, whereas for TRPV1, it was restricted to the postacrosomal region. Capsazepine (CPZ), a selective antagonist of TRPV1, inhibited progesterone (P)-enhanced sperm/oocyte fusion, as evaluated by the hamster egg penetration test. This inhibition was due to a reduction of the P-induced AR rate above the spontaneous AR rate, which was instead increased. The sperm exposure to OMDM-1, a specific inhibitor of EMT, prevented the promoting effect of CPZ on spontaneous AR rate and restored the sperm responsiveness to P. No significant effects could be observed on sperm motility. In conclusion, this study provides unprecedented evidence that human spermatozoa exhibit a completely functional endocannabinoid system related to AEA and that the AEA-binding TRPV1 receptor could be involved in the sperm fertilizing ability.

Anandamide regulates keratinocyte differentiation by inducing DNA methylation in a CB1 receptor-dependent manner

Anandamide (arachidonoylethanolamide, AEA) belongs to an important class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids, collectively termed “endocannabinoids.” Recently we have shown that AEA inhibits differentiation of human keratinocytes, by binding to type-1 cannabinoid receptors (CB1R). To further characterize the molecular mechanisms responsible for this effect, we investigated the expression of epidermal differentiation-related genes after AEA treatment. We observed that keratin 1 and 10, transglutaminase 5 and involucrin are transcriptionally down-regulated by AEA. Most importantly, we found that AEA is able to decrease differentiating gene expression by increasing DNA methylation in human keratinocytes, through a p38, and to a lesser extent p42/44, mitogen-activated protein kinase-dependent pathway triggered by CB1R. An effect of AEA on DNA methylation because of CB1R-mediated increase of methyltransferase activity is described here for the first time, and we believe that the importance of this effect clearly extends beyond the regulation of skin differentiation. In fact, the modulation of DNA methylation by endocannabinoids may affect the expression of a number of genes that regulate many cell functions in response to these substances.

Evidence for the intracellular accumulation of anandamide in adiposomes

Abstract.Anandamide is a lipid messenger that carries out a wide variety of biological functions. It has been suggested that anandamide accumulation involves binding to a saturable cellular component. To identify the structure(s) involved in this process, we analyzed the intracellular distribution of both biotinylated and radiolabeled anandamide, providing direct evidence that lipid droplets, also known as adiposomes, constitute a dynamic reservoir for the sequestration of anandamide. In addition, confocal microscopy and biochemical studies revealed that the anandamide-hydrolase is also spatially associated with lipid droplets, and that cells with a larger adiposome compartment have an enhanced catabolism of anandamide. Overall, these findings suggest that adiposomes may have a critical role in accumulating anandamide, possibly by connecting plasma membrane to internal organelles along the metabolic route of this endocannabinoid.

Interplay between endocannabinoids, steroids and cytokines in the control of human reproduction.

The use of marijuana, which today is the most used recreational drug, has been demonstrated to affect adversely reproduction. Marijuana smokers, both men and women, show impaired fertility, owing to defective signalling pathways, aberrant hormonal regulation, or wrong timing during embryo implantation. Anandamide (N‐arachidonoylethanolamine, AEA) and 2‐arachidonoylglycerol (2‐AG) mimic Δ9‐tetrahydrocannabinol (THC), the psychoactive principle of Cannabis sativa, by binding to both the brain‐type (CB1) and the spleen‐type (CB2) cannabinoid receptors. These ‘endocannabinoids’ exert several actions either in the central nervous system or in peripheral tissues, and are metabolised by specific enzymes that synthesise or hydrolyse them. In this review, we shall describe the elements that constitute the endocannabinod system (ECS), in order to put in a better perspective the role of this system in the control of human fertility, both in females and males. In addition, we shall discuss the interplay between ECS, sex hormones and cytokines, which generates an endocannabinoid−hormone−cytokine array critically involved in the control of human reproduction.

