RESUMEN
The endocannabinoid system (ECS) refers to a complex cell-signaling system highly conserved among species formed by numerous receptors, lipid mediators (endocannabinoids) and synthetic and degradative enzymes. It is widely distributed throughout the body including the CNS, where it participates in synaptic signaling, plasticity and neurodevelopment. Besides, the olfactory ensheathing glia (OEG) present in the olfactory system is also known to play an important role in the promotion of axonal growth and/or myelination. Therefore, both OEG and the ECS promote neurogenesis and oligodendrogenesis in the CNS. Here, we investigated if the ECS is expressed in cultured OEG, by assessing the main markers of the ECS through immunofluorescence, western blotting and qRT-PCR and quantifying the content of endocannabinoids in the conditioned medium of these cells. After that, we investigated whether the production and release of endocannabinoids regulate the differentiation of oligodendrocytes co-cultured with hippocampal neurons, through Sholl analysis in oligodendrocytes expressing O4 and MBP markers. Additionally, we evaluated through western blotting the modulation of downstream pathways such as PI3K/Akt/mTOR and ERK/MAPK, being known to be involved in the proliferation and differentiation of oligodendrocytes and activated by CB1, which is the major endocannabinoid responsive receptor in the brain. Our data show that OEG expresses key genes of the ECS, including the CB1 receptor, FAAH and MAGL. Besides, we were able to identify AEA, 2-AG and AEA related mediators palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in the conditioned medium of OEG cultures. These cultures were also treated with URB597 10-9 M, a FAAH selective inhibitor, or JZL184 10-9 M, a MAGL selective inhibitor, which led to the increase in the concentrations of OEA and 2-AG in the conditioned medium. Moreover, we found that the addition of OEG conditioned medium (OEGCM) enhanced the complexity of oligodendrocyte process branching in hippocampal mixed cell cultures and that this effect was inhibited by AM251 10-6 M, a CB1 receptor antagonist. However, treatment with the conditioned medium enriched with OEA or 2-AG did not alter the process branching complexity of premyelinating oligodendrocytes, while decreased the branching complexity in mature oligodendrocytes. We also observed no change in the phosphorylation of Akt and ERK 44/42 in any of the conditions used. In conclusion, our data show that the ECS modulates the number and maturation of oligodendrocytes in hippocampal mixed cell cultures.
RESUMEN
Complex carbohydrate structures are essential molecules of infectious bacteria, parasites, and host cells and are involved in cell signaling associated with immune responses, glycoprotein homeostasis, and cell migration. The uptake of mannose-tailed glycans is usually carried out by professional phagocytes to trigger MHC class I- and MHC class II-restricted antigen presentation or, alternatively, to end inflammation. We have detected the mannose receptor (MR) in cultured olfactory ensheathing cells (OECs), so we investigated by flow cytometry whether recently dissociated cells of the olfactory bulb (OB) nerve fiber layer (ONL) could bind a mannosylated ligand (fluorescein conjugate of mannosyl bovine serum albumin; Man/BSA-FITC) in a specific manner. In addition, we estimated the relative proportion of ONL OECs, microglia, and astrocytes, tagged by 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), by the B4 isolectin of Griffonia simplicifonia (IB4), and by glial fibrillary acidic protein (GFAP), respectively, that were Man/BSA-FITC(+) . We also determined by histochemistry and/or immunohistochemistry whether Man/BSA-FITC or an anti-MR antibody (anti-C-terminal MR peptide; anti-cMR) labeled OECs and/or parenchymal microglia. In addition, we confirmed by Western blot with the K1K2 (against the entire MR molecule) antibody that a band of about 180 kDA is expressed in the OB. Our findings are compatible with a prospective sentinel role of OECs against pathogens of the upper airways and/or damage-associated glycidic patterns as well as with homeostasis of OB mannosylated glycoproteins.
Asunto(s)
Lectinas Tipo C/biosíntesis , Lectinas de Unión a Manosa/biosíntesis , Neuroglía/metabolismo , Bulbo Olfatorio/citología , Bulbo Olfatorio/metabolismo , Receptores de Superficie Celular/biosíntesis , Animales , Western Blotting , Citometría de Flujo , Inmunohistoquímica , Receptor de Manosa , Ratas , Ratas WistarRESUMEN
Olfactory ensheathing cells (OECs) are a special glia that ensheath olfactory receptor axons that enter the brain via olfactory phila, thus, providing a potential route for access of pathogens. Streptococcus pneumoniae (Sp), that has a capsule rich in mannosyl residues, is the most common cause of rhinosinusitis that may evolve to meningitis. We have tested whether OECs in vitro express the mannose receptor (MR), and could internalize Sp via MR. Cultures were infected by a suspension of Sp (ATCC 49619), recognized by an anti-Sp antibody, in a 100:1 bacteria:cells ratio. Competition assays, by means of mannan, showed around a 15-fold reduction in the number of internalized bacteria. To verify whether MR could be involved in Sp uptake, OECs were reacted with an antibody against the MR C-terminal peptide (anti-cMR) and bacteria were visualized with Sytox Green. Selective cMR-immunoreaction was seen in perinuclear compartments containing bacteria whereas mannan-treated cultures showed an extremely low percentage of internalized bacteria and only occasional adhered bacteria. Our data suggest the involvement of MR in adhesion of bacteria to OEC surface, and in their internalization. Data are also coherent with a role of OECs as a host cell prior to (and during) bacterial invasion of the brain.