Adenosine a2b receptors and carbonic anhydrase: new therapeutic targets for cerebral ischemia and demyelination.

Cerebral ischemia is a multifactorial pathology characterized by different events evolving in time. The acute injury, characterized by a massive increase of extracellular glutamate levels, is followed by activation of resident immune cells and production or activation of inflammation mediators. Although after ischemia precocious activation of immune cells may be neuroprotective and supportive for regeneration, protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. In this thesis, I investigated on the putative protective effects of the agonists at adenosine A2B receptor subtype and of the carbonic anhydrase inhibitors in a rat model of focal ischemia. Demyelination occurs in a variety of pathophysiological conditions of the Central Nervous System (CNS). The most serious demyelination occurs in multiple sclerosis but also following cerebral ischemia. Remyelination does occur but is limited especially in chronic disease stages. Therefore, strategies aimed at promoting remyelination, represent an attractive additional therapy in demyelinating pathologies. The remyelination process is mediated by oligodendrocyte progenitor cells (OPCs), a population of cycling cells which persists in the adult CNS, where they can differentiate into mature myelinating oligodendrocytes (OLs). Oligodendrocytes at all maturational stages, express each of the different adenosine receptor subtypes (A1R, A2AR, A2BR and A3R). A number of pathways have been identified that may contribute to ameliorate/impaired remyelination among them, the adenosinergic signaling and sphingosine kinase/sphingosine 1-phosphate signaling axis (SphK/S1P). Therefore, a first aim of my work was to investigate the role of A2BR and of SphK/S1P signaling in modulating cell proliferation and maturation in cultured OPCs and the presence of a possible cross-talk between S1P/SphK and A2BR signaling. We used two A2BR agonists: BAY60-6583 (BAY) and the newly synthesized drug P453, the S1P analog Fingolimod-phosphate (FTY720-P) and the inhibitors of SphK VPC96047 and VPC96091. In cultured OPCs, phosphorylation of the SphK1 (a SphK subtype), that is a hallmark of the activation state of the enzyme, was enhanced after 10 min treatment with BAY (10 µM). Chronic application (7 days) of BAY (1-10 µM) or of P453 (50-100 nM) in cultured medium reduced OPC differentiation, as indicated by the decrease of the two genes target MAG (myelin-associated glycoprotein) and Mbp3 (myelin basic protein 3), typically expressed by mature oligodendrocytes. FTY720-P (1 µM), mimicked the effect of 10 µM BAY on OPC maturation. On the contrary, VPC96047 (500 nM), a pan-SphK inhibitor, and VPC96091 (500 nM) a selective SphK1 inhibitor, increased MAG and Mbp3 levels. These effects were abolished in the presence of 10 µM BAY. After 48 hours A2BR silencing by RNA interference (RNAi), about 50% of the A2BR was downregulated. A2B downregulation increased OPC differentiation (as demonstrated by the CNPase increase). These data are support that A2BR inhibits OPC maturation. Moreover, cells transfected with A2B-siRNA showed a striking increase in S1P lyase levels, the enzyme responsible for of S1P catabolism. Our results demonstrated that the adenosine A2BR inhibit OPC differentiation in cultured OPCs. Moreover, the A2BR agonist BAY increases the expression of phosphorylated SphK1, indicating an interaction between SphK1 and A2BR activation. To date this is the first characterization of the role of adenosine A2BR in oligodendrocyte maturation and of a cross-talk between A2BR and SphK/S1P signaling axis in inhibiting OPC maturation. Extracellular adenosine concentration dramatically increases during cerebral ischemia and a protective role is recognized to adenosine by acting on A1 receptors. However, the use of adenosine A1 agonists is hampered by peripheral and central side effects. Few studies are present in literature on the role of A2B receptors in brain ischemia. A2B receptors are present on endothelial cells, neurons and astrocytes 24 hours after transient middle cerebral artery occlusion (tMCAo) in the rat. Data in the literature indicate that A2BR agonist BAY protect from endothelial leakage and blood brain barrier permeability 24 hours after focal ischemia To date there are no evidences in literature on the protective effects of A2B receptor agonists at more distant times from ischemia when a defined neuroinflammation develops. A further aim of my thesis was to investigate, in the model of focal transient cerebral ischemia (tMCAo) in the rat, the putative protective effects of the A2B receptor agonist, BAY 7 days after ischemia, when a clear inflammatory response has developed. Treatment with BAY, chronically administered (0.1 mg/kg i.p. for 7 days), improves the neurological deficit evaluated 1 and 5 and up to 7 days after tMCAo (p<0.0005-0.02). Seven days after ischemia, BAY has significantly reduced the infarct volume in cortex (p<0.001) and in striatum (p<0.05), has reconstituted the cortical and striatal cytoarchitecture and has reduced glial cell proliferation that was induced by the ischemic insult. BAY has significantly reverted the increase in number of damaged neurons (stained with the specific marker for neurons, NeuN+). Furthermore, BAY has reverted the strong pattern of microglia activation and reduced the loss of astrocyte. Seven days after ischemia, plasma inflammatory marker of brain damage TNF-α, is definitely increased while the levels of IL10 regulatory cytokine with anti-inflammatory action is decreased. Interestingly, BAY has reverted these modifications. Moreover, two days after ischemia, BAY has reduced granulocytes (evaluated as HIS-48+ cells) infiltration into brain ischemic areas. Our results demonstrated a protective effect of the chronic treatment of the A2BR agonist BAY 7 days after focal ischemia. The protective effects of BAY can be attributed to the stimulation of A2BR located both on central neural cells and on blood cells where A2BR are known to reduce activation and cytokine production thus attenuating neuroinflammation that develops days after ischemia. The evidence that hypoxic microenvironments elicit the expression of specific isoforms of carbonic anhydrase (CA), in particular CAIX and CAXII, through the hypoxia inducible factor, has allowed to hypothesize a possible CA relevance in ischemia. Recently it has been demonstrated that carbonic anhydrase inhibitors (CAIs), sulfonamide and coumarin, were able to improve neurological functionalities after cerebral ischemic insult. Preliminary data obtained in our laboratory in a model of in vitro ischemia demonstrated that two CAIs, acetazolamide and AN11-740 were able to prevent the appearance of anoxic depolarization (AD), a phenomenon strictly related to cell damage and death, 30 minutes after oxygen and glucose deprivation (OGD) condition in hippocampal slices. Based on this preliminary result, the aim of third study in my thesis was to investigate the putative protective effect of two CAIs, acetazolamide and AN11-740 in the in vivo model of permanent cerebral ischemia (pMCAo) in the rat. Sub-chronic treatment with acetazolamide and AN11-740 at the dose 4.4 mg/kg i.p. and 1.0 mg/kg i.p. significantly reduced the neurological deficit (p<0.0010.0001) and the infarct volume in cortex and striatum (p<0.001) 24 hours after ischemia. Treatment with the two CAIs, significantly reverted the decrease in the number of neurons (stained with the specific marker for neurons, NeuN+) induced by pMCAo. Twenty-four hours after focal ischemia, plasma inflammatory markers of brain damage TNF-α, is definitely increased while the levels of IL10 is decreased. The sub-chronic treatment with both carbonic anhydrase inhibitors, acetazolamide and AN11-740, didn’t modify significantly neither TNF-α or IL-10 plasma levels. Our results demonstrated a protective role of CA inhibitors at an early time (i.e. 24 hours) after in vivo ischemia. Likely protective effect of CAIs are attributable to a early direct effect of reduction of excitotoxicity in the first hours after brain ischemia.