ENDOCYTIC TRAFFIC OF THE G-PROTEIN COUPLED RECEPTOR 17, A DUALISTIC RECEPTOR IMPLICATED IN OLIGODENDROCYTE DIFFERENTIATION

Oligodendrocytes are glial cells that produce specialized membranes (myelin sheaths) that are wrapped around the neuronal axons of the central nervous system (CNS) which function is to ensure an efficient and fast conduction of electrical impulses along axons and the maintenance of axonal integrity. Several pathologies of the CNS are correlated to the loss of myelin, an event that can be a direct cause of disease like in multiple sclerosis or, in other situation, it can be a consequence of a more general damage like during ischemia. Within the CNS there are OPCs, that after appropriate stimulation, are able to migrate in the damaged area and completely mature producing new myelin sheaths. Among the factors necessary for the recruitment of precursors at the lesion, GPR17 seems to play a role as a sensor of the damage and regulator of OPC differentiation. GPR17 is a dualistic G-protein-coupled receptor that can be activated by both UDP-glucose and CysLTs, signaling molecules massively released in the brain upon different types of injury (Lecca et al., 2008; Fumagalli et al., 2011). Upon agonist binding the receptor expression, almost absent in OPCs, gradually increases in more mature precursors, it reaches a plateau in immature/preoligodendrocytes and then gradually decreases along with terminal differentiation. On the other hand inhibition of GPR17 expression causes impairment in oligodendrocyte differentiation and myelination in in vivo (Chen et al., 2009) and in vitro systems (Fumagalli et al., 2011). Altogether these data indicate that GPR17, acting in a specific time window, is implicated in OPCs maturation. We characterized the expression of GPR17 in an immortalized oligodendrocyte precursor cell line (Oli-neu) (Jung et al., 1995): biochemical and immunofluorescence analyses of Oli-neu differentiation in neuronal conditioning medium, showed that GPR17 is almost absent in undifferentiated Oli-neu cells and reaches a peak after 72h differentiation, while myelin proteins begin to appear after 96h, when GPR17 levels decrease. When GPR17 was knocked down by means of RNA interference, fewer Oli-neu cells were capable of differentiating. These data demonstrate that Oli-neu cells are an helpful model to a further biochemical and biological characterization of GPR17. Little information is available on the role of the agonists in regulating receptor availability at the cell surface and on GPR17 trafficking during cell-differentiation. As reported for many GPCRs, after ligand binding GPR17 may undergo to endocytosis and subsequent sorting into lysosomes for degradation and/or to recycling endosomes for delivery to the plasma membrane. In order to shed light on the mechanisms of GPR17 signaling and agonist induced activation, we investigate the endocytic trafficking of the native receptor in differentiating Oli-neu. To trace internalized GPR17, an antibody against an extracellular epitope of the receptor was generated to be used in internalization assays performed in Oli-neu cells. As shown by immunocytochemical and biotinylation assays, both UDP-glucose and LTD4 increase the clathrin-dependent internalization of GPR17, though with different efficiency. Preliminary results seem to confirm that GPR17 is also differently internalized in OPC primary cultures. Confocal images demonstrated that, at early times after internalization, GPR17 is colocalized with the endosome markers transferrin receptor and Rab5 whereas at later times it shows colocalization with the lysosomal marker Lamp1. The intracellular accumulation of GPR17 in Lamp1 positive organelles increased after incubation with chloroquine, a lysosomotropic agent known to affect lysosomal degradation. On the other hand, the receptor colocalized to a very minor degree with Rab11, a marker of the long loop-recycling pathway. In conclusion these results provide the first characterization of the endocytic fate of the native GPR17 in response to the two different classes of ligand activation and demonstrate that the receptor is mainly down regulated after stimulation in differentiated oligodendroglial cells.