Endocannabinoid signaling in oligodendroglia.

In the central nervous system, oligodendrocytes synthesize the myelin, a specialized membrane to wrap axons in a discontinuous way allowing a rapid saltatory nerve impulse conduction. Oligodendrocytes express a number of growth factors and neurotransmitters receptors that allow them to sense the environment and interact with neurons and other glial cells. Depending on the cell cycle stage, oligodendrocytes may respond to these signals by regulating their survival, proliferation, migration, and differentiation. Among these signals are the endocannabinoids, lipidic molecules synthesized from phospholipids in the plasma membrane in response to cell activation. Here, we discuss the evidence showing that oligodendrocytes express a full endocannabinoid signaling machinery involved in physiological oligodendrocyte functions that can be therapeutically exploited to promote remyelination in central nervous system pathologies.

Transcription Profile and Pathway Analysis of the Endocannabinoid Receptor Inverse Agonist AM630 in the Core and Infiltrative Boundary of Human Glioblastoma Cells.

BACKGROUND: We have previously reported that the endocannabinoid receptor inverse agonist AM630 is a potent inhibitor of isocitrade dehydrogenase-1 wild-type glioblastoma (GBM) core tumour cell proliferation. To uncover the mechanism behind the anti-tumour effects we have performed a transcriptional analysis of AM630 activity both in the tumour core cells (U87) and the invasive margin cells (GIN-8), the latter representing a better proxy of post-surgical residual disease. RESULTS: The core and invasive margin cells exhibited markedly different gene expression profiles and only the core cells had high expression of a potential AM630 target, the CB1 receptor. Both cell types had moderate expression of the HTR2B serotonin receptor, a reported AM630 target. We found that the AM630 driven transcriptional response was substantially higher in the central cells than in the invasive margin cells, with the former driving the up regulation of immune response and the down regulation of cell cycle and metastatic pathways and correlating with transcriptional responses driven by established anti-neoplastics as well as serotonin receptor antagonists. CONCLUSION: Our results highlight the different gene sets involved in the core and invasive margin cell lines derived from GBM and an associated marked difference in responsiveness to AM630. Our findings identify AM630 as an anti-neoplastic drug in the context of the core cells, showing a high correlation with the activity of known antiproliferative drugs. However, we reveal a key set of similarities between the two cell lines that may inform therapeutic intervention.

Pentraxin 3 mediates neurogenesis and angiogenesis after cerebral ischaemia.

BACKGROUND: The acute phase protein pentraxin 3 (PTX3) is a new biomarker of stroke severity and is a key regulator of oedema resolution and glial responses after cerebral ischaemia, emerging as a possible target for brain repair after stroke. Neurogenesis and angiogenesis are essential events in post-stroke recovery. Here, we investigated for the first time the role of PTX3 in neurogenesis and angiogenesis after stroke. METHODS: PTX3 knockout (KO) or wild-type (WT) mice were subjected to experimental cerebral ischaemia (induced by middle cerebral artery occlusion (MCAo)). Poststroke neurogenesis was assessed by nestin, doublecortin (DCX) and bromodeoxyuridine (BrdU) immunostaining, whereas angiogenesis was assessed by BrdU, vascular endothelial growth factor receptor 2 (VEGFR2) and PECAM-1 immunostaining. In vitro neurogenesis and angiogenesis assays were carried out on neurospheres derived from WT or interleukin-1ß (IL-1ß) KO mice, and mouse endothelial cell line bEnd.5 respectively. Behavioural function was assessed in WT and PTX3 KO mice using open-field, motor and Y-maze tests. RESULTS: Neurogenesis was significantly reduced in the dentate gyrus (DG) of the hippocampus of PTX3 KO mice, compared to WT mice, 6 days after MCAo. In addition, recombinant PTX3 was neurogenic in vitro when added to neurospheres, which was mediated by IL-1ß. In vivo poststroke angiogenesis was significantly reduced in PTX3 KO mice compared to WT mice 14 days after MCAo, as revealed by reduced vascular density, less newly formed blood vessels and decreased expression of VEGFR2. In vitro, recombinant PTX3 induced marked endothelial cellular proliferation and promoted formation of tube-like structures of endothelial cell line bEnd.5. Finally, a lack of PTX3 potentiated motor deficits 14 days after MCAo. CONCLUSIONS: These results indicate that PTX3 mediates neurogenesis and angiogenesis and contributes to functional recovery after stroke, highlighting a key role of PTX3 as a mediator of brain repair and suggesting that PTX3 could be used as a new target for stroke therapy.

