Pharmacological and functional characterization of adenosine "A2" receptors as emerging targets in cerebral ischemia and oligodendrogliogenesis: an in vitro study

The role of the neuromodulator adenosine and its receptors (A1AR, A2AAR, A2BAR and A3AR) on cerebral ischemia, synaptic activity, oligodendrogenesis and myelination processes was investigated by using different in vitro models, with the specific purpose to individuate new class of neuroprotective agents for different pathological conditions. The involvement of adenosine receptors in synaptic plasticity phenomena in the CA1 hippocampal region is well described. Paired-pulse facilitation (PPF) is an electrophysiological model of short-term synaptic plasticity, which is considered a clear index of neurotransmitter release probability. It is known that the activation of Gi-coupled A1AR increases PPF ratio, due to a reduction of glutamate release. On the contrary, the stimulation of A2BARs or A2AARs, which are expressed on the glutamatergic presynaptic terminals, reduces PPF by facilitating glutamate release. Both “A2” adenosinergic receptor subtypes are Gs-coupled receptors; furthermore, a coupling to Gq protein for A2BAR was also described. In a first part of my Thesis the role of “A2” receptor subtypes on PPF was further characterized using extracellular recordings of synaptic potentials evoked in the CA1 region of rat hippocampal slices. To this purpose we used different adenosinergic ligands and for the first time new multi-target compounds (agonist, MRS3997 and antagonist, P626), able to simultaneously affect A2AAR and A2BAR. Multi-target compounds are designed to activate several cellular targets simultaneously. Therefore, these molecules offer the possibility of allowing better pharmacokinetic and symptomatologic control in various pathological conditions by reducing side effects due to the administration of different molecules. We confirm that the selective A2AAR or A2BAR activation reduces PPF, an effect significantly antagonized in the presence of P626. The inhibitory effect on PPF was mimicked by the multi-target agonist, MRS3997, but, surprisingly, not when the selective A2AAR agonist, CGS21680, and A2BAR agonist BAY60-6583 were co-applied. To explain these results, we hypothesize that A2AAR or A2BAR signalling is context-dependent, i.e. heteromer structure and/or allosteric interactions with G-proteins and scaffolds. Certainly, we can affirm that stimulation of A2AAR or A2BAR induced an increase in glutamate release in the CA1 hippocampal slices. This increase plays different roles under physiological condition, facilitating neuronal excitability, synaptic plasticity (see in PPF) and, coordination of neural networks. However, under pathological conditions (such as cerebral ischemia), an increase in glutamate release contributes to excitotoxic damage. The early phases of a hypoxic-ischemic insult are characterized by a significant increase in extracellular glutamate levels, which triggers a hyper-activation of glutamate receptors, production of reactive oxygen species, pathological increase in intracellular Ca2+, rapid decrease in ATP reserves, and activation of various proteolytic enzymes. Contemporarily to the glutamate-increase, also the extracellular concentration of adenosine significantly raised, as demonstrated by both in vivo and in vitro experiments. It is known that the selective A2AAR or A2BAR antagonism delays or prevent the anoxic depolarization (AD) appearance during an in vitro ischemic-like insult (obtained by oxygen and glucose deprivation: OGD) in the CA1 region of rat hippocampus, a cerebral area particularly susceptible to this type of damage. AD is a clear sign of neuronal damage and it is well accepted that a pharmacological treatment that postpones its development helps to protect brain tissue after ischemia. Hence, we decided to test P626 during 30-min OGD, a condition that always induced AD appearance. We demonstrated that P626 significantly delayed AD onset. Hence, P626 could represent a favourable strategy to achieve neuroprotection by a simultaneous block of “A2” receptors subtypes during an acute ischemic insult In addition, with the aim of study some new multi-target “A2” ligands, in this Thesis our attention was focused on newly synthetized mixed A2AAR antagonist, formed by an A2AAR antagonistic component conjugated to an antioxidant molecule, (i.e edaravone (EDA), 2-oxothiazolidin-4-carboxylic acid or α-Lipoic acid). We demonstrated that the mixed A2AAR antagonists delayed AD appearance in the CA1 hippocampus, induced by a severe OGD. Meanwhile, when the antioxidant molecules OTC, EDA, and Acid Lipoic were tested alone, only EDA resulted efficacious in delaying AD. The second part of my Thesis was focused on pharmacological and functional characterization of adenosine “A2” receptors in oligodendrogliogenesis and myelination processes in cultured cells. Differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes is a key event for axonal myelination in the brain; this process fails during demyelinating pathologies, such as multiple sclerosis (MS) or after cerebral ischemia. Extracellular adenosine increases during ischemia or inflammation, suggesting adenosine receptors as valid therapeutic targets in these pathological conditions. Our research group previously demonstrated that the selective activation of A2AARs and A2BARs decreased in vitro OPC maturation by inhibiting potassium currents necessary to their differentiation. Therefore, we elucidate the role of “A2” receptors on potassium currents in primary OPC cultures by using electrophysiological patch-clamp recordings. Moreover, the role of these receptors subtypes was investigated on myelination processes by co-culturing OPCs with dorsal root ganglion neurons and using immunocytochemical analysis. To this purpose, we used the endogenous ligand adenosine, selective A2BAR agonist and the new multi-target A2A-A2BARs antagonist, P626. Similar to what observed in the presence of CGS21680 or BAY60-6583, adenosine inhibited outward-K+ currents in a concentration-independent manner. In particular, low concentration of adenosine reduced only the Tetraethylammonium(TEA)-sensitive delayed rectifying K+ currents (IK), consistent with the A2AAR activation, while the application of high concentration of adenosine inhibited also the, 4-aminopyridine (4-AP)-sensitive transient K+ (IA) conductance, in line with the relative differences in adenosine sensitivity of the “A2” receptor subtypes. Of note, 4-AP, a broad-spectrum potassium channel blocker, was approved to improve motor skills in MS patients by promoting axonal conduction. Hence, we tested the effects of the multi-target antagonist P626 on the reduction of K+ currents induced by A2AAR and/or A2BAR activation on cultured OPCs. We demonstrated that P626 was able to antagonize the effect of adenosine and BAY60-6583 on K+ currents in cultured OPCs. In line with data in literature, we could suppose that also the effects of adenosine may be related with a reduction of OPC in vitro differentiation. Hence, the multi-target antagonist, P626, could represent a possible new compound to test for its pro-myelinating actions, as it could be able to prevent the effects of endogenous adenosine released in pathological condition. On these bases, we evaluated the effects of the “A2” subtypes on OPC-DRG co-cultures chronically treated with selective A2AAR or A2BAR agonists, in order to evaluate their role in myelination. We found that BAY60-6583 significantly increased the myelination coefficient (M1), without affecting the total myelin basic protein expression. In order to explain these apparently contradictory results of A2BAR agonism, we tested the effect of BAY60-6583 on isolated DRG neurons. We observed that the selective A2BAR agonist increased the action potentials (APs) firing in DRG neurons and that this effect was blocked by the selective A2BAR antagonist, PSB603. Hence, this effect of A2BAR activation could be associated with the release of chemoattractant compounds (i.e. glutamate, adenosine, ATP) that are involved in OPCs differentiation and myelin deposition. In conclusion, we can postulate that the A2AAR and A2BAR are promising target for the treatment of different pathologies, such as cerebral ischemia or demyelinating disorders.