242 research outputs found
Endocannabinoids modulate neuroglial phenotype and proteotoxic stress
No full textABSTRACT
Neuronal survival in neurodegenerative diseases and brain damage is closely related to the cell populations of the environment and in particular to glial cells. Astrocytes, microglia and oligodendrocytes oversee brain homeostasis providing the intrinsic brain defence system. Damage to brain cells triggers a condition generally referred to as reactive gliosis, which includes astrogliosis and activation of microglia. Neuroglia is also thoroughly involved in pathogenesis of many chronic neurological disorders and in neurodegeneration. Endocannabinoids modulating the behaviour of microglia and astrocytes might act as possible targets for therapeutic intervention. Recent studies have indicated that endocannabinoid levels and metabolic enzymes change during the progression of Alzheimer's disease (AD) and that the inhibition of fatty acid amide hydrolase (FAAH), the main catabolic enzyme of anandamide (AEA), has beneficial effects in mice with AD. The aim of this study was to determine whether URB597, a FAAH inhibitor, targets microglia polarization by altering the cytoskeleton reorganization induced by amyloid-β peptide (Aβ) in BV-2 microglial cells. Evaluation of actin cytoskeleton showed that Aβ treatment increased the surface area of BV-2 cells, which acquired a flat and polygonal morphology. Although URB597 did not affect cell morphology only, it partially rescued the control phenotype in BV-2 cells incubated with the combined treatment. Rho family proteins have a critical role in the plasticity of the actin cytoskeleton, influencing morphological changes, migration and phagocytic activity of cells. We observed an increase of Rho protein activation in Aβ samples and a decrease in samples treated with URB597 alone or in combination with Aβ compared to controls, while an increase of Cdc42 protein activation was observed in all samples with respect to control. Aβ induced the migration of BV-2 cells up to 2 h after stimulation. We also found that by reducing Rho protein activity, URB597 was able to reduce the migration rate. URB597 also increased the number of BV-2 cells performing phagocytosis. Taken together, these data suggest that an increase of anandamide (AEA), due to FAAH inhibition, may induce cytoskeleton reorganization, regulating phagocytosis and cell migration processes, and promote microglial polarization towards an anti-inflammatory phenotype.
As most research worldwide has focused on neurons, there is a dearth of protocols to generate glial cells and to produce co-culture systems for biomedical research. The aim of this project has also been the generation of co-culture with neurons, astrocytes and microglia cells and the subsequent characterization of the resulting model, evaluating interspecies differences through the generation of co-cultures with murine microglia. We focused our interest on the repair functions during brain injury and on the interactions between microglia and astrocytes. The protective effect of astrocytes and microglia against neuronal cells in the presence of inflammatory and pro-apoptotic processes was investigated. Human astrocytes and human microglia cells were activated with TNF-, IL-1 and IFN- to evaluate the inflammatory response. The results showed an increase of inflammatory cytokines gene expression such as IL-6 and IL-8 in both cell lines examined. The astrocytes activation by TNF-, or by conditioned medium (CM) of activated microglia cells was confirmed by NF-kB nuclearization. Therefore, the arise of inflammatory process in astrocyte cells is driven not only by TNF- induction, but also by a synergic effect due to microglia activation. Neuroinflammation, oxidative stress, and progressive degeneration of specific brain regions is also driven by proteasomal impairment, promoting protein accumulations. Since LUHMES neurons are quite susceptible cells to proteotoxic stress and amino acid starvation, we investigated whether murine microglia and human astrocytes exerted a protective effect also when the cell lines were treated with URB597. The obtained data demonstrated that the astrocytes through the glutathione (GSH) release, were able to attenuate neuronal proteotoxic stress in LUHMES cells. URB597 contributed to GSH anti-oxidant effects modulating GSH metabolism. The overall data demonstrated that neuroglial cells play a pivotal role on neuronal protection from noxious stimuli
Endocannabinoids and their involvement in the neurovascular system
No full textEndocannabinoids are a new class of lipids, which include amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of Delta(9)-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. It is known that the activity of AEA is limited by cellular uptake through a specific membrane transporter, followed by intracellular degradation by a fatty acid amide hydrolase. Together with AEA and congeners these proteins form the "endocannabinoid system". The endogenous cannabinoids were identified in brain, and also in neuronal and endothelial cells, suggesting a potential role as modulators in the central nervous system and in the periphery. This review summarises the metabolic routes for the synthesis and degradation of AEA, and the latest advances in the involvement of this lipid in neurovascular biology. In addition, the therapeutic potential of the modulation of endocannabinoid metabolism for neuronal and vascular system will be also reviewed