Neuroimmmune interactions of cannabinoids in neurogenesis: focus on interleukin-1ß (IL-1ß) signalling.

Neuroimmune networks and the brain endocannabinoid system contribute to the maintenance of neurogenesis. Activation of cannabinoid receptors suppresses chronic inflammatory responses through the attenuation of pro-inflammatory mediators. Moreover, the endocannabinoid system directs cell fate specification of NSCs (neural stem cells) in the CNS (central nervous system). The aim of our work is to understand better the relationship between the endocannabinoid and the IL-1ß (interleukin-1ß) associated signalling pathways and NSC biology, in order to develop therapeutical strategies on CNS diseases that may facilitate brain repair. NSCs express functional CB1 and CB2 cannabinoid receptors, DAGLα (diacylglycerol lipase α) and the NSC markers SOX-2 and nestin. We have investigated the role of CB1 and CB2 cannabinoid receptors in the control of NSC proliferation and in the release of immunomodulators [IL-1ß and IL-1Ra (IL-1 receptor antagonist)] that control NSC fate decisions. Pharmacological blockade of CB1 and/or CB2 cannabinoid receptors abolish or decrease NSC proliferation, indicating a critical role for both CB1 and CB2 receptors in the proliferation of NSC via IL-1 signalling pathways. Thus the endocannabinoid system, which has neuroprotective and immunomodulatory actions mediated by IL-1 signalling cascades in the brain, could assist the process of proliferation and differentiation of embryonic or adult NSCs, and this may be of therapeutic interest in the emerging field of brain repair.

Synthesis, physical-chemical characterisation and biological evaluation of novel 2-amido-3-hydroxypyridin-4(1H)-ones: Iron chelators with the potential for treating Alzheimer's disease.

A novel class of 2-amido-3-hydroxypyridin-4-one iron chelators is described. These compounds have been designed to behave as suitable molecular probes which will improve our knowledge of the role of iron in neurodegenerative conditions. Neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson disease (PD), can be considered as diverse pathological conditions sharing critical metabolic processes such as protein aggregation and oxidative stress. Interestingly, both these metabolic alterations seem to be associated with the involvement of metal ions, including iron. Iron chelation is therefore a potential therapeutic approach. The physico-chemical (pK(a), pFe(3+) and logP) and biological properties (inhibition of iron-containing enzymes) of these chelators have been investigated in order to obtain a suitable profile for the treatment of neurodegenerative conditions. Studies with neuronal cell cultures confirm that the new iron chelators are neuroprotective against ß-amyloid-induced toxicity.

The constitutive production of the endocannabinoid 2-arachidonoylglycerol participates in oligodendrocyte differentiation.

Endocannabinoids have recently emerged as instructive cues in the developing central nervous system, and, based on the expression of their receptors, we identified oligodendrocytes as potential targets of these molecules. Here, we show that the enzymes responsible for the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG), diacylglycerol lipase alpha (DAGLα) and beta (DAGLß), and degradation, monoacylglycerol lipase (MAGL), can be found in oligodendrocytes at different developmental stages. Moreover, cultured oligodendrocyte progenitor cells (OPCs) express DAGLα and ß abundantly, resulting in the stronger production of 2-AG than in differentiated oligodendrocytes. The opposite is observed with MAGL. CB1 and CB2 receptor antagonists (SR141716 and AM630) impaired OPC differentiation into mature oligodendrocytes and likewise, inhibiting DAGL activity with RHC-80267 or tetrahydrolipstatin also blocked oligodendrocyte maturation, an effect reversed by the addition of exogenous 2-AG. Likewise, 2-AG synthesis disruption using specific siRNAs against DAGLα and DAGLß significantly reduced myelin protein expression in vitro, whereas a pharmacological gain-of-function approach by using cannabinoid agonists or MAGL inhibition had the opposite effects. ERK/MAPK pathway is implicated in oligodendrocyte differentiation because PD98059, an inhibitor of MEK1, abrogated oligodendrocyte maturation. The cannabinoid receptor antagonists and RHC-80267 all diminished basal ERK1/2 phosphorylation, effects that were partially reversed by the addition of 2-AG. Overall, our data suggest a novel role of endocannabinoids in oligodendrocyte differentiation such that constitutive release of 2-AG activates cannabinoid receptors in an autocrine/paracrine way in OPCs, stimulating the ERK/MAPK signaling pathway.

