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.
Main Points
In oligodendroglial cells there is an operational endocannabinoid signaling network.
2‐AG contributes to the proliferation, directional migration and maturation of OPCs.
Manipulation of the endocannabinoid system exert beneficial therapeutic effects.
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•We provide precise experimental guidelines for CB1 detection by Western blot and immunoprecipitation.•We describe the formation of high molecular weight CB1 aggregates after ...heating.•We identify n-dodecyl-β-d-maltoside, DDM, as a preferred detergent for CB1 detection by WB and IP.•Based on WB, IP and deglycosylation results, we propose a new interpretation of CB1 WB and IP data.
Cannabinoid receptor 1 (CB1) identification by western blot (WB) has generated a great deal of controversial data making the interpretation of the results difficult. Our purpose is to find the most adequate experimental conditions to detect CB1 by WB and immunoprecipitation (IP) as a first step towards the study of CB1 interactome.
We use CB1 knockout mice tissue as negative controls and describe appropriate sample handling conditions for CB1 detection by WB and IP from brain and cortical neuron cultures.
Sample heating above 65 °C greatly impaired CB1 detection by WB, since it favored the formation of high molecular weight aggregates. We also show the convenience of using n-dodecyl-β-d-maltoside (DDM) as a detergent for the detection of CB1 by WB and, mostly, for IP.
We obtain consistent and specific CB1 detection by WB and IP using four different commercial antibodies and KO tissue for an accurate CB1 identification. We clarify the identification of the receptor in complex samples compared with the diverse and unclear results obtained using standard WB methods.
We establish experimental guidelines for the detection of CB1 by WB and the study of CB1 interacting proteins by IP. We propose a new interpretation of CB1 WB and IP data based on the folding and packing state of the protein and the detergent used. The standardization of the most advantageous conditions for coimmunoprecipitation (CoIP) would be a useful tool for the future study of the interactome of CB1.
CB1 cannabinoid receptor is widely expressed in the central nervous system of animals from late prenatal development to adulthood. Appropriate activation and signaling of CB1 cannabinoid receptors in ...cortical interneurons are crucial during perinatal/postnatal ages and adolescence, when long‐lasting changes in brain activity may elicit subsequent appearance of disorders in the adult brain. Here we used an optimized immunoprecipitation protocol based on specific antibodies followed by shot‐gun proteomics to find CB1 interacting partners in postnatal rat GABAergic cortical neurons in vitro at two different stages of maturation. Besides describing new proteins associated with CB1 like dihydrolipoyllysine‐residue acetyltransferase component of pyruvate dehydrogenase complex (DLAT), fatty acid synthase (FASN), tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein zeta (YWHAZ), voltage‐dependent anion channel 1 (VDAC1), myosin phosphatase Rho‐interacting protein (MPRIP) or usher syndrome type‐1C protein‐binding protein 1 (USHBP1), we show that the signaling complex of CB1 is different between maturational stages. Interestingly, the CB1 signaling complex is enriched at the more immature stage in mitochondrial associated proteins and metabolic molecular functions, whereas at more mature stage, CB1 complex is increased in maturation and synaptic‐associated proteins. We describe also interacting partners specifically immunoprecipitated with either N‐terminal or C‐terminal CB1 directed antibodies. Our results highlight new players that may be affected by altered cannabinoid signaling at this critical window of postnatal cortical development.
Cannabinoids are lipid mediators that participate in brain development. Appropriate activation and signaling of CB1 cannabinoid receptor are crucial during perinatal/postnatal ages and adolescence when long‐lasting changes in brain activity may elicit subsequent appearance of disorders in the adult brain. Here we describe new molecular partners that associate with CB1 during neuronal postnatal maturation and may mediate the different cannabinoid actions along with the development of brain circuits.
Theiler's virus infection of the CNS induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). ...Cannabinoids may act as immunosuppressive compounds that have shown therapeutic potential in chronic inflammatory disorders. Using the Theiler's murine encephalomyelitis virus model, we report here that treatment with the synthetic cannabinoids WIN 55,212-2, ACEA, and JWH-015 during established disease significantly improved the neurological deficits in a long-lasting way. At a histological level, cannabinoids reduced microglial activation, abrogated major histocompatibility complex class II antigen expression, and decreased the number of CD4+ infiltrating T cells in the spinal cord. Both recovery of motor function and diminution of inflammation paralleled extensive remyelination. Overall, the data presented may have potential therapeutic implications in demyelinating pathologies such as MS; in particular, the possible involvement of cannabinoid receptor CB2 would enable nonpsychoactive therapy suitable for long-term use.
