ABSTRACT
Title
The recently identified P2Y-like receptor GPR17 modulates potassium currents in cultured oligodendrocyte progenitor cells
Authors
G. Maraula
Dottorato in Neuroscienze
Dipartimento di Farmacologia Preclinica e Clinica, Università di Firenze
Dottorato in Neuroscienze
Dipartimento di Farmacologia Preclinica e Clinica, Università di Firenze
Abstract
The recently identified G-protein-coupled receptor GPR17 is a new P2Y receptor activated by uracil nucleotides (UDP-glucose and UDP) and cysteinyl-leukotrienes (cysLTs: LTD4 and LTE4) and blocked either by P2Y antagonists (cangrelor and MRS2179) or CysLT blockers (montelukast and pranlukast) [1]. Both the human and the rodent receptor are highly expressed in the brain, both on neurons and on a subset of oligodendrocyte precursor cells (OPCs) identified as NG2+ and Olig2+ cells. An important role of this receptor has been described after brain ischemia, when extracellular concentrations of nucleotides and cysLTs are markedly increased. At early times after the insult, its activation contributes to neuronal death and tissue damage whereas, at later times, it stimulates parenchymal quiescent OPCs, known as the primary source of remyelinating cells after damage, to proliferate suggesting the initiation of remyelination processes [2].
In light of this pro-myelinating effect, we studied the expression and the role of GPR17 on OPCs by using an in vitro model of purified primary OPC cultures isolated from rat cortex (methods in [3, 4]).
After confirming that purified cultures contained more than 98% of NG2+ and Olig2+ cells, OPCs where allowed to differentiate by appropriated conditions (i.e. by withdrawal of growth factors) and the time course of expression and functional effect of GPR17 were studied by immunocytochemical labeling and electrophysiological recordings, respectively.
In accordance with Fumagalli et al. [4], we confirmed that a subpopulation of cultured OPCs expresses GPR17 receptor, with a peak of expression observed after 5-6 days in culture (59.4% of GPR17+ cells).
Then, we performed single-cell patch-clamp recordings from OPCs (2-6 days in culture) aimed at investigating the functional role of GPR17 in these cells by applying its selective agonist, UDP-glucose. OPC K+ currents were elicited by specific voltage-clamp protocols and were recorded before, during and after a 10 min UDP-glucose application. It has been described that the major component of membrane K+ currents in proliferating NG2+ OPCs is represented by delayed rectifier currents (KDR), 70% of cells express also the transient outward (KA) current, whereas inwardly rectifying (Kir) currents are almost absent in undifferentiated cells and become predominant in mature oligodendrocytes [5]. In the presence of 10 µM UPD-glucose we observed an increase in KDR currents (66.5 pA/pF increase at +80 mV) in 44% of cells. No apparent changes were observed in the KA component. The effect was maximal after 5-7 min application and was partially reversed after 5 min washout. Such currents were absent when intracellular and extracellular K+ ions were replaced by equimolar Cs+. 1 µM UDP-glucose was ineffective whereas at 100 µM the effect was observed in 67% of cells recorded. Pilot experiments performed by us indicated that UDP-glucose effect was blocked in the presence of the GPR17 antagonist MRS2179 (1 µM).
In conclusion, we demonstrated that a fraction of cultured OPCs express functional GPR17 receptors depending on cell differentiation, and that the activation of this receptor positively modulates KDR currents. Interestingly, it has been demonstrated that the blockade of such currents inhibits OPC differentiation in culture [6], whereas the stimulation of GPR17 increases OPC development toward mature oligodendrocytes [4]. Thus, we can hypothesize that the mechanism by which GPR17 facilitates OPC differentiation is due to KDR current activation.
1) Ciana P et al. (2006) - Embo J vol 25, 4615–4627.
2) Lecca D et al. (2008) - PLoS One vol 3, e3579.
3) Chen Y et al. (2007) - Nat Protoc vol 2, 1044-1051.
4) Fumagalli M et al. (2011) - J Biol.Chem, in press.
5) Sontheimer et al. (1989) - Neuron 2 vol 2, 1135-1145.
6) Gallo V et al. (1996) - J Neurosci 16 vol 8, 2659-2670.
In light of this pro-myelinating effect, we studied the expression and the role of GPR17 on OPCs by using an in vitro model of purified primary OPC cultures isolated from rat cortex (methods in [3, 4]).
After confirming that purified cultures contained more than 98% of NG2+ and Olig2+ cells, OPCs where allowed to differentiate by appropriated conditions (i.e. by withdrawal of growth factors) and the time course of expression and functional effect of GPR17 were studied by immunocytochemical labeling and electrophysiological recordings, respectively.
In accordance with Fumagalli et al. [4], we confirmed that a subpopulation of cultured OPCs expresses GPR17 receptor, with a peak of expression observed after 5-6 days in culture (59.4% of GPR17+ cells).
Then, we performed single-cell patch-clamp recordings from OPCs (2-6 days in culture) aimed at investigating the functional role of GPR17 in these cells by applying its selective agonist, UDP-glucose. OPC K+ currents were elicited by specific voltage-clamp protocols and were recorded before, during and after a 10 min UDP-glucose application. It has been described that the major component of membrane K+ currents in proliferating NG2+ OPCs is represented by delayed rectifier currents (KDR), 70% of cells express also the transient outward (KA) current, whereas inwardly rectifying (Kir) currents are almost absent in undifferentiated cells and become predominant in mature oligodendrocytes [5]. In the presence of 10 µM UPD-glucose we observed an increase in KDR currents (66.5 pA/pF increase at +80 mV) in 44% of cells. No apparent changes were observed in the KA component. The effect was maximal after 5-7 min application and was partially reversed after 5 min washout. Such currents were absent when intracellular and extracellular K+ ions were replaced by equimolar Cs+. 1 µM UDP-glucose was ineffective whereas at 100 µM the effect was observed in 67% of cells recorded. Pilot experiments performed by us indicated that UDP-glucose effect was blocked in the presence of the GPR17 antagonist MRS2179 (1 µM).
In conclusion, we demonstrated that a fraction of cultured OPCs express functional GPR17 receptors depending on cell differentiation, and that the activation of this receptor positively modulates KDR currents. Interestingly, it has been demonstrated that the blockade of such currents inhibits OPC differentiation in culture [6], whereas the stimulation of GPR17 increases OPC development toward mature oligodendrocytes [4]. Thus, we can hypothesize that the mechanism by which GPR17 facilitates OPC differentiation is due to KDR current activation.
1) Ciana P et al. (2006) - Embo J vol 25, 4615–4627.
2) Lecca D et al. (2008) - PLoS One vol 3, e3579.
3) Chen Y et al. (2007) - Nat Protoc vol 2, 1044-1051.
4) Fumagalli M et al. (2011) - J Biol.Chem, in press.
5) Sontheimer et al. (1989) - Neuron 2 vol 2, 1135-1145.
6) Gallo V et al. (1996) - J Neurosci 16 vol 8, 2659-2670.