Mending the broken brain: neuroimmune interactions in neurogenesis.

Neuroimmune networks and the brain endocannabinoid system contribute to the maintenance of neurogenesis. Cytokines and chemokines are important neuroinflammatory mediators that are involved in the pathological processes resulting from brain trauma, ischemia and chronic neurodegenerative diseases. However, they are also involved in brain repair and recovery. Compelling evidence obtained, in vivo and in vitro, establish a dynamic interplay between the endocannabinoid system, the immune system and neural stem/progenitor cells (NSC) in order to promote brain self-repair. Cross-talk between inflammatory mediators and NSC might have important consequences for neural development and brain repair. In addition, brain immune cells (microglia) support NSC renewal, migration and lineage specification. The proliferation and differentiation of multipotent NSC must be precisely controlled during the development of the CNS, as well as for adult brain repair. Although signalling through neuroimmune networks has been implicated in many aspects of neural development, how it affects NSC remains unclear. However, recent findings have clearly demonstrated that there is bi-directional cross-talk between NSC, and the neuroimmune network to control the signals involved in self-renewal and differentiation of NSC. Specifically, there is evidence emerging that neuroimmune interactions control the generation of new functional neurones from adult NSC. Here, we review the evidence that neuroimmune networks contribute to neurogenesis, focusing on the regulatory mechanisms that favour the immune system (immune cells and immune molecules) as a novel element in the coordination of the self-renewal, migration and differentiation of NSC in the CNS. In conjunction, these data suggest a novel mode of action for the immune system in neurogenesis that may be of therapeutic interest in the emerging field of brain repair.

The endocannabinoid system modulates a transient TNF pathway that induces neural stem cell proliferation.

Evidence is emerging that the tumour necrosis factor (TNF-alpha) is a potent signal that induces neural stem cell proliferation and migration. We show that NSC self-renewal is controlled by bi-directional cross-talk between the endocannabinoid system and the TNF signalling pathway. By blocking endogenous TNF-alpha activity, we demonstrate that the TNF system is critical for the proliferation of NSC. Furthermore, we show that pharmacological blockade of the CB1/CB2 cannabinoid receptors dramatically suppresses TNF-alpha-induced NSC proliferation. Interestingly, we found that CB1 or CB2 agonists induce NSC proliferation coupled to a significant increase in both TACE/ADAM 17 and TNF-alpha levels. Overall these data suggest a novel mode of action for the endocannabinoid system in NSC proliferation that is coupled to TNF signalling and that may be of therapeutic interest in the emerging field of brain repair.

A diacylglycerol lipase-CB2 cannabinoid pathway regulates adult subventricular zone neurogenesis in an age-dependent manner.

The subventricular zone (SVZ) is a major site of neurogenesis in the adult. We now show that ependymal and proliferating cells in the adult mouse SVZ express diacylglycerol lipases (DAGLs), enzymes that synthesise a CB1/CB2 cannabinoid receptor ligand. DAGL and CB2 antagonists inhibit the proliferation of cultured neural stem cells, and the proliferation of progenitor cells in young animals. Furthermore, CB2 agonists stimulate progenitor cell proliferation in vivo, with this effect being more pronounced in older animals. A similar response was seen with a fatty acid amide hydrolase (FAAH) inhibitor that limits degradation of endocannabinoids. The effects on proliferation were mirrored in changes in the number of neuroblasts migrating from the SVZ to the olfactory bulb (OB). In this context, CB2 antagonists reduced the number of newborn neurons appearing in the OB in the young adult animals while CB2 agonists stimulated this in older animals. These data identify CB2 receptor agonists and FAAH inhibitors as agents that can counteract the naturally observed decline in adult neurogenesis that is associated with ageing.

Cannabinoids and their actions: An update.

LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Effect of endocannabinoid signalling on cell fate: life, death, differentiation and proliferation of brain cells.