Effect of Lipid Rafts on Cb2 Receptor Signaling and 2-Arachidonoyl-Glycerol Metabolism in Human Immune Cells

Recently, we have shown that treatment of rat C6 glioma cells with the raft disruptor methyl-β-cyclodextrin (MCD) doubles the binding of anandamide (AEA) to type-1 cannabinoid receptors (CB1R), followed by CB1R-dependent signaling via adenylate cyclase and p42/p44 MAPK activity. In the present study, we investigated whether type-2 cannabinoid receptors (CB2R), widely expressed in immune cells, also are modulated by MCD. We show that treatment of human DAUDI leukemia cells with MCD does not affect AEA binding to CB2R, and that receptor activation triggers similar [35S]guanosine-5′-O-(3-thiotriphosphate) binding in MCD-treated and control cells, similar adenylate cyclase and MAPK activity, and similar MAPK-dependent protection against apoptosis. The other AEA-binding receptor transient receptor potential channel vanilloid receptor subunit 1, the AEA synthetase N-acyl-phosphatidylethanolamine-phospholipase D, and the AEA hydrolase fatty acid amide hydrolase were not affected by MCD, whereas the AEA membrane transporter was inhibited (∼55%) compared with controls. Furthermore, neither diacylglycerol lipase nor monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoylglycerol, were affected by MCD in DAUDI or C6 cells, whereas the transport of 2-arachidonoylglycerol was reduced to ∼50%. Instead, membrane cholesterol enrichment almost doubled the uptake of AEA and 2-arachidonoylglycerol in both cell types. Finally, transfection experiments with human U937 immune cells, and the use of primary cells expressing CB1R or CB2R, ruled out that the cellular environment could account per se for the different modulation of CB receptor subtypes by MCD. In conclusion, the present data demonstrate that lipid rafts control CB1R, but not CB2R, and endocannabinoid transport in immune and neuronal cells.

Characterization of the endocannabinoid system in human neuronal cells and proteomic analysis of anandamide-induced apoptosis.

Anandamide (AEA) is an endogenous agonist of type 1 cannabinoid receptors (CB1R) that, along with metabolic enzymes of AEA and congeners, compose the “endocannabinoid system.” Here we report the biochemical, morphological, and functional characterization of the endocannabinoid system in human neuroblastoma SH-SY5Y cells that are an experimental model for neuronal cell damage and death, as well as for major human neurodegenerative disorders. We also show that AEA dose-dependently induced apoptosis of SH-SY5Y cells. Through proteomic analysis, we further demonstrate that AEA-induced apoptosis was paralleled by an ∼3 to ∼5-fold up-regulation or down-regulation of five genes; IgG heavy chain-binding protein, stress-induced phosphoprotein-1, and triose-phosphate isomerase-1, which were up-regulated, are known to act as anti-apoptotic agents; actin-related protein 2/3 complex subunit 5 and peptidylprolyl isomerase-like protein 3 isoform PPIL3b were down-regulated, and the first is required for actin network formation whereas the second is still function-orphan. Interestingly, only the effect of AEA on BiP was reversed by the CB1R antagonist SR141716, in SH-SY5Y cells as well as in human neuroblastoma LAN-5 cells (that express a functional CB1R) but not in SK-NBE cells (which do not express CB1R). Silencing or overexpression of BiP increased or reduced, respectively, AEA-induced apoptosis of SH-SY5Y cells. In addition, the expression of BiP and of the BiP-related apoptotic markers p53 and PUMA was increased by AEA through a CB1R-dependent pathway that engages p38 and p42/44 mitogen-activated protein kinases. Consistently, this effect of AEA was minimized by SR141716. In conclusion, we identified BiP as a key protein in neuronal apoptosis induced by AEA.

Cholesterol-dependent modulation of type 1 cannabinoid receptors in nerve cells

Type 1 cannabinoid receptors (CB1R) are G‐protein‐coupled receptors that mediate several actions of the endocannabinoid anandamide (N‐arachidonoylethanolamine; AEA) in the central nervous system. Here we show that cholesterol enrichment of rat C6 glioma cell membranes reduces by approximately twofold the binding efficiency (i.e., the ratio between maximum binding and dissociation constant) of CB1R and that activation of CB1R by AEA leads to approximately twofold lower [35S]GTPγS binding in cholesterol‐treated cells than in controls. In addition, we show that CB1R‐dependent signaling via adenylate cyclase and p42/p44 mitogen‐activated protein kinase is almost halved by cholesterol enrichment. Unlike CB1R, the other AEA‐binding receptor TRPV1, the AEA synthetase NAPE‐PLD, and the AEA hydrolase FAAH are not modulated by cholesterol, whereas the catalytic efficiency (i.e., the ratio between maximal velocity and Michaelis‐Menten constant) of the AEA membrane transporter AMT is almost doubled compared with control cells. These data demonstrate that, among the proteins of the “endocannabinoid system,” only CB1R and AMT critically depend on membrane cholesterol content. This observation may have important implications for the role of CB1R in protecting nerve cells against (endo)cannabinoid‐induced apoptosis.