Adenosine is an endogenous neuromodulator acting in the central nervous system (CNS) via four receptors subtypes (ARs: Adenosine receptors): A1ARs, A2AARs, A2BARs and A3ARs. The bioactive lipid Sphingosine 1-phosphate (S1P) is synthesised by sphingosine kinases (two isoforms: SphK1 and SphK2) and it is reversibly catabolised by S1P phosphatases and irreversibly by S1P lyase (S1L). The bioactive lipid acts as an extracellular ligand for a family of G-protein coupled receptors (S1PRs: S1P1-5Rs). Both A2AAR and S1PRs are express in neurons, astrocytes, oligodendrocytes and microglia. In the first part of the thesis, we performed our experiments using an ex vivo model of cerebral ischemia: the hippocampal slice. It is well known that hippocampus is a brain area particularly vulnerable to the ischemic insult. During cerebral ischemia, both adenosine and glutamate are released in large amounts. Extracellular glutamate overload generates exaggerated neuronal depolarization resulting in excitotoxic damage, revealed by the appearance of anoxic depolarization (AD), an unequivocal sign of tissue damage. Interesting, during excitotoxicity phase, an increase in free radicals can be found. Furthermore, a link between irreversible neuronal damage in ischemia with the increase in glutamate which promotes further depolarization, accumulation of intracellular Ca2+ concentrations, free radicals’ generation and an impairment of mitochondrial function was described. Our research group previously demonstrated that the selective antagonism of A2ARs delays AD appearance induced by severe oxygen glucose deprivation (OGD) insults in the CA1 region of hippocampus. In a rat model of transient global cerebral ischemia, 24 hours after the insult a S1L decrease was found, with a marked downregulation after 48 hours. Furthermore, the S1L antagonism is protective in a model of ischemia–reperfusion injury of the heart. Of note, in cerebral ischemia, S1PRs are upregulated starting 45 minutes after Intraluminal Middle Cerebral Artery Occlusion (MCAo) in mice and among S1P receptor mRNA showing the highest upregulated receptor is S1P3R. In addition, S1P3R and S1P2R activation presents a harmful effect: in mice, after MCAO, their pharmacological block concurred in the reduction of infarction area and microglia activation, respectively. Firstly, the effects of new A2AAR antagonists, alone (NPD151) or hybridized with an antioxidant molecule (NPD168, NPD201 and NPD132 conjugated with Edaravone, eda, or L-2-oxothiazolidine-4-carboxylic acid, OTC, respectively), during a severe oxygen and glucose deprivation insult (OGD, 30 minutes duration) was evaluated by extracellular recordings of field excitatory postsynaptic potentials (fEPSPs) in the CA1 region of rat hippocampus. We demonstrated that the new A2AAR antagonists and eda, antioxidant in clinical use for cerebral ischemia, delayed AD appearance during a severe OGD insult. Then, in the same model, we observed the putative neuroprotective effects of a selective S1L inhibitor and of two selective S1P3R and S1P2R antagonists, CAY10444 and JTE013, respectively. During a severe OGD insult, the compounds were able to significantly delay AD latency. In conclusion, the new A2AAR antagonists and the S1P modulators may represent putative neuroprotective compounds for stroke treatment. Then, we evaluated the role of A2AARs and S1P signaling in the cuprizone (CPZ) mice model, widely in the study of demyelination diseases, such as Multiple Sclerosis (MS). The patho-mechanism of MS and its mouse model counterpart experimental autoimmune encephalomyelitis (EAE) is characterized by the inflammatory autoreactive leukocytes egress in the CNS, where axonal myelin is destroyed, contributing to neurodegeneration. Remyelination does occur, but is limited especially in chronic disease stages. Among pathways that may contribute to ameliorated/reduced remyelination in MS the adenosinergic and S1P signaling are identified. Oligodendrocytes (OLs) are myelinating cells, exerting an important role in myelin sheet formation. Their progenitors are the Oligodendrocytes Precursor Cells (OPCs) that in physiological conditions may migrate, proliferate and maturate in OLs replacing myelin. In some pathologies, such as MS, this process fails or the myelin is not enough to repair the damage. Interesting, ARs are express during all stages of oligodendrogliogenesis and may represent new interesting molecular targets for drugs useful in demyelinating pathologies, such as MS. Previous findings in our laboratory demonstrated that the selective A2AAR agonist, CGS21680, applied in the culture medium of OLs modulates IK currents and by this mechanism, inhibits OPC differentiation. Furthermore, S1PRs, except S1P4R, are present in OPCs and OLs with a different pattern of expression during all maturational stages. Fingolimod, the first approved oral agent for treating relapsing-remitting MS, acts on all S1PRs, except S1P2R, inducing their internalization. The drug trap peripheral lymphocytes in the lymph nodes, reducing the lymphocytes egress in CNS, acting on S1P1R. In our experiments, we evaluated the role of A2AAR and S1P in the CPZ mice model, a toxic model of demyelination. CPZ administration induces OLs death and astrocytes and microglia activation. We fed animals with 0.2 % CPZ to induce an acute demyelination and, after the withdrawal, a spontaneous remyelination occurs. The role of adenosine receptors in this model was described: in A1AR-/- mice, CPZ feeding (for 4 weeks), lead to severe hippocampal demyelination while mice A2AAR-/- present a significant increase in myelin in comparison to WT mice that show an intermediate demyelination. These results suggest a protective role of A1AR activation and conversely a deleterious involvement of A2AAR in the development of myelin damage in this experimental model. In literature, emerged that Fingolimod prevented demyelination induced by CPZ, as indicated by a reduction of OLs death and the increase in OPCs proliferation, without effects on remyelination processes. A2AAR and Fingolimod exert a role in OPCs proliferation, maturation and survival; furthermore, they play a role in myelin damage modulation. We studied the ability of a selective A2AAR agonist or antagonist (CGS21680 and SCH58261, respectively) and of Fingolimod to modulate myelin damage in this experimental model. In a first protocol, we fed animals for 5 weeks with CPZ to induce demyelination and then administered the selective A2AAR agonist, CGS21680 for 2 weeks, to evaluate the involvement of this receptor subtype activation during remyelination after CPZ withdrawal. We successfully induced demyelination in CPZ fed-mice, as demonstrated by myelin damage observed in the corpus callosum (CC) with Luxol Fast Blue (LFB) staining and also confirmed by immunohistochemistry experiments, performed using anti-Myelin Basic Protein (MBP) antibodies. CGS21680-treated mice presented an exacerbated damage in CC, striatum caudate nuclei and motor cortex, indicating that the administration of the selective A2AAR agonist exacerbate the myelin damage induced by CPZ intake during remyelination. Then, we performed a different set of experiments using a second protocol. Mice were fed for 5 weeks with 0.2 % CPZ and, at the end of the third week, time able to induce an acute demyelination, we administered the A2AAR agonist or antagonist (CGS21680 and SCH58261, respectively) or the S1P analogue Fingolimod. In CC (Figure 2) and striatum, we demonstrated that CGS21680- and SCH58261-treated mice presented a significant demyelination in both areas; SCH58261 reduces the CGS21680 effect, but further experiments will be necessary. Finally, we observed a neuroprotective effect of Fingolimod on myelin damage in CC and striatum. In conclusion, in the CPZ mice model of de- remyelination, we showed a harmful role of A2AAR during both demyelination and remyelination phases and we confirmed a protective role of Fingolimod when administered during demyelination phase.

Targeting Adenosine A2A receptors or Sphingosine-1-phosphate signaling as promising neuroprotective strategies to ameliorate brain damage induced by oxygen and glucose deprivation or cuprizone intake / Clara Santalmasi. - (2025).