Cell fate events are regulated by different endogenous developmental factors such as the cell micro-environment, external or remote signals and epigenetic factors. Among the many regulatory factors, endocannabinoid-associated signalling pathways are known to conduct several of these events in the developing nervous system and in the adult brain. Interestingly, endocannabinoids exert modulatory actions in both physiological and pathological conditions. Endocannabinoid signalling can promote cell survival by acting on non-transformed brain cells (neurons, astrocytes or oligodendrocytes) and can have either a protumoural or antitumoural effect on transformed cells. Moreover, endocannabinoids are able to attenuate the detrimental effects on neurogenesis and neuroinflammation associated with ageing. Thus, the endocannabinoid system emerges as an important regulator of cell fate, controlling cell survival/cell death decisions depending on the cell type and its environment. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Neuroprotective actions of deferiprone in cultured cortical neurones and SHSY-5Y cells.

Alzheimer's disease (AD) is a common neurodegenerative disorder, but the initiating molecular processes contributing to neuronal death are not well understood. AD is associated with elevated soluble and aggregated forms of amyloid beta (Abeta) and with oxidative stress. Furthermore, there is increasing evidence for a detrimental role of iron in the pathogenic process. In this context, iron chelation by compounds such as 3-hydroxypyridin-4-one, deferiprone (Ferriprox) may have potential neuroprotective effects. We have evaluated the possible neuroprotective actions of deferiprone against a range of AD-relevant insults including ferric iron, H(2)O(2) and Abeta in primary mouse cortical neurones. We have investigated the possible neuroprotective actions of deferiprone (1, 3, 10, 30 or 100 microM) in primary neuronal cultures following exposure to ferric iron [ferric nitrilotriacetate (FeNTA); 3 and 10 microM], H(2)O(2) (100 microM) or Abeta1-40 (3, 10 and 20 microM). Cultures were treated with deferiprone or vehicle either immediately or up to 6 h after the insult in a 24-well plate format. In order to elucidate a possible neuroprotective action of deferiprone against Parkinson's disease relevant insults another group of experiments were performed in the human neuroblastoma catecholaminergic SHSY-5Y cell line. SHSY-5Y cells were treated with MPP(+) iodide, the active metabolite of the dopaminergic neurotoxin MPTP and the neuroprotective actions of deferiprone evaluated. Cytotoxicity was assessed at 24 h by lactate dehydrogenase release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide turnover (FeNTA and hydrogen peroxide) and morphometric analysis of cell viability by Hoechst 33324/propidium iodide (FeNTA, Abeta and MPP(+)) or 6-carboxyfluorescein diacetate and annexin V-Cy3 (Abeta). The present study demonstrates that deferiprone protects against FeNTA, hydrogen peroxide, MPP(+) and Abeta1-40-induced neuronal cell death in vitro, which is consistent with previous in vitro and in vivo studies that have demonstrated similar protection with other iron chelators.

Iron chelation as a potential therapy for neurodegenerative disease.

Neurodegenerative disorders include a variety of pathological conditions, which share similar critical metabolic processes such as protein aggregation and oxidative stress, both of which are associated with the involvement of metal ions. Chelation therapy could provide a valuable therapeutic approach to such disease states, since metals, particularly iron, are realistic pharmacological targets for the rational design of new therapeutic agents.

Inflammatory mediators in the frontal lobe of patients with mixed and vascular dementia.

Vascular dementia (VaD) accounts for about 20% of all dementias, and vascular risk is a key factor in more than 40% of people with Alzheimer's disease (AD). Little is known about inflammatory processes in the brains of these individuals. We have examined inflammatory mediators (interleukin (IL)-1beta, IL-1alpha, IL-6 and tumour necrosis factor alpha) and chemokines (macrophage inflammatory protein 1, monocyte chemo-attractant protein (MCP)-1 and granulocyte macrophage colony-stimulating factor) in brain homogenates from grey and white matter of the frontal cortex (Brodmann area 9) from patients with VaD (n = 11), those with concurrent VaD and AD (mixed dementia; n = 8) and from age-matched controls (n = 13) using ELISA assays. We found a dramatic reduction of MCP-1 levels in the grey matter in VaD and mixed dementia in comparison to controls (55 and 66%, respectively). IL-6 decreases were also observed in the grey matter of VaD and mixed dementia (72 and 71%, respectively), with a more modest decrease (30%) in the white matter of patients with VaD or mixed dementia. In the first study to examine the status of inflammatory mediators in a brain region severely affected by white-matter lesions, our findings highlight - in contrast to previous reports in AD - that patients at the later stage of VaD or mixed dementia have a significantly attenuated neuro-inflammatory response.

