Adenosine A2A and A2B receptors as new attractive targets involved in myelination processes: in vitro and in vivo models of myelin development or damage

Oligodendrocytes (OLs) are neuroglial cells responsible in the brain of myelin sheath formation, which allow fast synaptic transmission. Remyelination requires the differentiation of oligodendrocyte progenitor cells (OPCs) into mature OLs but, in chronic neurodegenerative disorders, such as multiple sclerosis (MS), remyelination fails. In this regard, studies have shown that dysfunctional adenosinergic signalling plays a pivotal role, as patients with MS have altered levels of adenosine (ADO). Adenosine is gaining recognition as a significant factor in oligodendrogliogenesis, primarily through the activation of its metabotropic receptors (A1R, A2AR, A2BR, A3R). In the first part of the Thesis, we evaluated the effects of the endogenous ligand ADO on K+ currents in rat cultured OPCs and, for the first time, we tested the effects of P626, a dual adenosine A2A/A2B receptors antagonist able to simultaneously block both A2Rs. It has been demonstrated that Gs-coupled A2BRs and A2ARs decrease OPC differentiation by inhibiting TEA-sensitive potassium currents in primary OPC cultures. Of note, 4-aminopyridine (4-AP), a broad-spectrum potassium channel blocker, was approved to improve motor skills in MS patients by promoting axonal conduction. Our group, previously demonstrated, that selective stimulation of A2AR or A2BR inhibit OPC maturation by reducing voltage-dependent K+ currents. Among these currents, IK were sensitive to both A2AR or A2BR agonists, while IA were only inhibited in the presence of selective A2B agonist. In order to isolate A2 response all these experiments were carried out in the presence of selective A1 and A3 antagonists. When applied at nanomolar (0.1 μM) concentration, necessary to activate only high affinity A2AR, ADO was able to only inhibit IK currents, an effect blocked in the presence of SCH 58621 (0.1 μM), a selective A2AR antagonist. On the other hand, ADO, applied at micromolar concentrations (50 μM), usually reached during a pathological condition, was able to activate also A2BR and to reduce both IK and IA. This effect was reverted by PSB 603 (0.1 μM) and by the dual antagonist P626 (0.1 μM). However, these monocultures do not represent a myelination model due to the absence of neuronal axons, so in the second part of the Thesis we focused our attention on the role of A2B subtypes in neuron-OL interactions and the myelination process. These receptor subtype among the others are activated only during pathological conditions. We examined the effects of various A2BR ligands in a dorsal root ganglion (DRG) neuron/OPC co-culture system. Using confocal microscopy, we found that the selective A2BR agonist BAY60-6583 significantly decreased myelin basic protein levels While simultaneously increasing the myelination index in DRG/OPC co-cultures, the last effect was prevented by the selective A₂B₁R antagonists, PSB 603 and MRS1706. To clarify this unexpected data, we wondered whether A₂B₁Rs could play a functional role on DRG neurons. We firstly demonstrated by immunocytochemistry that primary DRG monoculture expressed A₂B₁Rs. Their selective activation by BAY60-6583 enhanced DRG neuronal excitability, as demonstrated by the increase in action potential firing, the decrease in rheobase, and the depolarization of resting membrane potential. All these effects were prevented by PSB-603. Throughout this mechanism, DRG neurons could facilitate myelination processes. In order to prove this theory, we downregulated A₂B₁Rs by infecting DRG neurons with AAV2/5 carrying shRNA designed to target A₂B₁R. Under these conditions, BAY60-6583 still caused a reduction in MBP expression but failed to enhance myelination of shA₂B₁R-DRGN axons. These findings indicate that the increase in myelination was mediated by A₂B₁Rs on DRG neurons, while the decrease in MBP levels resulted from the activation of this receptor subtype on OPCs. Last but not least, we set up an in vivo model, the cuprizone (CPZ) model, to study whether our adenosinergic ligands could play a role in the process of remyelination and demyelination after myelin damage. In protocol I, animals were fed with 0.2% CPZ for 5 weeks in order to induce a myelin damage. Using immunohistochemistry, we confirmed that CPZ was able to significantly reduce MBP fluorescence in three distinct areas of the brain: (i) corpus callosum, (ii) motor cortex, and (iii) striatum. During this time period, we monitored body weight variation, and a diminished weight gain over time was observed in animals administered the CPZ-based diet compared to controls. Moreover, behavioral tests were conducted to investigate the role of the toxin on motor locomotion, using the rotarod test and on anxiety-like behavior, through the hole board test. Both these parameters were significantly impaired in animals fed with CPZ. It is known from the literature that this copper chelator is able to only affect mature OLs but not OPCs, which are still able to migrate to the damaged area. Taking that into account, in order to study the process of spontaneous remyelination, a group of animals, after 5 weeks of CPZ-based diet, were returned to normal chow and pharmacologically treated with BAY60-6583 or PSB 603 for the following 2 weeks. Neither BAY60-6583 nor PSB 603 were able to modulate the body weight variation of the animals. Furthermore, no statistical significance was found in MBP intensity after the treatment with the two compounds. At this point, in order to study demyelination, we arranged protocol II. Animals were fed 0.2% CPZ for 5 weeks but, during the last two weeks, together with the diet, we intraperitoneally administered the above-mentioned adenosinergic compounds. Even in this case, neither BAY60-6583 nor PSB 603 were able to modify the variation in body weight. However, the treatment with PSB 603 was able to restore the levels of MBP fluorescence, showing a protective effect. A2B receptors may represent a valid molecular target for the development of new therapeutic options for demyelinating diseases.