Regulation of endocannabinoid system by lipid rafts along the neuroimmune axis
No full textL’anandamide (arachidonoiletanolammide, AEA), insieme con l’altro endocannabinoide 2arachidonoilglicerolo (2-AG), lega ed attiva i due recettori accoppiati a proteine G inibitorie, (GPCR), chiamati recettori dei cannabinoidi di tipo-1 (CB1R) e di tipo-2 (CB2R). I CB1R sono localizzati principalmente nel sistema nervoso centrale, ma sono anche espressi in tessuti periferici come le cellule immunitarie. Mentre i CB2R sono maggiormente espressi a livello periferico, ma sono anche presenti nel cervello. Quindi l’attivazione dei recettori CB1 o CB2 da parte dell’AEA e del 2-AG ha molti effetti sia a livello centrale che periferico. Queste azioni sono controllate attraverso un non ancora caratterizzato meccanismo cellulare, che regola il rilascio degli endocannabinoidi da precursori di membrana, il loro trasporto all’interno delle cellule, ed infine la loro eliminazione. L’agente chiave nella sintesi dell’AEA è la N-acilfosfatidiletanolammine (NAPE)-fosfolipasi D (NAPE-PLD), mentre la sua degradazione avviene attraverso un trasportatore di membrana per AEA (AMT), ed una fatty acid amide hydrolase (FAAH). Oltre i recettori CB, l’AEA lega anche il recettore dei vanilloidi di tipo 1 (TRPV1). Il 2-AG è invece rilasciato da lipidi di membrana per azione di una lipasi specifica, sn-1-diacilglicerolo (DAGL), ed è idrolizzato da una specifica monoacilglicerolo lipasi (MAGL). E’ stato dimostrato che il trasporto del 2-AG attraverso la membrana cellulare è saturabile ed energia-indipendente e che può occorrere attraverso lo stesso trasportatore dell’AEA. L’AEA ed il 2-AG, con altri congeneri, le proteine che legano, trasportano, sintetizzano ed idrolizzano questi lipidi formano il “sistema endocannabinoide”. I lipid rafts sono subdomini della membrana plasmatica che contengono alte concentrazioni di colesterolo e di sfingolipidi, e sono ben conosciuti modulatori dell’attivitità di un numero di GPCR. Infatti, essi modulano il segnale ed il “trafficking” in molti tipi cellulari. La crescente evidenza che i lipid rafts possono modulare il segnale degli endocannabinoidi ci ha spinto ad investigare il possibile effetto della loro integrità sui recettori CB, sul metabolismo dell’AEA e del 2-AG in cellule neuronali e del sistema immunitario. A tale scopo abbiamo utilizzato la metil-β-ciclo destrina (MCD), un depletore del colesterolo, un composto largamente utilizzato per distruggere l’integrità dei lipid rafts. Abbiamo utilizzato le cellule di glioma di ratto C6, perché esse hanno un ben caratterizzato sistema endocannabinoide. Abbiamo poi esteso lo studio alla linea cellulare di neuroblastoma umano CHP100, la quale ha la stessa abilità delle cellule C6 di metabolizzare l’AEA, ma sono prive del CB1R e quindi sono più sensibili all’attività pro-apoptotica dell’AEA. Abbiamo inoltre esteso lo studio agli enzimi che degradano e sintetizzano il 2-AG. Inoltre, abbiamo studiato la linea cellulare umana DAUDI, perché possiede l’AMT e l’enzima FAAH ed esprime un CB2R funzionale. Le cellule DAUDI, inoltre, regolano importanti funzione in cui sono coinvolti i lipid rafts, come la secrezione di esosomi e l’arresto della crescita indotta dai farmaci antitumorali. Inoltre abbiamo valutato l’effetto della deplezione e dell’arrichimento di colesterolo sul metabolismo degli endocannabinoidi. In conclusione, questo studio ha monitorato l’effetto dell’integrità dei lipid rafts sulle principali proteine che legano e metabolizzano l’AEA ed il 2-AG, sia in cellule neuronali che in cellule del sistema immunitario. Tale studio indica che il CB1R ed il trasportatore degli endocannabinoidi sono probabilmente localizzati all’interno dei lipid rafts a differenza del CB2R e delle altre proteine che compongono il sistema endocannabinoide.Anandamide (arachidonoylethanolamide, AEA) and the other endocannabinoid 2-arachidonoylglycerol (2-AG) bind to and activate two G protein-coupled receptors (GPCR), namely type-1 (CB1R) and type-2 (CB2R) cannabinoid receptors. CB1R are localized mainly in the central nervous system, but are also expressed in peripheral tissues like immune cells. Conversely CB2R are predominantly expressed peripherally, but they are also present in the brain. Therefore, activation of CB1 or CB2 receptors by AEA or 2-AG has many central and peripheral effects. These actions are controlled through not yet fully characterized cellular mechanisms, that regulate the release of endocannabinoids from membrane precursors, their uptake by cells, and finally their intracellular disposal. The key agent in AEA synthesis is the N-acylphosphatidylethanolamines (NAPE)-hydrolyzing phospholipase D (NAPE-PLD), whereas degradation occurs through an AEA membrane transporter (AMT), and a fatty acid amide hydrolase (FAAH). Besides CB receptors, AEA binds also to type 1 vanilloid receptors (now called transient receptor potential channel vanilloid receptor subunit 1, TRPV1). On the other hand, 2-AG is released from membrane lipids by means of a sn-1-specific diacylglycerol lipase (DAGL), and is hydrolyzed by a specific monoacylglycerol lipase (MAGL). The transport of 2-AG through the cellular membrane has been shown to be saturable and energyindependent, and might occur through the same AMT that