Neurospheres are clonal cellular aggregates of neural stem/precursor cells that grow in culture as free‐floating clusters. Activation of CB1 cannabinoid receptors, which are expressed by these cells, ...promotes proliferation. In the present study we investigated the expression of CB2 cannabinoid receptors and the effect of exogenous cannabinoids on neural stem/precursor cell proliferation. Neurospheres containing nestin‐positive and sn‐1 diacylglycerol lipase α‐positive cells expressed both CB1 and CB2 receptors, which were maintained through several passages. Application of the non‐selective cannabinoid agonist (HU‐210, 0.5 µm) stimulated bromodeoxyuridine incorporation and neurosphere formation. This action involved both CB1 and CB2 receptors as neurosphere formation was stimulated by either selective CB1 arachidonyl‐2’chloroethylamide/(all Z)‐N‐(2‐cycloethyl)‐5,8,11,14‐eicosatetraenamide (ACEA), 200 nm and 1 µm or CB2 (JWH‐056, 0.5 µm) agonists. In addition, CB1 or CB2 antagonists (1 µm SR‐141716A and SR‐144528, respectively) blocked basal proliferation, suggesting that endogenous cannabinoids are implicated in neurosphere proliferation. In addition, cannabinoid agonist‐stimulated proliferation was reduced by the Akt translocation inhibitor BML‐257 (12.5 µm), suggesting a role for phosphoinositide‐3 kinase signalling. Together, our results suggest that cannabinoids stimulate proliferation of neural stem/precursor cells acting on both CB1 and CB2 cannabinoid receptors through a phosphoinositide‐3 kinase/Akt pathway.
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.
The subventricular zone (SVZ) is a source of post‐natal glial precursors that can migrate to the overlying white matter, where they may differentiate into oligodendrocytes. We showed that, in the ...post‐natal SVZ ependymocytes, radial glia and astrocyte‐like cells express cannabinoid receptor 1 (CB1), whereas cannabinoid receptor 2 (CB2) is found in cells expressing the polysialylated neural cell adhesion molecule. To study CB1 and CB2 function, post‐natal rats were exposed to selective CB1 or CB2 agonists (arachidonyl‐2‐chloroethylamide and JWH‐056, respectively) for 15 days. Accordingly, we found that CB1 activation increases the number of Olig2‐positive cells in the dorsolateral SVZ, whereas CB2 activation increases polysialylated neural cell adhesion molecule expression in this region. As intense myelination occurs during the first weeks of post‐natal development, we examined how modulating these factors affected the expression of myelin basic protein. Pharmacological administration of agonists and antagonists of CB1 and CB2 showed that the activation of both receptors is needed to augment the expression of myelin basic protein in the subcortical white matter.
Proinflammatory mediators have been implicated in demyelinating disorders, including multiple sclerosis, whereas it has been proposed that the anti‐inflammatory cytokines interleukin‐ (IL‐) 4 and ...IL‐10 participate in disease recovery. The present study analysed the effect of interferon‐γ (IFN‐γ) and bacterial endotoxin (lipopolysaccharide, LPS) on proliferation and survival of progenitors and differentiated oligodendrocytes. We also investigated the presence of receptors for IL‐4 and IL‐10 in oligodendroglial cells and explored a possible protective action of IL‐4 and IL‐10 in cultures following LPS/IFN‐γ. Finally, the role of endogenous nitric oxide (NO) on cell viability and the modulatory action of IL‐4 and IL‐10 on inducible nitric oxide synthase (iNOS) expression were also analysed. We report that LPS and/or IFN‐γ reduced proliferation and viability of oligodendroglial cells. Cell death, presumably by apoptosis as evidence by TUNEL and Annexin V binding, was observed following LPS/IFN‐γ, progenitors being more sensitive than differentiated cells. At both developmental stages, LPS/IFN‐γ‐treated cultures expressed iNOS protein and released micromolar concentrations of NO. In progenitors, LPS/IFN‐γ‐mediated cell damage was partially dependent on endogenous NO production, whereas NO was fundamental for cytotoxicity of differentiated oligodendrocytes. Both cell types expressed mRNA for IL‐4 and IL‐10 receptors and expression of IL‐10 receptors at the protein level was also demonstrated. Treatment with either cytokine inhibited the expression of iNOS resulting from the proinflammatory stimulation. IL‐10 was more effective than IL‐4 in suppressing iNOS expression and, interestingly, IL‐10 conferred protection against oligodendroglial death evoked by LPS/IFN‐γ. Our data raise the question of whether IL‐10 may play a protective role in demyelinating diseases, not only downregulating the function of inflammatory cells but also promoting survival of progenitors and differentiated oligodendrocytes.
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