Spicing Up Pharmacology: A Review of Synthetic Cannabinoids From Structure to Adverse Events.

Recreational use of synthetic cannabinoids (SCB), a class of novel psychoactive substances is an increasing public health problem specifically in Western societies, with teenagers, young adults, and the prison population being the most affected. Some of these SCB are analogs of tetrahydrocannabinol, aminoalkylindoles, and other phytocannabinoid analogs have been detected in herbal preparations generically called "Spice." Spice, "K2" or "fake cannabis" is a general term used for variable herbal mixtures of unknown ingredients or chemical composition. SCB are highly potent CB1 cannabinoid receptor agonists falsely marketed and sold as safe and legal drugs. Here, we present an overview of the endocannabinoid system, CB, and SCB chemical structures and activity at CB receptors. Finally, we highlight the psychological effects of SCB, particularly on learning and memory, and adverse clinical effects including on the cardiovascular system, kidneys, and CNS, including psychosis. Taken together, it is clear that many SCB are extremely dangerous and a major public health problem.

Endotoxin preconditioning protects neurones from in vitro ischemia: role of endogenous IL-1beta and TNF-alpha.

We have examined whether changes in the expression of several inflammatory factors mediate the neuroprotective action of LPS preconditioning on cerebellar granule neurones (CGN) exposed to the mitochondrial toxin 3-nitropropionic acid (3-NP), chosen as an in vitro ischemic model. CGN were either directly pre-treated with LPS or indirectly by exposure to conditioned medium (CM) from LPS-treated mixed glial cultures obtained from wild type or IL-1beta-knock out mice. Following this pre-treatment CGN were incubated with 3-NP and cell viability assessed. Our results show that LPS preconditioning in neurones, promotes neuronal survival against 3-NP-induced cell death and that endogenous TNF-alpha is a critical mediator for the neuroprotective actions of LPS independently of the presence of endogenous IL-1beta after 3-NP exposure.

Cannabinoids promote oligodendrocyte progenitor survival: involvement of cannabinoid receptors and phosphatidylinositol-3 kinase/Akt signaling.

Cannabinoids exert pleiotropic actions in the CNS, including the inhibition of inflammatory responses and the enhancement of neuronal survival after injury. Although cannabinoid receptors are distributed widely in brain, their presence has not been investigated previously in oligodendrocytes. This study examined the expression of cannabinoid type 1 (CB1) receptors in rat oligodendrocytes in vivo and in culture and explored their biological function. Expression of CB1 receptors by oligodendrocytes was demonstrated immunocytochemically in postnatal and in adult white matter as well as in oligodendrocyte cultures. Reverse transcription-PCR and Western blotting further confirmed the presence of CB1 receptors. Oligodendrocyte progenitors undergo apoptosis with the withdrawal of trophic support, as determined by TUNEL assay and caspase-3 activation, and both the selective CB1 agonist arachidonyl-2'-chloroethylamide/(all Z)-N-(2-cycloethyl)-5,8,11,14-eicosatetraenamide (ACEA) and the nonselective cannabinoid agonists HU210 and (+)-Win-55212-2 enhanced cell survival. To investigate intracellular signaling involved in cannabinoid protection, we focused on the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. HU210, (+)-Win-55212-2, and ACEA elicited a time-dependent phosphorylation of Akt. Pertussis toxin abolished Akt activation, indicating the involvement of G(i)/G(o)-protein-coupled receptors. The CB1 receptor antagonist SR141716A partially inhibited Akt phosphorylation in response to HU210 and (+)-Win-55212-2 and abolished the effects of ACEA. Trophic support deprivation downregulated Akt activity, and cannabinoids recovered phospho-Akt levels. Inhibition of PI3K abrogated the survival action and the recovery of Akt activity in response to cannabinoids. SR141716A prevented only the protection conferred by ACEA. Nevertheless, SR141716A and the selective CB2 receptor antagonist SR144528 in combination inhibited the prosurvival action of HU210, which is in accordance with the finding of CB2 receptor expression by oligodendroglial cells. These data identify oligodendrocytes as potential targets of cannabinoid action in the CNS.

Endogenous interleukin-1 receptor antagonist mediates anti-inflammatory and neuroprotective actions of cannabinoids in neurons and glia.

