Implementation of the stem cell properties of NG2-expressing neural precursor cells by purinergic signaling: an innovative reparative approach to neurodegeneration

Cells expressing the proteoglycan NG2 are multipotent neural progenitors with lineage plasticity able to primarily generate myelinating oligodendrocytes. They are also known as oligodendrocyte precursors cells (OPCs), are the only slowly proliferating cells in the adult brain, and react to injury. Under some circumstances, they can also generate neurons (Kondo & Raff, Science 289:1754, 2000); however, this first sensational paper showing that NG2 cells can be reprogrammed to neurons was followed by only few in vitro (Liu et al., J Neurosci 27:7339, 2007) and in vivo confirmations (Richardson et al, Neuron 70:661, 2011). The latter study suggests that NG2 cells lineage plasticity may be markedly widened by insults and epigenetic agents that profoundly change their reactivity and the surrounding local mileu. Previous studies from our laboratory have identified the purinergic receptor GPR17 as a new marker of early stages of NG2 cell differentiation, showing that GPR17 activation accelerates NG2 cells’ oligodendrocyte fate (Fumagalli et al., JBC 25:10593, 2011; Ceruti et al., Glia, 59:363, 2011; Boda et al., Glia 59:1958, 2011). Based on these premises, aim of this work is to unveil the stem cell properties of NG2+ precursor cells and to induce their differentiation towards a neuronal lineage, also through the modulation of the GPR17 receptor. Primary OPCs from the cerebral cortex of P2 rat pups were kept in culture for 6-8 days as mixed astrocytes-OPCs cultures and purified by vigorous shaking of flasks, and immunopanning selection. OPCs have been then cultured accordingly to two published protocols (Kondo & Raff, 2000; Liu et al., 2007) both able to unveil their stem cell properties. In either protocol, we have verified if and how the exposure to various pharmacological agents (including GPR17 receptor ligands) can modulate OPCs plasticity and their differentiation to neurons. In particular, we have utilized the non-selective GPR17 agonist UDP-glucose (10 µM) and antagonist cangrelor (10 µM), in parallel to the anticonvulsant agent valproic acid (VPA, 500 µM). VPA is known as an epigenetic modulator, which inhibits histone deacethylases (HDAC) and consequently induces transcription changes which favor neurogenesis (Yu et al., Neuropharm 56:473, 2009). In both culturing conditions, we observed an increase in the percentage of cells expressing the neuronal marker β-tubulinIII (βtubIII) upon treatment with cangrelor and VPA with respect to control. Interestingly GPR17, which is normally expressed only by NG2+ cells, was surprisingly detected in a subset of βtubIII+ cells already under control differentiative condition, suggesting its potential involvement in neurogenesis. The appearance of this cell population was further incremented by the exposure to VPA and cangrelor, and slightly reduced by UDP-glucose, which likely promotes receptor down-regulation and drove NG2 cell specification towards a “classical” oligodendrocyte fate. Taken together, our results suggest that antagonizing GPR17 functions can address the fate of NG2 cells towards the generation of new neurons, as observed with epigenetic modulators like VPA. We are currently testing whether a combination of the two agents may further enhance the neurogenic shift of NG2 cells, a paradigm that could be exploited in neuroregeneration in vivo.