transports AEA. Altogether AEA and 2-AG, with other congeners, the proteins that bind, transport, synthesize and hydrolyze these lipids, form the “endocannabinoid system”. Lipid rafts are subdomains of the plasma membrane that contain high concentrations of cholesterol and glycosphingolipids, and are well-known modulators of the activity of a number of GPCR. In fact, they modulate signaling and membrane trafficking in many cell types. The growing evidence suggesting that lipid rafts might modulate the endocannabinoid signaling prompted us to investigate also the possible effect of lipid rafts integrity on CB receptors, on AEA metabolism in neuronal and immune cells and on the proteins that synthesize, transport and degrade 2-AG. We have used the methyl--cyclodextrin (MCD), a membrane cholesterol depletor that is widely used to disrupt the integrity of lipid rafts. We have chosen rat C6 glioma cells, because they have a well characterized endocannabinoid system. We extended the study to human CHP100 neuroblastoma cells, which have the same ability as C6 cells to metabolize AEA, but are devoid of CB1R and hence are more sensitive to the pro-apoptotic activity of AEA. We did not further extend this study to 2-AG and the enzymes that degrade and synthesize it, because 2-AG does not have pro-apoptotic activity toward C6 cells or CHP100 cells, in keeping with the observation that it does not activate TRPV1 receptors. Furthermore, we have chosen human DAUDI leukemia cells, because they have active AMT and FAAH, and express functional CB2R. On the other hand, in DAUDI cells lipid rafts regulate important functions like exosome secretion, or growth arrest induced by antitumor drugs. In addition, we checked for the first time the effect of membrane cholesterol depletion or enrichment on 2-AG metabolism in C6 cells and DAUDI cells. In conclusion, this study monitor the effect of lipid rafts integrity on all the major proteins that bind and metabolize AEA and 2-AG, both in neuronal and immune cells. The results point out that CB1R and endocannabinoid transporters are probably localized within lipid rafts, at variace with CB2R and the other proteins of the endocannabinoid system
Radiometric Assay of NAPE-PLD Activity
No full textN-Acyl-phosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) is a prominent enzyme involved in the biosynthesis of fatty acid amides, a family of bioactive lipids including anandamide as the prototypical member. here, we describe a NAPE-PLD assay based on radioactive substrates and product separation by thin layer chromatography (TLC)
Endocannabinoids and their involvement in the neurovascular system
No full textEndocannabinoids are a new class of lipids, which include amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of Delta(9)-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. It is known that the activity of AEA is limited by cellular uptake through a specific membrane transporter, followed by intracellular degradation by a fatty acid amide hydrolase. Together with AEA and congeners these proteins form the "endocannabinoid system". The endogenous cannabinoids were identified in brain, and also in neuronal and endothelial cells, suggesting a potential role as modulators in the central nervous system and in the periphery. This review summarises the metabolic routes for the synthesis and degradation of AEA, and the latest advances in the involvement of this lipid in neurovascular biology. In addition, the therapeutic potential of the modulation of endocannabinoid metabolism for neuronal and vascular system will be also reviewed
LIPID RAFTS, CB2 RECEPTOR SIGNALING AND METABOLISM OF 2-ARACHIDONOYL-GLYCEROL IN HUMAN IMMUNE CELLS
No full textRecently we have shown that treatment of rat C6 glioma cells with the membrane cholesterol depletor and 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 (AC) and p42/p44 mitogen activated protein kinase (MAPK) activity (Bari, M., Battista, N., Fezza, F., Finazzi-Agrò, A. and Maccarrone, M. (2005) J. Biol. Chem. 280, 12212-12220). Here, we investigated whether also type-2 cannabinoid receptors (CB2R), widely expressed inimmune cells, are modulated by MCD. We show that treatment of human DAUDI leukemia cells with MCD does not affect AEA binding to CB2R. The activation of CB2R by AEA triggers a similar [35S]GTPγS binding in MCD-treated and controlcells, and thus a similar effect on AC and MAPK activity, and on MAPK-dependent protection against apoptosis. The other AEA-binding receptor TRPV1, the AEA synthetase NAPE-PLD and the AEA hydrolase FAAH were not affected by MCD,whereas the activity of the AEA membrane transporter AMT was reduced to ~55% of the controls. Furthermore, neither diacylglycerol lipase nor monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoylglycerol, were affected by MCD, whereas the transport of 2-arachidonoylglycerol was reduced to ~50%. Instead, membrane cholesterol enrichment almost doubled the uptake of 2-arachidonoylglycerol and AEA. Transient expression of CB1R and CB2R in human immune cells ruled out that the different effect of raft disruption on the two receptor subtypes might be due to the gross cellular environment. Altogether, the present data demonstrate that lipid rafts control the activity of CB1R, but not that of CB2R, and endocannabinoid transport across the plasma membranes.[...
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