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Endocannabinoids modulate neuroglial phenotype and proteotoxic stress
Full text linkABSTRACT
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
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.[...
In silico and in vitro analysis of major cannabis-derived compounds as fatty acid amide hydrolase inhibitors
Full text linkAccumulated evidence suggests that enhancing the endocannabinoid (eCB) tone, in partic-ular of anandamide (N-arachidonoylethanolamine, AEA), has therapeutic potential in many human diseases. Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme principally responsible for the degradation of AEA, and thus it represents a relevant target to increase signaling thereof. In recent years, different synthetic and natural compounds have been developed and tested on rat FAAH, but little is known of their effect on the human enzyme. Here, we sought to investigate six major cannabis-derived compounds to compare their action on rat and human FAAHs. To this aim, we combined an in silico analysis of their binding mode and affinity, with in vitro assays of their effect on enzyme activity. This integrated approach allowed to disclose differences in efficacy towards rat and human FAAHs, and to highlight the role of key residues involved in the inhibition of both enzymes. This study suggests that the therapeutic efficacy of compounds targeted towards FAAH should be always tested in vitro on both rat and human enzymes
The endocannabinoid system in neurodegeneration
No full textEndocannabinoids are bioactive lipids, that comprise amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the best studied endocannabinoids, and act as agonists of cannabinoid receptors. Thus, AEA and 2-AG mimic several pharmacological effects of the exogenous cannabinoid delta9-tetrahydrocannabinol, the psychoactive principle of hashish and marijuana. It is known that the activity of endocannabinoids at their receptors is limited by cellular uptake through specific membrane transporters, followed by intracellular degradation by a fatty acid amide hydrolase (for AEA and partly 2-AG) or by a monoacylglycerol lipase (for 2-AG). Together with AEA, 2-AG and congeners, the proteins that bind, transport and metabolize these lipids form the "endocannabinoid system". This new system will be briefly presented in this review, in order to put in a better perspective the role of the endocannabinoid pathway in neurodegenerative disorders, like Parkinson's disease, Huntington's disease, and multiple sclerosis. In addition, the potential exploitation of antagonists of endocannabinoid receptors, or of inhibitors of endocannabinoid metabolism, as next-generation therapeutics will be discussed
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