Targeting Adenosine A2A receptors or Sphingosine-1-phosphate signaling as promising neuroprotective strategies to ameliorate brain damage induced by oxygen and glucose deprivation or cuprizone intake

Clara Santalmasi
2025

Abstract

Adenosine is an endogenous neuromodulator acting in the central nervous system (CNS) via four receptors subtypes (ARs: Adenosine receptors): A1ARs, A2AARs, A2BARs and A3ARs. The bioactive lipid Sphingosine 1-phosphate (S1P) is synthesised by sphingosine kinases (two isoforms: SphK1 and SphK2) and it is reversibly catabolised by S1P phosphatases and irreversibly by S1P lyase (S1L). The bioactive lipid acts as an extracellular ligand for a family of G-protein coupled receptors (S1PRs: S1P1-5Rs). Both A2AAR and S1PRs are express in neurons, astrocytes, oligodendrocytes and microglia. In the first part of the thesis, we performed our experiments using an ex vivo model of cerebral ischemia: the hippocampal slice. It is well known that hippocampus is a brain area particularly vulnerable to the ischemic insult. During cerebral ischemia, both adenosine and glutamate are released in large amounts. Extracellular glutamate overload generates exaggerated neuronal depolarization resulting in excitotoxic damage, revealed by the appearance of anoxic depolarization (AD), an unequivocal sign of tissue damage. Interesting, during excitotoxicity phase, an increase in free radicals can be found. Furthermore, a link between irreversible neuronal damage in ischemia with the increase in glutamate which promotes further depolarization, accumulation of intracellular Ca2+ concentrations, free radicals’ generation and an impairment of mitochondrial function was described. Our research group previously demonstrated that the selective antagonism of A2ARs delays AD appearance induced by severe oxygen glucose deprivation (OGD) insults in the CA1 region of hippocampus. In a rat model of transient global cerebral ischemia, 24 hours after the insult a S1L decrease was found, with a marked downregulation after 48 hours. Furthermore, the S1L antagonism is protective in a model of ischemia–reperfusion injury of the heart. Of note, in cerebral ischemia, S1PRs are upregulated starting 45 minutes after Intraluminal Middle Cerebral Artery Occlusion (MCAo) in mice and among S1P receptor mRNA showing the highest upregulated receptor is S1P3R. In addition, S1P3R and S1P2R activation presents a harmful effect: in mice, after MCAO, their pharmacological block concurred in the reduction of infarction area and microglia activation, respectively. Firstly, the effects of new A2AAR antagonists, alone (NPD151) or hybridized with an antioxidant molecule (NPD168, NPD201 and NPD132 conjugated with Edaravone, eda, or L-2-oxothiazolidine-4-carboxylic acid, OTC, respectively), during a severe oxygen and glucose deprivation insult (OGD, 30 minutes duration) was evaluated by extracellular recordings of field excitatory postsynaptic potentials (fEPSPs) in the CA1 region of rat hippocampus. We demonstrated that the new A2AAR antagonists and eda, antioxidant in clinical use for cerebral ischemia, delayed AD appearance during a severe OGD insult. Then, in the same model, we observed the putative neuroprotective effects of a selective S1L inhibitor and of two selective S1P3R and S1P2R antagonists, CAY10444 and JTE013, respectively. During a severe OGD insult, the compounds were able to significantly delay AD latency. In conclusion, the new A2AAR antagonists and the S1P modulators may represent putative neuroprotective compounds for stroke treatment. Then, we evaluated the role of A2AARs and S1P signaling in the cuprizone (CPZ) mice model, widely in the study of demyelination diseases, such as Multiple Sclerosis (MS). The patho-mechanism of MS and its mouse model counterpart experimental autoimmune encephalomyelitis (EAE) is characterized by the inflammatory autoreactive leukocytes egress in the CNS, where axonal myelin is destroyed, contributing to neurodegeneration. Remyelination does occur, but is limited especially in chronic disease stages. Among pathways that may contribute to ameliorated/reduced remyelination in MS the adenosinergic and S1P