Gene regulation of the P2Y-like receptor GPR17 in oligodendrocytes, and pathological alterations in a mouse model of multiple sclerosis

The P2Y-like receptor GPR17 is a G protein-coupled receptor activated by uracil nucleotides and cysteinyl-leukotrienes (Ciana et al., 2006. EMBO J. 25:4615-27). We have previously demonstrated that GPR17 is a key regulator of oligodendroglial differentiation. Under physiological conditions, the receptor starts to be expressed in oligodendrocyte precursor cells (OPCs) also positive for the early marker NG2, it reaches a peak of expression in immature oligodendrocytes, and it is progressively downregulated during terminal maturation (Lecca et al., 2008. PLoS One. 3:e3579; Fumagalli et al., 2011. J Biol Chem. 286:10593-604). At early differentiation stages, activation of GPR17 with agonists induced, while inhibition with antagonists or specific interfering RNAs impaired OPC maturation. At later stages, the forced overexpression of the receptor also led to impaired maturation. All these data suggest that the expression of the receptor GPR17 needs to be time regulated either at transcriptional, or post-transcriptional level. The present work was aimed at understanding what is the molecular mechanism at the basis of Gpr17 gene expression, and to assess if receptor expression is altered in spinal cord of mice with the experimental autoimmune encephalomyelitis (EAE), a widely used model of demyelinating disease. To answer to the first issue, we cloned a putative promoter region of the Gpr17 into a reporter vector upstream to the firefly luciferase gene; then, we transfected this construct in Oli-neu cells, an immortalized oligodendroglial cell line, and we set up a dual luciferase reporter assay to evaluate the bioluminescence produced in response to an array of stimuli, including nucleotides, hormones and growth factors. Interestingly, incubation of cells with conditioned medium from cortical neurons significantly induced Gpr17 promoter activity (Fratangeli et al., 2013. J Biol Chem. 288:5241-56), but we still don\u2019t know which is the released factor responsible of this effect. Signals released by axons can profoundly influence the expression pattern of oligodendrocytes, and this is indeed responsible of their behaviour both in physiological and in pathological conditions. For this reason, we assessed the expression of GPR17 in mouse spinal cord in EAE. As already described for brain, immunohistochemical analysis revealed that, also in spinal cord, GPR17 specifically decorates a subset of OPCs both in grey and white matter. After EAE induction, both the total number of CC1-positive (representing the maturing oligodendrocytes) and GPR17-positive cells was reduced, whereas the number of NG2-positive cells was increased, suggesting disease-induced recruitment and proliferation of early progenitors. Interestingly, qRT-PCR showed that the mRNA of GPR17 is increased in EAE, and this seems to be in relation with the clinical score of the mice. We hypothesize that, as demonstrated in brain ischemia, the expression of GPR17 is up-regulated in the disease to counteract demyelination, but this response is insufficient and may lead to dysregulations contributing to the disease. Understanding how the expression of GPR17 is finely regulated will not only advance basic knowledge in oligodendrogliogenesis, but will also help developing new strategies to enhance the reparative potential of the quiescent OPCs that are still present in the adult brain. Sponsored by FISM 2010/R2 to MPA and partially supported by Cariplo Foundation (Rif. 2012.0546) to PR