Interleukin-1 receptor antagonist (IL-1ra) is an important anti-inflammatory cytokine that blocks all known actions of IL-1 and markedly protects against experimentally induced ischemic, excitotoxic, and traumatic brain insults. Cannabinoids (CBs) also exert potent anti-inflammatory and neuroprotective effects, but the mechanisms of their actions are unknown. Here we tested the hypothesis that the actions of CBs are mediated by endogenous IL-1ra. We report for the first time that both CB1 and CB2 receptors modulate release of endogenous IL-1ra from primary cultured glial cells. Activation of CB1 or CB2 receptors increased lipopolysaccharide-induced IL-1ra release, and specific CB1 or CB2 antagonists blocked lipopolysaccharide-induced production of IL-1ra from glial cells. Comparison of neuronal cultures from wild-type mice and mice lacking IL-1ra (knock-out) indicates that endogenous IL-1ra is essential for the neuro-protective effects of CBs against excessive activation of glutamate receptors (excitotoxicity) in response to S-AMPA or NMDA. Similarly, analysis of mixed glial cultures from IL-1ra knock-out mice indicates that endogenous IL-1ra is required for the CB-induced inhibition of nitric oxide production in response to bacterial lipopolysaccharide. These data suggest a novel neuroprotective mechanism of action for CBs in response to inflammatory or excitotoxic insults that is mediated by both CB1 and CB2 receptor-dependent pathways.

The role of cannabinoid system on immune modulation: therapeutic implications on CNS inflammation.

There is a growing amount of evidence suggesting that cannabinoids may be neuroprotective in CNS inflammatory conditions. Advances in the understanding of the physiology and pharmacology of the cannabinoid system have increased the interest of cannabinoids as potential therapeutic targets. Cannabinoid receptors and their endogenous ligands, the endocannabinoids, have been detected in cells of the immune system, as well as in brain glial cells. In the present review it is summarized the effects of cannabinoids on immune reactivity and on the regulation of neuroinflammatory processes associated with brain disorders with special attention to chronic inflammatory demyelinating diseases such as multiple sclerosis.

A Basal Tone of 2-Arachidonoylglycerol Contributes to Early Oligodendrocyte Progenitor Proliferation by Activating Phosphatidylinositol 3-Kinase (PI3K)/AKT and the Mammalian Target of Rapamycin (MTOR) Pathways.

A basal tone of the endocannabinoid 2-arachidonoylglycerol (2-AG) enhances late oligodendrocyte progenitor cell (OPC) differentiation. Here, we investigated whether endogenous 2-AG may also promote OPC proliferation in earlier stages. We found that the blockade of 2-AG synthesizing enzymes, sn-1-diacylglycerol lipases α and ß (DAGLs), with RHC-80267 or the antagonism of either CB1 or CB2 cannabinoid receptors with AM281 and AM630, respectively, impaired early OPC proliferation stimulated by platelet-derived growth factor (PDGF-AA) and basic fibroblast growth factor (bFGF). On the contrary, increasing the levels of endogenous 2-AG by blocking the degradative enzyme monoacylglycerol lipase (MAGL) with JZL-184, significantly increased OPC proliferation as did agonists of cannabinoid receptor CB1 (ACEA), CB2 (JWH133) or both (HU-210). To elucidate signaling pathways underlying OPC proliferation, we studied the involvement of phosphatidylinositol 3-kinase (PI3K)/Akt and its downstream target mammalian target of rapamycin (mTOR). We show that phosphorylation of Akt and mTOR is required for OPC proliferation stimulated by growth factors (PDGF-AA and bFGF) or by CB1/CB2 agonists (ACEA/JWH133), since it was strongly decreased after LY294002 or rapamycin treatment. In line with this, blockade of CB1 (AM281), CB2 (AM630) or DAGLs (RHC-80267), decreased phosphorylation of Akt, mTOR and 4E-BP1, diminished cyclin E-cdk2 complex association and increased p27(kip1) levels. Our data suggest that proliferation of early OPCs stimulated by PDGF-AA and bFGF depends on the tonic activation of cannabinoid receptors by endogenous 2-AG and provide further evidence on the role of endocannabinoids in oligodendrocyte development, being important for the maintenance and self-renewal of the OPCs. The results highlight the therapeutic potential of the endocannabinoid signaling in the emerging field of brain repair.