signaling are identified. Oligodendrocytes (OLs) are myelinating cells, exerting an important role in myelin sheet formation. Their progenitors are the Oligodendrocytes Precursor Cells (OPCs) that in physiological conditions may migrate, proliferate and maturate in OLs replacing myelin. In some pathologies, such as MS, this process fails or the myelin is not enough to repair the damage. Interesting, ARs are express during all stages of oligodendrogliogenesis and may represent new interesting molecular targets for drugs useful in demyelinating pathologies, such as MS. Previous findings in our laboratory demonstrated that the selective A2AAR agonist, CGS21680, applied in the culture medium of OLs modulates IK currents and by this mechanism, inhibits OPC differentiation. Furthermore, S1PRs, except S1P4R, are present in OPCs and OLs with a different pattern of expression during all maturational stages. Fingolimod, the first approved oral agent for treating relapsing-remitting MS, acts on all S1PRs, except S1P2R, inducing their internalization. The drug trap peripheral lymphocytes in the lymph nodes, reducing the lymphocytes egress in CNS, acting on S1P1R. In our experiments, we evaluated the role of A2AAR and S1P in the CPZ mice model, a toxic model of demyelination. CPZ administration induces OLs death and astrocytes and microglia activation. We fed animals with 0.2 % CPZ to induce an acute demyelination and, after the withdrawal, a spontaneous remyelination occurs. The role of adenosine receptors in this model was described: in A1AR-/- mice, CPZ feeding (for 4 weeks), lead to severe hippocampal demyelination while mice A2AAR-/- present a significant increase in myelin in comparison to WT mice that show an intermediate demyelination. These results suggest a protective role of A1AR activation and conversely a deleterious involvement of A2AAR in the development of myelin damage in this experimental model. In literature, emerged that Fingolimod prevented demyelination induced by CPZ, as indicated by a reduction of OLs death and the increase in OPCs proliferation, without effects on remyelination processes. A2AAR and Fingolimod exert a role in OPCs proliferation, maturation and survival; furthermore, they play a role in myelin damage modulation. We studied the ability of a selective A2AAR agonist or antagonist (CGS21680 and SCH58261, respectively) and of Fingolimod to modulate myelin damage in this experimental model. In a first protocol, we fed animals for 5 weeks with CPZ to induce demyelination and then administered the selective A2AAR agonist, CGS21680 for 2 weeks, to evaluate the involvement of this receptor subtype activation during remyelination after CPZ withdrawal. We successfully induced demyelination in CPZ fed-mice, as demonstrated by myelin damage observed in the corpus callosum (CC) with Luxol Fast Blue (LFB) staining and also confirmed by immunohistochemistry experiments, performed using anti-Myelin Basic Protein (MBP) antibodies. CGS21680-treated mice presented an exacerbated damage in CC, striatum caudate nuclei and motor cortex, indicating that the administration of the selective A2AAR agonist exacerbate the myelin damage induced by CPZ intake during remyelination. Then, we performed a different set of experiments using a second protocol. Mice were fed for 5 weeks with 0.2 % CPZ and, at the end of the third week, time able to induce an acute demyelination, we administered the A2AAR agonist or antagonist (CGS21680 and SCH58261, respectively) or the S1P analogue Fingolimod. In CC (Figure 2) and striatum, we demonstrated that CGS21680- and SCH58261-treated mice presented a significant demyelination in both areas; SCH58261 reduces the CGS21680 effect, but further experiments will be necessary. Finally, we observed a neuroprotective effect of Fingolimod on myelin damage in CC and striatum. In conclusion, in the CPZ mice model of de- remyelination, we showed a harmful role of A2AAR during both demyelination and remyelination phases and we confirmed a protective role of Fingolimod when administered during demyelination phase.
2025
Anna Maria Pugliese
Clara Santalmasi
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